dispositivo medico chondrogrid · ricostruzione del tessuto cartilagineo articolare. per queste...
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Dispositivo Medico ChondroGrid®
Report di valutazione clinica: profilo di efficacia e sicurezza
Revisione 01 del 24.07.2016
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Sommario
Introduzione ......................................................................................................................................... 4
Standard seguiti per la realizzazione del documento. ...................................................................... 5
Autori................................................................................................................................................ 5
Caratteristiche del dispositivo .............................................................................................................. 6
Il dispositivo ChondroGrid ............................................................................................................... 6
Il meccanismo d’azione: il collagene e le sue potenzialità terapeutiche .......................................... 6
Le indicazioni d’uso e l’applicazione ............................................................................................... 7
Il ventaglio di indicazioni ................................................................................................................. 7
Un’alternativa sicura, almeno parziale, all’impiego degli antidolorifici .......................................... 8
La qualità farmaceutica della materia prima .................................................................................... 9
La sterilizzazione terminale e l’integrità dei peptidi ...................................................................... 10
Il dispositivo ChondroGrid: classificazione CE ............................................................................. 10
Bioteck: il Fabbricante ................................................................................................................... 11
Materiali e metodi di reperimento delle informazioni ....................................................................... 12
Reperimento degli articoli significativi .......................................................................................... 13
Creazione di un database bibliografico di riferimento ................................................................... 15
Analisi del database di riferimento ................................................................................................. 15
ChondroGrid: Profilo di sicurezza ..................................................................................................... 16
Considerazioni generali sulla sicurezza dell’impiego del collagene in ambito biomedico ............ 16
Valutazione dei rischi connessi all’uso di ChondroGrid ................................................................ 17
Possibile trasmissione di patogeni animali ..................................................................................... 18
Possibile trasmissione di encefalopatie spongiformi ...................................................................... 18
Possibile immunogenicità o mancanza di biocompatibilità ........................................................... 18
Test di citotossicità..................................................................................................................... 20
Test di sensibilizzazione allergica .............................................................................................. 20
Test di reattività intracutanea ..................................................................................................... 20
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Test di tossicità sistemica acuta ................................................................................................. 21
Pirogenicità ................................................................................................................................ 21
Test di tossicità subacuta............................................................................................................ 21
Test di genotossicità ................................................................................................................... 22
Test d’impianto .......................................................................................................................... 22
Valutazione del rischio residuo complessivo ................................................................................. 23
Valutazione da parte della autorità competenti degli altri Paesi Europei: ..................................... 23
Gli standard tecnici impiegati nella progettazione e fabbricazione del dispositivo ....................... 24
ChondroGrid: profilo di efficacia ...................................................................................................... 26
Effetti biologici del collagene e contestualizzazione dell’impiego di ChondroGrid ..................... 26
Studi in vitro ................................................................................................................................... 30
Studi in vivo ................................................................................................................................... 32
Studi clinici..................................................................................................................................... 33
Conclusioni ........................................................................................................................................ 36
Riepilogo dei risultati ......................................................................................................................... 37
Razionale biologico dell'utilizzo del dispositivo ............................................................................ 37
Razionale clinico dell'utilizzo del dispositivo ................................................................................ 37
Livello di evidenza relativo alla sicurezza dell'impiego clinico del dispositivo ............................ 37
Tabelle Riassuntive ............................................................................................................................ 39
Sicurezza del dispositivo ChondroGrid .......................................................................................... 39
Efficacia del dispositivo ChondroGrid. .......................................................................................... 40
Domande Frequenti – Frequently Asked Questions (FAQ) ............................................................... 42
Bibliografia ........................................................................................................................................ 45
Appendici ......................................................................................................................................... 130
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Introduzione
ChondroGrid è un dispositivo medico innovativo, destinato all’uso professionale, per il trattamento
sintomatologico e funzionale di numerose affezioni a carico delle articolazioni, dei tendini e dei
legamenti. ChondroGrid è innovativo in quanto è progettato e realizzato per permettere al paziente
di beneficiare al meglio delle proprietà terapeutiche del collagene, una proteina ampiamente diffusa
nel regno animale, nonché quella maggiormente rappresentata nel corpo umano ed il principale
costituente della matrice cartilaginea, dei tendini e dei legamenti.
Le due caratteristiche principali di ChondroGrid che permettono l’ottenimento del migliore effetto
terapeutico consistono a) nell’impiego di collagene in forma idrolizzata, a basso peso molecolare e
b) nella modalità di somministrazione, che avviene tramite iniezione intra- o peri-articolare.
Avere idrolizzato il collagene realizza due importanti vantaggi: a) il collagene in forma idrolizzata
si può facilmente sospendere in fase acquosa e b) esso può diffondere rapidamente, essendo i
peptidi molecole di dimensione limitata, negli spazi intra- e peri-articolari, distribuendosi su tutta la
superficie raggiunta dal liquido stesso.
La modalità di somministrazione, attraverso iniezione, permette di portare direttamente il collagene
idrolizzato al sito di interesse, ove è necessario che esso esplichi la sua azione. ChondroGrid,
quindi, rende possibile un’azione molto semplice: arricchire di collagene tutto l'ambiente intra-
articolare o peri-articolare soggetto a trauma o degenerazione o altra patologia.
Poiché le componenti articolari (matrice cartilaginea) e peri-articolari (tendini e legamenti) hanno,
come loro costituente principale, il collagene stesso e poiché, come sarà più oltre spiegato in
dettaglio, il collagene e i peptidi di origine collagenica sono in grado di unirsi fra loro
spontaneamente, il collagene idrolizzato sarà in grado immediatamente di fornire rinforzo
strutturale alla componente lesionata.
Inoltre, è stato provato che la presenza del collagene in forma peptidica è in grado, ad esempio nelle
cartilagini affette da osteoartrosi, di ridurre l’infiammazione e quindi il dolore a livello
dell’articolazione, contribuendo allo stesso tempo a migliorare il trofismo cellulare.
Scopo del presente report è fornire al lettore gli elementi necessari alla comprensione del
meccanismo d’azione del dispositivo ChondroGrid, e di illustrarne – nel contesto di una puntuale e
dettagliata valutazione clinica – i profili di sicurezza e di efficacia.
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Standard seguiti per la realizzazione del documento.
La valutazione clinica del dispositivo medico ChondroGrid descritta nel presente report è stata
eseguita secondo le indicazioni contenute nelle linee guida MEDDEV 2.7.1 Rev.3 del Dicembre
2009, pubblicate dal Direttorato Generale della Commissione Europea, settore beni di consumo,
cosmetici e dispositivi medici: “Guidelines on medical devices. Clinical evaluation: a guide for
manufacturers and notified bodies” (Linee guida sui dispositivi medici. Valutazione clinica: una
guida per i fabbricanti e gli organismi notificati).
La valutazione è basata su di un’analisi esaustiva dei dati clinici disponibili sul dispositivo in
relazione al suo uso inteso, al fine di evidenziare come il dispositivo soddisfi i requisiti di sicurezza
ed efficacia espressi dal suo fabbricante, sulla base di dati scientifici e clinici oggettivi,
evidenziando come l’impiego del dispositivo ChondroGrid per il suo inteso realizzi una situazione
per il paziente nella quale il beneficio supera abbondantemente il rischio residuo.
Autori
Il presente report è stato redatto da Clariscience S.r.l. – www.clariscience.com – azienda di
consulenza scientifica specificamente rivolta ai fabbricanti e distributori di dispositivi medici.
All'interno di Clariscience opera personale di estrazione biomedica ed esperti in affari regolatori
con esperienza più che decennale nel settore.
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Caratteristiche del dispositivo
Il dispositivo ChondroGrid
ChondroGrid è un dispositivo medico composto costituito da collagene idrolizzato a basso peso
molecolare, di origine bovina, liofilizzato, per iniezione intra- e peri-articolare.
CHondroGrid è indicato per il trattamento della sintomatologia dolorosa e della perdita di
funzionalità delle maggiori articolazioni (ginocchio, spalla, anca, polso e caviglia) e delle strutture
muscolo-tendinee e legamentose ad esse annesse, causate da patologie degenerative o dovute a esito
traumatico o a sovraccarico.
In Appendice A è riportato il foglietto illustrativo del dispositivo.
Il meccanismo d’azione: il collagene e le sue potenzialità terapeutiche
Il meccanismo d’azione di ChondroGrid si basa sulle molteplici caratteristiche possedute dal
collagene, struttura proteica come si è detto ubiquitaria la cui funzione biologica è sia strutturale che
funzionale. Da un punto di vista strutturale, in quanto proteina fibrillare in grado di autoassemblarsi
in modo gerarchico in fibre mano a mano più complesse (Kadler et al., 2008) esso garantisce la
creazione di strutture estremamente elastiche e, allo stesso tempo resistenti: sia nella costituzione
della matrice amorfa che compone la cartilagine, sia nelle strutture organizzate di connettivi
specializzati quali tendini e legamenti, sia infine in tessuti di sostegno quale quello osseo. A livello
funzionale, la sua azione si esplica in molteplici modi. Alcuni di questi, struttura-mediati,
consistono nella capacità di interazione con molecole diffusibili quali piccole proteine o ioni: in
questo senso il collagene può agire da filtro ionico o molecolare, facilitando la diffusione di alcuni
ioni o molecole e impedendo quella di altre (in funzione sia della carica che della dimensione
molecolare); esempio limite di questa proprietà del collagene di interagire con altri composti è la
capacità che esso dimostra nel tessuto osseo di autocatalizzare la propria mineralizzazione,
favorendo la formazione e deposizione sulla sua stessa fibra di cristalli di apatite ossea. Altri effetti,
invece, sono basati su precisi meccanismi biomolecolari e prevedono l'interazione tra porzioni della
molecola collagenica (anche di pochi amminoacidi) con specifici recettori localizzati a livello della
membrana cellulare di differenti tipi cellulari, siano essi precursori di cellule differenziate o cellule
già al termine del processo differenziativo. Si può quindi a ragione parlare del collagene, sia in
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senso lato che nello specifico ambito della medicina rigenerativa, come di una molecola ad azione
pleiotropica.
Il meccanismo d’azione del dispositivo Chondrogrid, quando iniettato per via intra-articolare
consiste nel distribuirsi del collagene a basso peso molecolare sulla superficie dell’articolazione
rinforzando la matrice cartilaginea, deteriorata dai processi patologici in atto, la cui impalcatura
sono formate da un fitto intreccio di fibre collagene. Il dispositivo svolge quindi un’azione
meccanica di rinforzo diretto di strutture collageniche indebolite e/o deteriorate, migliorando la
mobilità e contribuendo a ridurre la sintomatologia dolorosa a carico dell’articolazione. Il
meccanismo per via peri-articolare è simile: il collagene idrolizzato a basso peso molecolare agisce
quale rinforzo diretto all’impalcatura collagenica danneggiata delle strutture peri-articolari, quali
tendini e/o legamenti, la cui componente più abbondante è il collagene stesso - contribuendo ad una
riduzione del dolore e ad una più veloce ripresa funzionale.
Le indicazioni d’uso e l’applicazione
Le più comuni indicazioni di utilizzo di ChondroGrid sono il trattamento sintomatologico e
funzionale di: osteoartrosi, artrosinoviti acute o croniche secondarie ad artrosi o artrite reumatoide,
esiti di traumi o lesioni, affaticamento da sovraccarico a carico delle articolazioni sopra indicate.
ChondroGrid è indicato inoltre in caso di meniscopatie e in preparazione e come terapia di
mantenimento a seguito di interventi di meniscectomia, di ricostruzione legamentosa o di pulizia e/o
ricostruzione del tessuto cartilagineo articolare. Per queste indicazioni l’applicazione è eseguita per
via intra-articolare.
Per esiti di traumi e/o lesione peri-articolari o a carico di strutture muscolo-tendinee-ligamentose si
procede invece tramite iniezione peri-articolare e infiltrazione delle strutture coinvolte.
Il ventaglio di indicazioni
ChondroGrid, date le indicazioni per l’uso ed il meccanismo d’azione presentati nei paragrafi
precedenti, è indicato per il trattamento della sintomatologia dolorosa a carico di differenti
articolazioni in differenti distretti corporei, quali:
- l’anca
- le articolazioni vertebrali lombo-sacrali
- il ginocchio
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- la spalla
- le articolazioni minori, quali il gomito, la caviglia, il polso
sia che il dolore abbia origine dall’invecchiamento, da difetti posturali, patologie croniche o
cronico-degenerative, o traumi. Il dispositivo ha pertanto un ampio ventaglio di indicazioni, e può
essere d’ausilio nel trattamento di patologie particolarmente comuni, la cui incidenza nella
popolazione è destinata ad aumentare a causa dell’incremento della vita media cui si assiste, e si
assisterà, nel corso degli anni. Ulteriormente, per lo meno nei Paesi occidentali e industrializzati si
osserva un aumento di attenzione della popolazione alla qualità della vita, aumento che spinge i
pazienti a chiedere trattamenti più efficaci per tutte quelle patologie che sono potenzialmente
debilitanti e che possono mettere a rischio il benessere complessivo della persona.
Un’alternativa sicura, almeno parziale, all’impiego degli antidolorifici
In questo contesto, il dispositivo ChondroGrid fornisce all’utilizzatore medico la possibilità di
ridurre in modo sicuro l’impiego dei farmaci antidolorifici da banco o da prescrizione. Infatti,
ChondroGrid:
- agisce in modo locale nelle zone dolenti (e non per via sistemica)
- esibisce un tropismo naturale verso le strutture danneggiate (il collagene lega
spontaneamente altre strutture collageniche per formare strutture composte quali fibrille e
fibre)
- inoltre, e soprattutto, il dispositivo non presenta effetti collaterali o allergenicità (salvo il
caso in cui il paziente presenti un’ipersensibilità al collagene, per quanto rara) in quanto gli
xeno-collageni, un volta idrolizzati e sottoposti ad eliminazione dei telopeptidi N- e C-
terminali, perdono le loro proprietà immunogeniche (Cooperman L et al., 1984; De Lustro F
et al., 1987; DeLustro F et al., 1990).
L’impiego del dispositivo può quindi contribuire a ridurre la somministrazione di farmaci,
analgesici e FANS, che pur attenuando la sintomatologia dolorosa non esercitano alcuna azione sui
fattori scatenanti, ed espongono inoltre il paziente a potenziali effetti avversi quali la tossicità
gastrointestinale, il possibile aumento di effetti a carico del sistema cardiovascolare, renale ed
epatico. ChondroGrid, invece, fornisce in modo diretto piccole porzioni ed aminoacidi specifici
della struttura collagenica stessa, il che contribuisce al recupero strutturale e funzionale delle
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strutture intra- (cartilagine) e peri- (tendini e legamenti) articolari, migliorando così la
sintomatologia dolorosa e la funzionalità in modo sicuro ed efficace.
La qualità farmaceutica della materia prima
Da un punto di vista prettamente molecolare, il collagene (o, meglio, le famiglie dei collageni) non
differisce in modo sostanziale tra le diverse specie di Mammiferi, mostrando la sequenza
amminoacidica del protocollagene solo minime variazioni da una specie all’altra (Wada H et al.,
2006; Stover DA et al., 2011). Il dispositivo, dunque, potrebbe essere teoricamente fabbricato a
partire da collagene proveniente da qualunque specie di Mammifero. ChondroGrid è fabbricato a
partire da gelatina di collagene bovino, di grado farmaceutica e di altissima purezza. La scelta
dell’impiego di gelatina di collagene di origine bovina risiede nel fatto che la tecnologia alla base
della sua estrazione è oggi ampiamente collaudata in termini di qualità di prodotto finito, le cui
caratteristiche sono tali da superare tutti i requisiti estremamente stringenti che si applicano in
ambito farmaceutico.
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La sterilizzazione terminale e l’integrità dei peptidi
Il dispositivo ChondroGrid è sterilizzato, alla fine del processo per la sua fabbricazione, attraverso
irraggiamento beta (elettroni accelerati) a 25 kGy. Questo processo assicura che il prodotto finale
sia sterile, con un fattore di sicurezza conforme agli standard internazionali di riferimento uguale a
10-6. Gli elettroni accelerati sono radiazioni ionizzanti, ovvero radiazioni in grado di degradare gli
acidi nucleici batterici e/o virali, uccidendo così i microorganismi. Potrebbe quindi sorgere il
dubbio che tali radiazioni ionizzanti possano anche degradare la miscela peptidica di cui è composto
ChondroGrid. Questo non accade: il fabbricante ha infatti verificato, attraverso opportuni test
cromatografici, che il trattamento di sterilizzazione attraverso irraggiamento beta non è in grado di
degradare i peptidi presenti in ChondroGrid, come illustrato dall’immagine che segue.
Figura1. Il grafico mostra il profilo di eluizione in HPLC (High Performance Liquid Chromatography) di ChondroGrid
prima (rosso) e dopo (blu) la sterilizzazione attraverso irraggiamento beta. Ogni picco nel grafico corrisponde ad un
particolare peptide. Si può osservare come non vi sia differenza nella distribuzione dei picchi tra le due curve, a
dimostrazione del fatto che nessun peptide subisce degradazione a causa del processo di sterilizzazione.
Il dispositivo ChondroGrid: classificazione CE
Il dispositivo è un dispositivo impiantabile, di origine animale e destinato ad essere assorbito
completamente a seguito di impianto. Per questo motivo è classificato in classe III secondo la
direttiva di riferimento europea 93/42/EEC e s.m.i., relativa ai dispositivi medici. Il dispositivo è
registrato presso il Repertorio dei Dispositivi Medici del Ministero della Salute con numero di
iscrizione 1412112/R.
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Bioteck: il Fabbricante
ChondroGrid è prodotto da Bioteck S.p.A., azienda italiana leader mondiale nella fabbricazione di
dispositivi medici impiantabili di origine animale per rigenerazione tessutale. Bioteck opera in
questo settore dal 1995. Azienda a compagine sociale interamente italiana, applica processi
biotecnologici avanzati per la creazione di sostituti tessutali e dispositivi coadiuvanti per la
rigenerazione tessutale all’avanguardia. Attualmente, Bioteck è presente in più di 50 Paesi. I
dispositivi medici fabbricati da Bioteck sono impiegati con successo in diversi ambiti,
dall’Ortopedia, alla Neurochirurgia, alla Chirurgia Oro-maxillo-facciale.
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Materiali e metodi di reperimento delle informazioni
Le informazioni utilizzate per la redazione del presente report sono state raccolte attraverso due
canali.
Per tutto quanto riguarda il processo di fabbricazione di ChondroGrid, la tecnologia impiegata per
la sua realizzazione, le caratteristiche degli ambienti di fabbricazione, i test di biocompatibilità
eseguiti prima della sua immissione in commercio, si è analizzata e valutata la documentazione
tecnica, messa gentilmente a disposizione dal fabbricante, che lo stesso ha presentato in sede di
procedura di marcatura CE all’Organismo Notificato di riferimento (CE 0373, Istituto Superiore di
Sanità). Tali informazioni, analizzate, sono confluite principalmente nella definizione del profilo di
sicurezza del dispositivo.
Per quanto riguarda invece l’approfondimento relativo al profilo di efficacia del dispositivo si è
proceduto, oltre ad analizzare le informazioni fornite dal fabbricante, ad una specifica ricerca
bibliografica in merito, secondo le modalità di seguito descritte.
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Reperimento degli articoli significativi
Al fine di identificare i dati ritenuti pertinenti in letteratura è stata eseguita una ricerca bibliografica
interrogando il maggiore database biomedicale mondiale, PubMed, gestito dal National Institute of
Health (NIH) americano (www.pubmed.com ).
La ricerca, visto l'elevato numero di lavori sull'impiego del collagene in ambito biomedico e la
conseguente mole di articoli da analizzare, ha seguito un approccio "top-down" ove si è operato
inserendo nel motore di ricerca parole chiave sempre più stringenti in una procedura di "narrowing
down" al fine di identificare gli articoli realmente pertinenti secondo un processo di natura logico-
deduttiva.
Per ogni parola/frase chiave, constatata la dimensione del corrispondente insieme di articoli
corrispondenti, si è deciso di a) eseguire un "narrowing down" con parole chiave più stringenti
oppure b) analizzare gli abstract degli articoli reperiti nel database per identificare gli articoli
inerenti. Si è quindi proceduto al recupero, ove possibile, degli articoli stessi e all’analisi della
bibliografia in essi citata per il reperimento di ulteriori articoli inerenti. La soglia di cut-off relativa
all'operatività a) o b) è stata decisa arbitrariamente in 150 pubblicazioni. E' da notare che, nella
strategia di ricerca "narrowing down" anche i risultati delle ricerche ove non si sono analizzati gli
abstract relativi portano con sé un'informazione significativa, ovvero il numero di lavori inerenti.
Gli abstract degli articoli ritenuti pertinenti sono presentati in Bibliografia. Gli articoli scientifici
contenuti nel database di riferimento sono stati letti e valutati criticamente in funzione alla loro
relazione con ChondroGrid, identificando in primo luogo le analogie o le differenze di
composizione chimica tra i prodotti e/o i composti testati nelle pubblicazioni con quelle del
dispositivo; in secondo luogo – per gli studi su animale e clinici – si sono anche valutate le analogie
o le differenze con il dispositivo del Fabbricante per quanto riguarda la modalità di impiego del
dispositivo e, chiaramente, le indicazioni cliniche proposte dal Fabbricante in funzione dei risultati
attesi.
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La tabella seguente riporta le parole chiave utilizzate e i risultati della ricerca dopo il narrowing
down.
Parola/frase chiave
Numero di articoli reperiti
Numero di articoli inerenti
Ulteriori articoli reperiti dall’analisi della bibliografia degli articoli inerenti
Totale articoli inerenti
Low molecular weight hydrolyzed collagen
19 13 6 18
Intra-articular collagen injection
296 5 0 5
Tabella 1. Articoli reperiti sul sito http://www.ncbi.nlm.nih.gov/pubmed. La ricerca è stata eseguita in data 20 giugno 2016.
Ulteriormente, sono stati consultati database già in possesso degli autori raccoglienti le
pubblicazioni più significative, apparse su riviste indicizzate ed impattate, riguardanti la struttura
molecolare e le azioni biologiche principali del collagene in quanto componente delle matrici
extracellulari dei tessuti di origine connettivale.
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Creazione di un database bibliografico di riferimento
Gli articoli scientifici considerati inerenti al dispositivo del Fabbricante, sono stati quindi
raggruppati in un database generale, consultabile in bibliografia, e possono essere distinti per
tipologia di studio reperito come descritto nella tabella che segue.
Tipologia di studio Sottotipologia Numero di articoli inerenti
Studio in vitro 21
Studio su animale 25
Studio clinico (*) Case reports 1
Cross sectional surveys 1
Case-control studies 1
Cohort studies 2
Randomised controlled trials with non-definitive results (a point estimate that suggests a clinically significant effect but with confidence intervals overlapping the threshold for this effect)
0
Randomised controlled trials (RCT) with definitive results (confidence intervals that do not overlap the threshold clinically significant effect)
7
Systematic reviews and meta-analyses
28
Articoli reperiti e analizzati 84
Tabella 2. Composizione del database di riferimento.
(*) La classificazione degli studi clinici utilizzata è quella descritta in: Guyatt GH, Sackett DL, Sinclair JC, Hayward R, Cook DJ,
Cook RJ. Users' guides to the medical literature. IX. A method for grading health care recommendations. JAMA 1995; 274:1800-4.
Analisi del database di riferimento
Gli articoli scientifici e clinici contenuti nel database di riferimento sono stati letti e valutati
criticamente in funzione alla loro relazione con il dispositivo, identificando in primo luogo le
analogie o le differenze di composizione chimica tra i prodotti e/o i composti testati nelle
pubblicazioni con quelle del dispositivo stesso; in secondo luogo – per gli studi su animale e clinici
– si sono anche valutate le analogie o le differenze con il dispositivo ChondroGrid per quanto
riguarda la modalità di impiego e, chiaramente, le indicazioni cliniche proposte in funzione dei
risultati attesi.
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ChondroGrid: Profilo di sicurezza
Considerazioni generali sulla sicurezza dell’impiego del collagene in ambito biomedico
Un’analisi ad ampio respiro dell’impiego del collagene come biomateriale mostra che esso investe
vari ambiti quali l’impiego come materiale con attività osteogenica, per la sua attività pro-
coagulante, come rivestimento per la guarigione di ferite e ustioni. Recentemente è stato usato
anche come veicolo per numerosi farmaci; in questi casi il collagene viene usato sotto forma di
microsfere e reti la cui struttura è costituita appunto da tale sostanza; da ricordare inoltre l’esistenza
di membrane costituite da collagene sintetico in oftalmologia e i composti costituiti da collagene
liposomiale destinato al rilascio di farmaci (Wakitani et al., 1989). Il collagene è anche utilizzato
dall’ingegneria biomedica come substrato fisico per la creazione in vitro di pelle, vasi sanguigni e
delle valvole cardiache. I film di collagene sono usati anche come matrice per la valutazione della
calcificazione tissutale negli studi sulle neoplasie (Lee et al., 2001). Sono descritti in letteratura usi
del collagene iniettabile anche in ambito urologico (reflusso vescicouretrale, trattamento
dell’incontinenza urinaria) e in ambito gastroenterico (trattamento del reflusso gastroesofageo
trattamento dell’insufficienza della glottide) (Aragona et al., 1998). Il collagene è usato in ambito
chirurgico in vari campi quali la chirurgia cardiovascolare, la neurochirurgia, la chirurgia plastica,
nonché nella fase ricostruttiva delle ferite e come emostatico. Il collagene di tipo I è usato
comunemente nella costituzione dei dispositivi medici impiantabili (Yunus Basha et al., 2015), ad
esempio in interventi di rigenerazione ossea, sotto forma di materiale spugnoso, anche per la
costituzione di matrici atte a prolungare la permanenza delle proteine morfogenetiche dell’osso
(bone morphogenetic proteins, BMPs), o come scaffold per dare sostegno all’invasione dei vasi e
delle cellule osteoprogenitrici (Geiger et al., 2003) anche in combinazione con l’idrossiapatite per la
costituzione innesti ossei (Wahl et al., 2006). Non sono riportati in letteratura problemi di
interazione del collagene con farmaci o eccipienti utilizzati per la loro fabbricazione (Lucey P et al,
2014).
Il collagene idrolizzato è considerato in primis un alimento, per ragioni storiche connesse al suo
impiego, e per questo tossicità ed effetti collaterali sono già stati ampiamente valutati. La FDA lo
definisce infatti come “Generally Recognized As Safe” (GRAS), ponendolo nella categoria più
elevata di sicurezza, la stessa categoria degli aminoacidi essenziali, ma anche del sale, dello
zucchero, delle vitamine. Nessun ente nazionale o internazionale impone restrizioni all’uso del
collagene, riconoscendo l’impiego del collagene idrolizzato come additivo alimentare sicuro e come
integratore autorizzato in ambito medico. La tossicità del collagene si osserva a dosi così elevate
17
(Takeda et al., 1982) da potere affermare con sicurezza che non sussiste la possibilità di una
tossicità sistemica nell’impiego del dispositivo ChondroGrid. Ulteriori studi sul collagene
idrolizzato condotti su Salmonella, E. coli e midollo osseo di Hamster attraverso il Test di Ames
non hanno mostrato né effetti di mutagenicità né carcinogenicità (come si vedrà poi tali risultati
sono confermati da tutta la batteria di test di biocompatibilità condotti sul dispositivo
ChondroGrid).
Il collagene impiantato, idrolizzato o meno, è degradato dall’azione di una serie di enzimi
collagenolitici rilasciati da diverse popolazioni cellulari, compresi i granulociti neutrofili e i
macrofagi (Patino et al., 2002). I casi di infiammazione indotta dal collagene, tuttavia, sono
estremamente rari. In ambito di impianto dermico, ad esempio, è stata riscontrata un frequenza di
infiammazione indotta da collagene in soli 72 casi su oltre 1 milione presi in considerazione (Cukier
et al, 1993). L’impiego ormai più che decennale di numerosi dispositivi medici costituiti da
collagene per la rigenerazione e la riparazione dei tessuti sia molli che duri confermano come
l’impianto non crei fenomeni né di irritazione locale né di tossicità sistemica: evidenziando che
un’eventuale risposta immunitaria nei confronti dei loro costituenti collagenici non predispone
comunque a fenomeni di ipersensibilità successivi (DeLustro F et al., 1990). Come per tutte le
proteine, esiste anche per il collagene ed i suoi derivati la possibilità, per quanto rara (Charriere G et
al. 1989), di causare sensibilizzazione e reazioni allergiche in singoli pazienti predisposti. Tale
eventualità, opportunamente segnalata nella documentazione che accompagna il dispositivo, deve
essere accertata attraverso un’accurata anamnesi nei confronti del paziente.
Valutazione dei rischi connessi all’uso di ChondroGrid
I possibili rischi connessi all’impiego del dispositivo sono stati valutati attraverso l’esecuzione di
opportuni test e l’attenta analisi del processo produttivo, dalla materia prima di origine animale fino
alla sterilizzazione terminale.
Per quanto riguarda i due rischi maggiori connessi all’origine animale, e specificamente bovina,
della materia prima si devono considerare due fattori: 1) il processo di produzione dell’idrolizzato
di collagene è un processo virus- e prione-inattivante e 2) la materia prima da cui è estratto
l’idrolizzato proviene da Paesi non considerati a rischio di BSE. Inoltre, gli animali le cui parti sono
impiegate per la produzione dell’idrolizzato di collagene sono soggetti a tutti i controlli europei
previsti per la tracciabilità degli animali destinati al consumo umano. In sintesi, dato un lotto di
prodotto è sempre possibile risalire all’animale da cui la materia prima derivava, e viceversa. Più in
18
particolare, per i diversi rischi connessi alla sicurezza del dispositivo valgono le considerazioni che
seguono:
Possibile trasmissione di patogeni animali
Il processo produttivo prevede il trattamento con basi ed acidi forti a concentrazioni tali
da inattivare qualunque virus/patogeno eventualmente presente nella materia prima.
Ulteriormente, il processo terminale di sterilizzazione a raggi beta a 25 kGy introduce un
ulteriore fattore di riduzione della carica microbiologica pari a 10-6 (che rappresenta già
un fattore di sicurezza adeguato in funzione degli standard tecnici di riferimento, quali
lo standard ISO11137:2007).
Possibile trasmissione di encefalopatie spongiformi
Il processo produttivo, sempre per la presenza di basi e acidi forti ad alta concentrazione,
è in grado di inattivare eventuali prioni presenti nella materia prima (cfr. ad esempio
Grobben AH et al, 2004).
In più, gli animali utilizzati per l’estrazione del collagene provengono da Paesi
considerati intrinsecamente sicuri per la presenza di BSE. Tutto questo è certificato
dall’EDQM (European Directorate for the Quality of Medicine) con apposita
certificazione, disponibile su richiesta
Possibile immunogenicità o mancanza di biocompatibilità
è noto che il collagene è utilizzato da tempo in medicina come materiale impiantabile, e
si è dimostrato intrinsecamente sicuro nella sua lunga storia d’impiego (a meno di
ipersensibilità individuale al collagene, peraltro rare e facilmente diagnosticabili –
Charriere G et al. 1989).
Il dispositivo è stato inoltre oggetto di una serie di prove di biocompatibilità nel quadro
concettuale di quanto prescritto dalla ISO10993, Biological evaluation of medical
devices – Part 1: Evaluating and testing e più precisamente:
19
Prova Standard tecnico Esito
Test di Citotossicità ISO 10993-5
Superati positivamente
Test di Pirogenicità/Lal Test Eu.Ph. § 2.6.14
Test di Mutagenesi (Ames) ISO 10993-3 ISO 10993-12
Test di Reattività cutanea (intradermica) ISO 10993-10
Test di Ipersensibilità ritardata ISO 10993-10
Test di Tossicità subacuta ISO 10993-2 ISO 10993-11 ISO 10993-12
Test di Tossicità sistemica acuta ISO 10993-11
Test di impianto ISO 10993-6
Ciascun test è descritto con maggior dettaglio nei paragrafi che seguono:
20
Test di citotossicità
Obiettivo del test: tramite l’uso della tecnica di coltura cellulare, il test di citotossicità
determina l’eventuale lisi cellulare, la morte cellulare, l’inibizione alla crescita cellulare
e altri effetti indotti dal rilascio di eventuali sostanze dal dispositivo medico.
Metodo utilizzato: Cytotoxicity test MEM elution - UNI EN ISO 10993-5: Tests for in
vitro cytotoxicity; Protocollo interno PI 07 “Test di citotossicità in vitro”.
Test di sensibilizzazione allergica
Obiettivo del test: il test di sensibilizzazione allergica valuta il potenziale sensibilizzante
dell’estratto del dispositivo medico sottoposto ad analisi tramite contatto diretto con la
cute. Il test di sensibilizzazione allergica è attendibile poiché valuta un’eventuale
sensibilizzazione indotta dal rilascio di sostanze sensibilizzanti dopo un’esposizione
prolungata.
Metodo utilizzato: Guinea Pig Magnusson Kligman Maximisation Test - ISO 10993-10:
Tests for irritation and delayed-type hypersensitivity
Test di reattività intracutanea
Obiettivo del test: il test di reattività intracutanea valuta la reazione locale cutanea
all’estratto di un dispositivo medico; questo test è utilizzabile allorché la determinazione
di reattività dermica e mucosale risulti essere inapplicabile.
Metodo utilizzato: Intracutaneous reactivity test in the rabbit - ISO 10993-10: Tests for
irritation and delayed-type hypersensitivity.
21
Test di tossicità sistemica acuta
Obiettivo del test: il test di tossicità sistemica acuta valuta i potenziali effetti dannosi di
singole o multiple esposizioni, durante l’arco delle 72 h, agli estratti del dispositivo
medico in analisi, su di un modello animale. Il test è indicato nel caso in cui il contatto
col dispositivo permetta il rilascio di sostanze o di prodotti di degradazione.
Metodo utilizzato: Acute systemic toxicity test in the mouse - ISO 10993-11: Tests for
systemic toxicity.
Pirogenicità
Obiettivo del test: il test di pirogenicità rileva reazioni pirogeniche indotte dagli estratti
dei dispositivi medici.
Metodo utilizzato: Pyrogen test in the rabbit – Ph. Eur., § 2.6.14 Bacterial endotoxins.
Method D (Kinetic-chromogenic test).
Test di tossicità subacuta
Obiettivo del test: Il Test di tossicità subacuta valuta gli effetti di esposizione multipla o
di contatto prolungato con l’estratto del dispositivo medico su di un modello animale
per un periodo protratto di giorni. Questo studio dovrebbe garantire una base razionale
per stabilire un’ analisi del rischio in ambito umano.
Metodo utilizzato: Rabbit sub-acute systemic toxicity test- ISO 10993-11: Tests for
systemic toxicity; Rabbit sub-acute systemic toxicity
22
Test di genotossicità
Obiettivo del test: Tale batteria di test usa colture cellulari di mammiferi e non, oppure
anche altre tecniche per determinare mutazioni genetiche, alterazioni nel numero dei
cromosomi o delle strutture cromosomiche, e del DNA; viene indagata anche tossicità
genetica causata dagli estratti dei dispositivi medici.
Metodo utilizzato: Ames test - UNI EN ISO 10993-3: Tests for genotoxicity,
carcinogenicity and reproductive toxicity; OECD 471.
Metodo utilizzato: MLA Test - UNI EN ISO 10993-3: Tests for genotoxicity,
carcinogenicity and reproductive toxicity; OECD 476.
Test d’impianto
Obiettivo del test: Questo test determina l’effetto locale patologico dell’impianto del
dispositivo medico su tessuti vivi, similari a quelli ai quali è destinato il dispositivo, sia
a livello macroscopico/necroscopico che a livello istologico.
Metodo utilizzato: Implantation, 28 days, in the rabbit - UNI EN ISO 10993-6: Tests for
local effects after implantation.
23
Valutazione del rischio residuo complessivo
Il rischio residuo complessivo relativo all’utilizzo del dispositivo per il suo uso inteso è
stato oggetto di analisi del rischio secondo gli standard tecnici:
• ISO 22442:2015 Animal tissues and their derivatives utilized in the
manufacture of medical devices
• ISO 14971:2012 Medical devices - Application of risk management to
medical devices
concludendo che il rischio residuo complessivo è risultato trascurabile in
rapporto al beneficio che il dispositivo apporta al paziente.
Valutazione da parte della autorità competenti degli altri Paesi Europei:
Il dispositivo soddisfa inoltre i requisiti espressi nel Regolamento Europeo 722/2012
(COMMISSION REGULATION (EU) No 722/2012 - of 8 August 2012 - concerning
particular requirements as regards the requirements laid down in Council Directives
90/385/EEC and 93/42/EEC with respect to active implantable medical devices and
medical devices manufactured utilising tissues of animal origin).
Si noti che, in virtù del suddetto Regolamento, il profilo di sicurezza ed il rapporto
beneficio/rischio del dispositivo ChondroGrid sono stati valutati con esito positivo non
solo dall’Organismo Notificato che ha rilasciato le certificazione CE di conformità ai
requisiti della Direttiva sui dispositivi medici (Istituto Superiore di Sanità, Organismo
Notificato 0373) ma anche da ciascuna Autorità Competente (ovvero, Ministeri della
Salute) dei Paesi che seguono: Austria, Belgio, Bulgaria, Croazia, Repubblica Ceca,
Cipro, Danimarca, Estonia, Finlandia, Francia, Germania, Grecia, Ungheria, Islanda,
Lituania, Lussemburgo, Malta, Olanda, Norvegia, Polonia, Portogallo, Romania,
Slovacchia, Spagna, Svezia, Slovenia, Spagna, Svizzera, Turchia, Gran Bretagna.
24
Gli standard tecnici impiegati nella progettazione e fabbricazione del dispositivo
Ad ulteriore supporto della sicurezza intrinseca al dispositivo ChondroGrid, si citano di seguito gli
standard tecnici seguiti nella progettazione e fabbricazione del dispositivo. Gli standard tecnici
definiscono dei metodi condivisi a livello mondiale per verificare, da un punto di vista tecnico, i
requisiti di sicurezza ed efficacia relativi ai dispositivi medici, in ogni loro parte. L’adozione di
precisi standard tecnici è ulteriore garanzia che gli specifici requisiti siano effettivamente verificati,
e che la conformità del dispositivo alla Direttiva europea di riferimento – in termini di sicurezza ed
efficacia - sia effettivamente provata
Standard tecnico Titolo
ICH guidelines, 06-2003 Q1A (R2) Stability Testing of New Drug Substances and Products
ASTM F1929-98:2004 Standard Test Method for Detecting Seal Leaks in Porous Medical Packaging by Dye Penetration
ASTM F1608-00:2009 Standard Test Method for Microbial Ranking of Porous Packaging Materials ( Exposure Chamber Method).
ASTM F1886-09:2009 Standard Test Method for Determining Integrity of Seals for Flexible Packaging by Visual Inspection
ASTM F1980-07:2011 Standard Guide for Accelerated Aging of Sterile Barrier Systems for Medical Devices
EU-GMP:2008, Annex 1 Good manufacturing practice (GMP) Guidelines
ISO 11737-1:2006 Sterilization of medical devices -- Microbiological methods -- Part 1: Determination of a population of microorganisms on products
ISO 11737-2:2009 Sterilization of medical devices -- Microbiological methods -- Part 2: Tests of sterility performed in the definition, validation and maintenance of a sterilization process
ISO 14698-1:2003 Cleanrooms and associated controlled environments -- Biocontamination control -- Part 1: General principles and methods
ISO 14698-2:2003 Cleanrooms and associated controlled environments -- Biocontamination control -- Part 2: Evaluation and interpretation of biocontamination data
ISO 187:1990 Paper, board and pulps -- Standard atmosphere for conditioning and testing and procedure for monitoring the atmosphere and conditioning of samples
ISO/ASTM 51261:2002 Practice for calibration of routine dosimetry systems for radiation processing
ISO/ASTM 51707:2005 Guide for estimation of measurement uncertainty in dosimetry for radiation processing
Italian Ph. Ed. XII, § 2.6.1 Italian Pharmacopea
OECD 471 Ed. 1997 Bacterial Reverse Mutation Test
OECD 476 Ed. 1997 In vitro Mammalian Cell Gene Mutation Test
Ph. Eur. Ed. VII, § 2.6.1 European Pharmacopea
Ph. Eur. Ed. VII, § 2.6.14 European Pharmacopea
UNI CEI EN 1041:2013 Information supplied by the manufacturer of the medical devices
UNI CEI EN ISO 14971:2012 Medical devices - Application of risk management to medical devices
UNI CEI EN ISO 15223-1:2012 Medical devices - Symbols to be used with medical device labels, labelling and information to be supplied - Part 1: General requirements
UNI EN 556-1:2002 Sterilization of medical devices - Requirements for medical devices to be designated "STERILE" - Part 1: Requirements for terminally sterilized medical devices
UNI EN 556-2:2005 Sterilization of medical devices - Requirements for medical devices to be designated "STERILE" - Part 2: Requirements for aseptically processed medical devices
UNI EN 868-5:2009 Packaging for terminally sterilized medical devices - Part 5: Sealable pouches and reels of porous materials and plastic film construction - Requirements and test methods
UNI EN ISO 10993-1:2009 Biological evaluation of medical devices -- Part 1: Evaluation and testing within a risk management process
25
UNI EN ISO 10993-10:2002 Biological evaluation of medical devices -- Part 10: Tests for irritation and skin sensitization
UNI EN ISO 10993-11:2006 Biological evaluation of medical devices -- Part 11: Tests for systemic toxicity
UNI EN ISO 10993-12:2007 Biological evaluation of medical devices -- Part 12: Sample preparation and reference materials
UNI EN ISO 10993-2: 2006 Biological evaluation of medical devices -- Part 2: Animal welfare requirements
UNI EN ISO 10993-5:2009 Biological evaluation of medical devices -- Part 5: Tests for in vitro cytotoxicity
UNI EN ISO 10993-6:2009 Biological evaluation of medical devices -- Part 6: Tests for local effects after implantation
UNI EN ISO 11137-1:2006 Sterilization of health care products -- Radiation -- Part 1: Requirements for development, validation and routine control of a sterilization process for medical devices
UNI EN ISO 11137-2:2012 Sterilization of health care products -- Radiation -- Part 2: Establishing the sterilization dose
UNI EN ISO 11137-3:2006 Sterilization of health care products -- Radiation -- Part 3: Guidance on dosimetric aspects
UNI EN ISO 11138-1:2006 Sterilization of health care products -- Biological indicators -- Part 1: General requirements
UNI EN ISO 11607-1:2009 Packaging for terminally sterilized medical devices -- Part 1: Requirements for materials, sterile barrier systems and packaging systems
UNI EN ISO 11607-2:2006 Packaging for terminally sterilized medical devices -- Part 2: Validation requirements for forming, sealing and assembly processes
UNI EN ISO 13485: 2012 Medical devices -- Quality management systems -- Requirements for regulatory purposes
UNI EN ISO 14644-1:2001 Cleanrooms and associated controlled environments -- Part 1: Classification of air cleanliness by particle concentration
UNI EN ISO 14644-2:2001 Cleanrooms and associated controlled environments -- Part 2: Monitoring to provide evidence of cleanroom performance related to air cleanliness by particle concentration
UNI EN ISO 14644-3:2006 Cleanrooms and associated controlled environments -- Part 3: Test methods
UNI EN ISO 22442-1:2008 Medical devices utilizing animal tissues and their derivatives -- Part 1: Application of risk management
UNI EN ISO 9001:2008 Quality management systems -- Requirements
26
ChondroGrid: profilo di efficacia
L'efficacia di un dispositivo medico è comprovata da tutto l'insieme di dati che dimostrano come,
per l'uso inteso del dispositivo, la sua applicazione conduca efficacemente al risultato atteso. I dati
possono essere diretti, derivanti cioè dall'applicazione pre-clinica e clinica dello specifico
dispositivo oggetto d'esame, o indiretti – a loro volta suddivisibili concettualmente in a) evidenza
dell'equivalenza del dispositivo oggetto di esame con uno o più dispositivi equivalenti e b) analisi
della letteratura inerente l'applicazione dei costituenti di cui il dispositivo è composto. Nel presente
report si è voluto dare un quadro generale dei dati disponibili, comprendendo anche quelli indiretti,
in quanto si ritiene – nel caso particolare del dispositivo in esame – che essi permettano anche di
meglio comprendere quello che è il meccanismo d'azione del dispositivo e quindi di fornire al
lettore un quadro maggiormente esaustivo ed utile a comprendere l'effettiva utilità del preparato.
Effetti biologici del collagene e contestualizzazione dell’impiego di ChondroGrid
Gli effetti biologici del collagene sulle strutture coinvolte nella patologia degenerative, traumatica o
da sovraccarico e sulle cellule che le compongono sono molteplici. Importante è la funzione che il
collagene esercita di filtro molecolare, come componente della matrice extracellulare, modulando
la diffusione di molecole diffusibili (Toroian D et al. 2007). A livello di orientamento di
popolazioni cellulari è noto che il collagene possiede sequenze segnale in grado di favorire
l'adesione ad esso di diversi tipi di popolazioni cellulari (Ruoslahti E et al. 1987). Vi è un indubbio
effetto sull'adesione di cellule mesenchimali, anche di derivazione midollare (Liu G et al, 2004,
Chan CK et al. 2009; Beitzel K et al. 2014). Tale adesione, che gioca un ruolo chiave nei primi
eventi di ripopolamento del sito lesionato, avviene attraverso il riconoscimento, come si è detto, di
specifiche sequenze amminoacidiche presenti nella catena collagenica. Tali sequenze sono
riconosciute da una specifica famiglia di recettori a livello della membrana cellulare delle cellule
mesenchimali, le integrine. Il legame integrina-collagene, oltre a permettere l'adesione della cellula
alla matrice funge inoltre da segnale per ulteriori eventi intracellulari, prime fra tutti le
modificazioni delle strutture citoscheletriche (Shen B et al. 2012; Das M et al.2014) responsabili
della migrazione cellulare. A livello sopramolecolare, il collagene costituisce il componente
principale delle matrici extracellulari, tanto che esso è largamente impiegato in ingegneria tessutale
nella produzione di sostituti tessutali che, mimino la matrice extracellulare naturale al fine di
favorire e guidare lo sviluppo di nuovo tessuto funzionale (Cunniffe GM et al. 2011, Aigner T et al,
27
2003) ed è stato oggetto per questo di numerosi studi relativi alla sua applicazione in ingegneria
tessutale nella rigenerazione di diversi tessuti, (Lee YB et al. 2010; Chen Z et al. 2011; Sumita Y et
al. 2006, Zhang Y et al. 2006; Gungormus, et al 2004;(Ruszczak Z, 2003), (Davidenko N et al.
2010),data anche la sua abbondanza nel regno animale ed il fatto che le porzioni antigeniche
specie-specifiche, che consistono nelle sole porzioni terminali della molecola (Davison PF et al.
1967; Michaeli D et al. 1969; Furthmayr et al. 1971), possono facilmente essere eliminate per
digestione enzimatica tramite pepsina. E’ in questo contesto più ampio che si colloca l’impiego
medico del collagene idrolizzato iniettabile: il collagene, anche nella sua forma idrolizzata, gioca un
ruolo fondamentale nel trattamento sintomatologico e funzionale delle cartilaginopatie e nelle
affezioni muscolo-tendinee in quanto le strutture anatomiche coinvolte ne sono costituite in maggior
parte: così che gli eventi di danno degenerativo o traumatico coinvolgono sempre, in qualche
misura, questa molecola. La cartilagine articolare è un tessuto altamente specializzato, non
vascolarizzato e privo di terminazioni nervose, la cui matrice è sintetizzata da cellule specializzate, i
condrociti. A livello sopramolecolare, la matrice cartilagine consiste di due componenti principali,
una fibrillare ed una non-fibrillare. La componente fibrillare è una fitta rete costituita
principalmente di collagene di tipo II, e in minore quantità di collagene di tipo IX e XI. La
componente non fibrillare consiste di monomeri di aggrecano, acido ialuronico, proteine in forma di
aggregati polianionici. E’ la componente fibrillare collagenica che conferisce alla cartilagine le sue
proprietà tensili di resistenza alla compressione, mentre le molecole di aggrecano ne modulano,
assorbendo e rilasciando la componente liquida, le proprietà viscoelastiche (Aigner et al, 2002). Nel
corso dell’instaurarsi della patologia artrosica, si osserva sia la degradazione delle componenti
molecolari sia la destabilizzazione delle strutture sopramolecolari, compresa la distruzione della
matrice collagenica visibile prima solo microscopicamente e poi anche a livello macroscopico. La
degradazione avviene sia per usura meccanica che per degradazione enzimatica, in presenza di
processi anabolici della matrice che divengono alterati, così come il profilo di espressione genica
dei condrociti. L’importanza di un aumentato apporto di collagene nel momento dell’instaurarsi
della patologia, anche nelle prime fasi di destabilizzazione ancora osservabili solo per via
microscopica, è evidenziata dal fatto che gli stessi condrociti, in questa condizione, aumentano la
loro attività di sintesi di collagene di tipo II (Aigner et al., 2002). E’ chiaro quindi che già nelle
prime nelle prime fasi della patologia, ove si assiste al cosiddetto “network loosening” della
struttura fibrillare collagenica, l’apporto di collagene esogeno è da considerarsi favorevole in
quanto rende possibile il rinforzo della struttura di matrice fibrillare in corso di deterioramento,
mimando un evento riparativo (la biosintesi di collagene da parte dei condrociti) naturale. Nel
tessuto tendineo/legamentoso la sequenza degli eventi al sito di danno è tradizionalmente divisa in
28
tre fasi sovrapposte (Yang et al. 2013, Hope e Saxby, 2007) - (1) infiammazione, (2) la
proliferazione / riparazione, e (3) rimodellamento Nella fase infiammatoria, il coagulo di sangue
che si forma immediatamente dopo la rottura dei vasi del tendine attiva il rilascio di fattori
chemiotattici e serve come impalcatura preliminare per l'invasione. Cellule infiammatorie, tra cui
neutrofili, monociti e linfociti migrano dai tessuti circostanti nel sito della ferita, dove digeriscono
tramite fagocitosi i detriti necrotici (Voleti et al., 2012). Inoltre, in questa fase inizia il reclutamento
e l'attivazione di tenociti inizia, che raggiungeranno un picco nella fase successiva. La seconda fase,
detta fase proliferativa o riparativa, inizia circa due giorni dopo il verificarsi del danno. I fibroblasti
del paratenon o della circostante guaina sinoviale sono reclutati verso la zona lesionata e
proliferano, in particolare nel epitenon (Garner et al., 1989). Allo stesso modo, tenociti intrinseci
localizzati nell'endotenon ed epitenon migrano verso il sito lesionato ed iniziano a proliferare.
Entrambe le fonti di tenociti sono importanti nella sintesi della matrice extracellulare e nella
creazione di una rete interna neovascolare (James et al., 2008). Allo stesso tempo, i livelli di
neutrofili diminuiscono mentre i macrofagi continuano a rilasciare fattori di crescita che dirigono il
reclutamento e l'attività dei diversi tipi cellulari (Voleti et al., 2012). In questa fase iniziale di
guarigione, la matrice sintetizzata dai tenociti è composta di una maggiore quantità di collagene di
tipo III (Juneja et al., 2013). Le concentrazioni di contenuto d'acqua e glicosamminoglicani in
questa fase sono particolarmente elevate (Sharma e Maffulli, 2006). Infine, 1-2 mesi dopo il
verificarsi della lesione ha inizio la fase di rimodellamento. Tenociti e fibre collagene si allineano
alla direzione del carico e viene sintetizzata una maggiore percentuale di collagene di tipo I è
(Abrahamsson, 1991), con una corrispondente riduzione della cellularità, del collagene di tipo III e
del contenuto di glicosamminoglicani (Sharma e Maffulli, 2006). Dopo 10 settimane, il tessuto
fibroso diviene gradualmente un tessuto tendineo simil-cicatriziale, un processo che continua per
anni. Il tessuto riparato non riacquista mai completamente le proprietà biomeccaniche che aveva
prima del danno e le caratteristiche biochimiche e ultrastrutturali rimangono anormali anche a 12
mesi dalla lesione (Miyashita et al., 1997). Appare dunque chiaro che, tra gli eventi principali che
conducono alla guarigione dei tendini e dei legamenti, vi è la sintesi di matrice extracellulare che,
come si è visto ha come componente principale il collagene di tipo I. Anche in questo caso, quindi,
l’apporto di collagene esogeno, in modo mirato e localizzato al sito di danno, realizza una
condizione favorevole al rinforzo strutturale della parte affetta. Ne è conferma la presenza di studi
in cui altri dispositivi impiantabili a base di collagene, anche se non in forma diffusibile, sono stati
utilizzati con successo come adiuvanti nel trattamento di patologie a carico della cartilagine (Riesle
J et al. 1998; Aigner T et al. 2003) e dei tendini (Chen JL et al, 2010).
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I risultati di questi studi concordano tutti nell'evidenziare che l'applicazione locale al sito di innesto
di collagene esogeno induce la riduzione della sintomatologia algico-infiammatoria causata dalla
lesione. come permesso da ChondroGrid, il quale dà all’operatore, essendo iniettabile, un modo
semplice e rapido per arricchire il sito di impianto di un componente fondamentale della matrice
extracellulare, fornendo così
a) supporto meccanico diretto alle strutture sofferenti, come già detto e
b) un substrato già ottimale all'azione degli elementi cellulari coinvolti nella
rigenerazione tessutale al sito lesionato, favorendo la modificazione degli eventi
riparativi in eventi rigenerativi.
Esempi che illustrano questa tipologia di modificazione si possono ritrovare in diversi studi
preclinici e clinici inerenti la rigenerazione di tessuti anche differenti fra loro: esempi di effetti
positivi nell'utilizzo di collageni sono reperibili nell'ambito della rigenerazione del tessuto nervoso
(Itoh S et al. 2001, Fukushima K et al. 2008), dei dischi intervertebrali (Sato M et al. 2003; Sakai D
et al. 2006; Halloran DO et al. 2008; Lee KI et al. 2012) di lesioni dermiche refrattarie a trattamento
(Nakanishi A et al. 2005); di lesioni muscolari sperimentalmente indotte (Kin S et al. 2007); di
difetti cartilaginei ed ossei (Jeong IH et al. 2013, Hamanishi M et al. 2013; Kagawa R et al. 2012;
Yamaoka H et al. 2010; Sato M et al. 2007; Masuoka K et al. 2006; Stone KR et al. 1997).
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Studi in vitro
Per quanto riguarda gli studi in vitro, considerati inerenti, riguardanti il collagene, e in particolare il
collagene idrolizzato di tipo I, si è constatato che l’applicazione di collagene idrolizzato su colture
cellulari di condrociti (le cellule deputate alla produzione e al mantenimento della cartilagine)
induce una stimolazione della produzione del collagene stesso, probabilmente come possibile
meccanismo di regolazione a feedback positivo della stessa produzione di collagene o come segnale
di presenza di collagene degradato e, quindi, come segnale di danno e di necessità di procedere alla
conseguente sintesi riparativa (Oesser et al, 2003); inoltre, particolari sequenze peptidiche
certamente presenti negli idrolizzati di collagene a basso peso molecolare, quali il dipeptide Prolina-
Idrossiprolina (Pro-Hyp), hanno mostrato un effetto pro-differenziante su colture condrocitarie e
l’induzione di un aumento della sintesi di glicosaminoglicani, un componente fondamentale della
matrice extracellulare cartilaginea (Nakatani et al, 2009). Questo effetto, misurabile in un
incremento del 300% quando era impiegato il dipeptide come sostanza test, era osservato dagli
stessi autori, anche se in misura minore, quando la sostanza test era l’intero idrolizzato collagenico
(+200%). Nell’ambito dello stesso studio, Nakatani e colleghi hanno quindi proceduto all’impiego
del dipeptide e dell’idrolizzato collagenico in un modello animale di osteoartrosi inducibile
(attraverso eccesso di fosfato in dieta), verificando che entrambi i composti erano in grado di inibire
sia la perdita condrocitaria che l’assottigliamento della cartilagine articolare. Risultati del tutto
simili sono stati ottenuti nel 2010 dal gruppo di Ohara (Ohara et al, 2010) su cellule sinoviali in
coltura dove il dipeptide Prolina-Idrossiprolina si è mostrato in grado di stimolare la produzione di
acido ialuronico (un altro costituente fondamentale della matrice extracellulare) da parte di questo
tipo cellulare. Studi simili sono stati condotti anche con una particolare forma di collagene di tipo I,
ottenuto da pepsinizzazione di tessuti connettivali porcini (Furuzawa-Carballeda J et al, 2009(1)), su
colture cartilaginee e sinoviali ottenute da biopsie di pazienti affetti da osteoartrosi. Gli autori, oltre
ad evidenziare una stimolazione sia della proliferazione condrocitaria che della produzione di
proteine della matrice extracellulare, hanno anche verificato la diminuzione del livello di numerosi
marcatori biochimici dell’infiammazione. Nel caso specifico dell’osteoartrosi, infatti, che come
noto è una patologia infiammatoria caratterizzata da un progressivo deterioramento della cartilagine
articolare e delle articolazioni sinoviali, le principali citochine maggiormente coinvolte nella
fisiopatologia della malattia risultano essere l’interleuchina (IL)-1β, il tumor necrosis factor (TNF)-
α, e l’IL-6. Altri fattori biomolecolari coinvolti includono le IL-15, IL-18, IL-21, ed il leukemia
inhibitory factor (LIF) Recentemente, si sono compiuti molti progressi nella comprensione dello
sviluppo della patologia e dei meccanismi responsabili del suo decorso. Una nuova strategia
terapeutica consiste nello sviluppare agenti in grado di modificare la progressione strutturale della
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patologia e, quindi, di agire a livello del lenimento della sintomatologia. Recenti studi in vitro
hanno visto coinvolti anche gli idrolizzati di collagene: in uno studio su condrociti di pazienti affetti
da osteoartrosi, l’esposizione anche a collagene idrolizzato non ha mostrato effetti sulla vitalità
cellulare, indicando un’assenza di citotossicità del composto, mentre a seguito di stimolazione con
IL1β, una condizione simulante uno stato infiammatorio, il collagene idrolizzato contribuiva a
ridurre i marcatori tipici dell’infiammazione quali l’NO, l’IL-6, la metalloproteinasi 3 (Comblain F
et al, 2015).Uno studio ulteriore degli stessi autori ha mostrato come ulteriori potenziali bersagli
molecolari possono essere il l’IL-1β stimulated chemokine ligand 6, la metalloproteinase di matrice
MM13, la bone morphogenetic protein-2 (BMP2) e la stanniocalcina-1. In tutti i casi la presenza
dell’idrolizzato contribuiva a diminuire sia l’espressione genica che la sintesi proteica di questi
fattori. Per alcuni fattori, l’effetto era specificamente quello inverso a quello dell’IL-1β,
confermando quindi un potenziale effetto antinfiammatorio diretto dell’idrolizzato (Comblain et al,
2016). Allo stesso tempo, studi meno recenti, hanno dimostrato che un idrolizzato di collagene,
ulteriormente idrolizzato (ad esempio se assunto per via orale) origina una concentrazione ematica
di peptide contenenti prolina-idrossiprolina: questi sono in grado, in vitro, di stimolare cellule
sinoviali a produrre acido ialuronico e, in un modello animale di artrosi, di stimolare
significativamente la sintesi di proteoglicani a livello delle articolazioni epifisarie. E’ ragionevole
supporre che la somministrazione intra-articolare di un idrolizzato di collagene renda disponibili,
immediatamente e poi nel corso del tempo mano a mano che i polipeptidi sono idrolizzati dagli
enzimi tessutali una certa quantità di dipeptidi Pro-Hyp che potrebbe portare ai benefici effetti
osservati nei modelli animali in cui il dipeptide risulta presente a livello ematico, e quindi
disponibile agli elementi cellulari coinvolti (Ohara et al, 2010).
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Studi in vivo
Gli studi su animale reperiti riguardano sia il profilo di sicurezza che l’attività anti-infiammatoria
del collagene idrolizzato specialmente in modelli animali di osteoartrosi. Per quanto riguarda il
profilo di sicurezza, si è osservato che studi sull’immunogenicità del collagene di tipo I iniettabile
di origine animale sono presenti in letteratura a partire dal 1987 (De Lustro F et al, 1987), con
risultati che mostrano la scarsa o nulla antigenicità di questa tipologia di preparati. Un elegante
studio su animale di Oesser et al, 1999 mostra chiaramente come il collagene idrolizzato mostri un
particolare tropismo per le cartilagini articolari: quando gelatina di collagene marcato
radioattivamente è stata somministrata alle cavie per via orale si è osservato un accumulo
significativamente maggiore di collagene nelle articolazioni di quanto osservato nel gruppo
controllo. In relazione agli studi sui modelli di osteoartrosi, oltre ai già citati studi di Nakatani e
Ohara ricordiamo anche il lavoro di Naraoka et al. (2013), che hanno mostrato attraverso indagine
istologica ed immunoistochimica come l’iniezione intra-articolare ripetuta di un tripeptide
collagenico di tipo Gly-X-Y in un modello animale affetto da osteartrosi artificialmente indotta
permetta una significativa riduzione della degradazione della cartilagine articolare e l’incremento
del numero di condrociti positivi per i test immunoistochimici di nuova sintesi di collagene di tipo
II, a dimostrare che esiste almeno una possibile frazione peptidica proveniente dal collagene
idrolizzato che potrebbe indurre fenomeni riparativi da parte del sistema condrocitario.
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Studi clinici
Gli studi clinici analizzati forniscono innanzitutto evidenza che il collagene iniettabile è poco o per
nulla immunogenico (Cooperman L et al, 1984) in linea con quanto osservato su animale (De
Lustro F et al, 1987). Il primo uso clinico di collagene iniettabile per alleviare la sintomatologia
dolorosa delle articolazioni risale al 1982 (Batyrov TU et al., 1982). Più recentemente sono stati
apparsi studi clinici che dimostrano come l’iniezione intra-articolare di collagene sia efficace per il
trattamento sintomatico delle patologie degenerative articolari. Uno studio di Furuzawa-Carballeda
e collaboratori nel 2009 (Furuzawa-Carballeda J et al, 2009(2)) su 53 pazienti affetti da osteoartrosi
del ginocchio, divisi in gruppo controllo (27 pazienti) a cui veniva iniettato un placebo e gruppo test
(26 pazienti) a cui veniva iniettato collagene di tipo I, mostra che l’iniezione di collagene di tipo I
porta alla riduzione significativa di tutti i parametri di misura della sintomatologia dolorosa e
funzionale, quali il Western Ontario and McMaster University Osteoarthritis Index, il Lequesne
index, e l’analogo visivo del dolore (scala VAS). Anche parametri secondari, quali il punteggio
globale del paziente e del ricercatore, e la quantità di farmaci, tra cui gli antidolorifici, assunti
migliorano significativamente quando il paziente è trattato tramite iniezione intra-articolare di
collagene. Lo stesso studio è stato recentemente ripetuto dagli stessi autori (Furuzawa-Carballeda et
al, 2012) in un trial clinico randomizzato in doppio cieco, confermando i risultati precedentemente
descritti. Risultati simili erano stati ottenuti dallo stesso gruppo nel 2006 (Furuzawa-Carballeda et
al., 2006) in uno studio clinico randomizzato su pazienti affetti da artrite reumatoide. Ulteriormente,
vi è notevole evidenza che integratori alimentari a base di collagene realizzano una condizione per
cui a seguito dell’attività gastrica e dell’assorbimento intestinale si osserva un picco ematico di
peptidi del collagene, tra cui il dipeptide Pro-Hyp, con effetto tropico e biologico sulla popolazione
condrocitaria delle cartilagini come già precedentemente descritto. Si noti che dei tre enzimi
proteolitici operanti nel succo gastrico, pepsina, tripsina e chemotripsina, la pepsina è l’enzima
principalmente impiegato per la produzione dei collageni idrolizzati, e tra questi per la produzione
del dispositivo ChondroGrid. L’enzima pepsina è più specificamente una proteasi idrolitica acida in
grado di recidere il legame amidico tra gli aminoacidi che formano le catene polipeptidiche
preferenzialmente a valle, in direzione N terminale, degli aminoacidi aromatici quali fenilalanina,
triptofano e tirosina (Cox et al., 2008). La sua azione, così come quella degli altri enzimi, a livello
delle catene polipeptidiche del collagene realizza una miscela di peptidi a diverso peso molecolare
che vengono successivamente ulteriormente degradati e assorbiti a livello intestinale per entrare
infine nel circolo ematico. L’introduzione per via orale di integratori a base di collagene realizza
quindi una condizione simile, sebbene con diverse cinetiche di esposizione delle strutture e delle
componenti cellulari coinvolte ai frammenti collagenici, simile a quella che si realizza attraverso
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l’iniezione diretta intra-articolare di collagene idrolizzato che – tramite quest’ultima modalità –
viene portato in situ a maggiori concentrazioni e con un più ampio spettro di pesi molecolari (cioè:
dimensioni) di frammenti peptidici. In questo senso i risultati degli studi condotti su paziente
pubblicati sull’effetto antiinfiammatorio dell’assunzione orale di integratori a base di collagene
sono una dimostrazione indiretta dell’efficacia di ChondroGrid. Si consideri a questo riguardo la
revisione di letteratura di Bello ed Oesser del 2006, ove già si riepilogava come il collagene
idrolizzato assunto per via orale, in seguito ad assorbimento intestinale, è in grado di accumularsi
nella cartilagine, stimolando un aumento significativo della sintesi della matrice extracellulare da
parte dei condrociti, suggerendo una possibile spiegazione per i dati osservati di riduzione del
dolore e miglioramento della funzionalità in pazienti affetti sia da artrosi che da altre patologie
articolari. Ulteriori studi sono stati condotti sull’uso del collagene in pazienti sia geriatrici che
sportivi, affetti da osteoartrite e osteoartrosi a carico delle articolazioni; per citare i più noti si
ricordino Moskowitz et al. (2000) e Clark et al. (2008): in questi studi il collagene idrolizzato risulta
essere somministrato per via orale alla dose di 10 g al dì, per periodi variabili dai 3 fino ai 6 mesi,
con miglioramento della sintomatologia dolorosa rispetto al gruppo di controllo negativo. Più
recentemente, uno studio clinico randomizzato di Schauss AG et al, 2012 ha mostrato come pazienti
affetti da osteoartrosi che assumevano collagene per via orale evidenziavano una significativa
riduzione della patologia dolorosa dopo 70 giorni di trattamento e un miglioramento funzionale
(misurato attraverso la scala WOMAC), nonché della propria attività fisica generale, già dopo 35
giorni di trattamento. Analogo risultato era stato ottenuto da Benito Ruiz e colleghi (2009) in un
trial clinico randomizzato su 250 pazienti affetti da osteoartrite del ginocchio cui sono stati
somministrati 10 g di collagene idrolizzato per os una volta al giorno per 6 mesi: ove i pazienti che
assumevano collagene mostravano un miglioramento significativo del dolore articolare, misurato
attraverso la scala VAS e la sottoscala WOMAC relativa al dolore. I soggetti col maggiore danno
articolare risultavano essere quelli con il maggiore beneficio. Un trial clinico randomizzato in
doppio cieco con gruppo di controllo su pazienti affetti da osteoartrosi del ginocchio ha dato
recentemente ulteriore conferma di questi risultati (Kumar S et al., 2015): i pazienti del gruppo di
studio, trattati per via orale con collagene idrolizzato estratto o da derma suino o da osso bovino
sperimentavano, dopo 13 settimane, un significativo miglioramento dei punteggi ottenuti secondo le
tre scale utilizzate: WOMAC, VAS e QOL (Quality of Life), sia rispetto alla baseline sia rispetto il
gruppo controllo. Questi studi, in congiunzione a quelli già precedentemente citati relativi
all’iniezione intra-articolare di collagene, suggeriscono che sia plausibile supporre che l’iniezione
locale di collagene idrolizzato possa rappresentare un trattamento avente quanto meno degli
outcome simili e potenzialmente più favorevoli all’ingestione, data la maggiore concentrazione
35
locale ottenibile tramite l’iniezione in situ, ferme restando le differenze sulla via di
somministrazione che portano le due condizioni a non essere direttamente confrontabili.
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Conclusioni L’analisi della letteratura reperita porta a concludere che a) gli studi in vitro dimostrano che cellule
cimentate con idrolizzato collagenico non subiscono alcun tipo di danno ma, anzi, tutti i parametri
di vitalità e trofismo cellulare misurati negli studi considerati risultano aumentati (e, in studi
specificamente mirati, marcatori dell’infiammazione cellulare risultano significativamente
diminuiti); il razionale biologico secondo il quale ChondroGrid è in grado esercitare le prestazioni
previste è quindi coerente e robusto. A loro volta, b) gli studi su animale confermano a livello
istologico che l’applicazione del collagene idrolizzato migliora il metabolismo cellulare in modelli
sperimentali delle patologie indicate come indicazioni d’uso del dispositivo ChondroGrid, e che si
ottiene una riduzione dei marcatori considerati coerente con la prestazione attesa in ambito clinico.
Risulta di particolare interesse l’osservazione che i peptidi che compongono gli idrolizzati di
collagene mostrano un particolare tropismo per le strutture articolari quali la cartilagine, ove –
oltretutto – si accumulano. Inoltre, c) applicazioni cliniche di preparati simili, se non identici, a
ChondroGrid per le stesse applicazioni ed indicazioni permettono di ottenere i risultati clinici dati
come risultati attesi per ChondroGrid stesso (trattamento con esito positivo della sintomatologia
dolorosa e funzionale). Gli scriventi concludono, quindi, che la valutazione clinica di sicurezza ed
efficacia del dispositivo ChondroGrid dia esito completamente positivo. Il presente report sarà
aggiornato quando saranno disponibili i risultati degli studi clinici post-market che attualmente il
fabbricante ha comunicato essere in procinto di attivazione.
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Riepilogo dei risultati
Razionale biologico dell'utilizzo del dispositivo
Il dispositivo ChondroGrid permette all’operatore di apportare direttamente alla struttura coinvolta
nell’affezione dolorosa sia essa di origine degenerativa, traumatica o da sovraccarico, un’elevata
concentrazione di collagene idrolizzato a basso peso molecolare, ovvero una miscela di diversi
peptidi collagenici, sotto forma di sospensione. Poiché al diminuire della dimensione molecolare
aumentano i coefficienti di diffusione delle molecole stesse in fase acquosa, la miscela peptidica
contenuta i ChondroGrid è in grado di diffondere efficacemente e in modo uniforme in tutto il
volume raggiunto dall’iniezione, distribuendosi su tutta la superficie della struttura anatomica
coinvolta. La particolare proprietà di autoassemblaggio del collagene fa sì che il collagene
idrolizzato, legandosi alle strutture esposte – anch’esse costituite per la maggior parte di collagene,
eserciti un’azione di supporto e sostegno meccanico. Ulteriori evidenze, presentate nel presente
report, suggeriscono effetti ancillari di tipo biologico quali la stimolazione di alcuni tipo cellulari
alla produzione di ulteriore collagene, o di acido ialuronico o di glicosaminoglicani, e l’inibizione
dell’attività o dell’espressione di alcune citochine mediatrici dei processi infiammatori.
Razionale clinico dell'utilizzo del dispositivo
Visti gli effetti biologici che il dispositivo è in grado di esercitare, risulta evidente che l'impiego
clinico realizza una condizione al sito di iniezione estremamente favorevole per la creazione di uno
stato di trofismo locale in grado di migliorare la percezione dolorosa – e quindi favorire il recupero
funzionale – del paziente. i
Livello di evidenza relativo alla sicurezza dell'impiego clinico del dispositivo
Il dispositivo ChondroGrid può ritenersi intrinsecamente sicuro. La sicurezza del dispositivo è
comprovata direttamente, in quanto è dimostrato attraverso test condotti da enti terzi che
ChondroGrid è completamente biocompatibile secondo lo standard di riferimento, accettato a livello
mondiale, ISO 10993. Ulteriormente, la possibilità che il dispositivo possa essere veicolo di
patologie trasmissibili da animale all'uomo, ad esempio a causa di agenti patogeni virali e prioni, è
nulla in quanto la materia prima animale subisce un’esposizione prolungata sia a basi che acidi forti
condizioni alle quali è stato dimostrato nessun virus possa mantenere la propria capacità replicativa,
né possano permanere molecole prioniche in forma mutata. Prioni patogenici la cui presenza è
comunque da ritenere estremamente improbabile nella materia d’origine come certificato dalle
38
Autorità Competenti europee con apposito certificato EDQM. Infine, conferma della sicurezza
dell'efficacia del dispositivo è apportata dalla certificazione CE che esso ha ottenuto da Organismo
Notificato per la valutazione della Conformità dei Dispositivi Medici (Istituto Superiore di Sanità,
Organismo Notificato CE 0373) alle normative europee di riferimento.
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Tabelle Riassuntive
Sicurezza del dispositivo ChondroGrid
ChondroGrid è stato oggetto di saggi e test che hanno valutato diversi parametri relativi alla
sicurezza, secondo stringenti standard internazionali:
Parametro Test Standard
Biocompatibilità
Citotossicità ISO 10993-5
Reattività intracutanea ISO10993 -10
Tossicità sistemica ISO10993-11
Pirogenicità Eu.Ph. § 2.6.14
Tossicità subacuta ISO10993-11
Impianto ISO10993-6
Possibilità di trasmissione di
encefalopatie spongiformi
La materia prima (tessuti animali) utilizzata per la
fabbricazione di ChondroGrid proviene da Paesi non a
rischio di BSE: la sicurezza è certificata anche
dall’European Directorate for the Quality of Medicines and
Healthcare (EDQM).
Il processo produttivo di ChondroGrid prevede inoltre alcuni
passaggi (impiego di basi e acidi forti) che sono in grado di
eliminare un’eventuale contaminazione prionica del
materiale d’origine.
Sterilità Sterilità ISO11137
Inattivazione virale Gli stessi passaggi del processo produttivo prima menzionati
in merito all’inattivazione e rimozione di possibili prioni
sono in grado anche di eliminare un’eventuale
contaminazione virale presente nel materiale d’origine.
Ulteriormente, il prodotto subisce la sterilizzazione
terminare per irraggiamento beta.
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Efficacia del dispositivo ChondroGrid.
I dati a sostegno dell'efficacia di ChondroGrid possono essere riassunti come segue:
Parametro di efficacia Livello di evidenza
Azione a livello cellulare Gli studi in vitro mostrano che il collagene idrolizzato è in
grado di stimolare il trofismo cellulare, ad esempio
condrocitario, inducendo un aumento sia della sintesi del
collagene stesso, sia dei glucosaminoglicani.
Le cellule sinoviali sono in grado, quando in contatto con
peptidi derivati da collagene idrolizzato di produrre
maggiori quantità di acido ialuronico.
I condrociti, in particolare, sono stimolati ad una maggiore
proliferazione.
Non si osserva alcun tipo di citotossicità indotta dal
collagene idrolizzato.
Azione a livello di signaling Il collagene idrolizzato inibisce in modo significativo
l’espressione di alcune interleuchine e di altri marcatori
dell’infiammazione.
Azione clinica – studi
preclinici
Gli studi preclinici mostrano che l’iniezione di collagene
idrolizzato intra- e peri-articolare è, prima di tutto, sicura
non osservandosi nei modelli sperimentali sensibilizzazione,
irritazione, tossicità né acuta né ritardata.
Ulteriormente, gli studi preclinici mostrano che il collagene
in forma di peptidi, come è ChondroGrid, ha un forte
tropismo per le strutture articolari e peri-articolari, ove si
accumula preferenzialmente, concentrandosi naturalmente
nei siti oggetto del trattamento.
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Gli studi pre-clinici suggeriscono inoltre che il collagene
idrolizzato, grazie all’azione di specifici peptidi nella
miscela, possa avere anche ulteriori azioni quali la
stimolazione di attività riparativa e rigenerativa da parte di
alcuni specifici elementi cellulari.
Azione clinica – casistica su
paziente
Per quanto riguarda la sicurezza di ChondroGrid, anche gli
studi su paziente mostrano che l’iniezione di collagene intra-
e peri-articolare non dà reazioni immunitarie indesiderate,
né a breve né a lungo termine.
Per quanto concerne l’efficacia di ChondroGrid, gli studi
reperiti, alcuni dei quali anche randomizzati e in doppio
cieco, mostrano inequivocabilmente che quando il collagene
idrolizzato, come è ChondroGrid, raggiunge le articolazioni
di pazienti affetti da osteoartrosi, si assiste ad una
significativa diminuzione della sintomatologia dolorosa e a
un più rapido recupero funzionale, a prescindere dalla via di
somministrazione.
I pazienti in cura con questo tipo di approccio terapeutico,
inoltre, assumono una minore quantità di farmaci analgesici
ed antinfiammatori.
42
Domande Frequenti – Frequently Asked Questions (FAQ)
Cosa contiene ChondroGrid?
ChondroGrid è composto esclusivamente di collagene idrolizzato e liofilizzato a basso peso
molecolare, altamente purificato, estratto da cute bovina.
Cosa è il collagene idrolizzato?
Si tratta di collagene che è stato sottoposto ad un processo di frammentazione attraverso
l’impiego di metodi enzimatici e chimico-fisici. Come risultato si producono peptidi di
diverso peso molecolare, ovvero con diversa lunghezza.
Perché è importante il collagene idrolizzato?
I peptidi, avendo dimensione minore rispetto quella dell’intera molecola, diffondono
facilmente nei liquidi, e quindi si prestano ad essere iniettati negli spazi intra- e peri-articolari
ove, per diffusione, raggiungono l’intera superficie dell’articolazione o dei tendini e dei
legamenti.
E' possibile una sensibilità individuale al collagene idrolizzato?
E' da considerarsi un evento estremamente raro.
Quali sono le indicazioni d'uso di ChondroGrid?
Chondrogrid è indicato per il trattamento sintomatologico e funzionale di: osteoartrosi,
artrosinoviti acute o croniche secondarie ad artrosi o artrite reumatoide, esiti di traumi o
lesioni, affaticamento da sovraccarico a carico delle diverse articolazioni nonché in caso di
meniscopatie e in preparazione e come terapia di mantenimento a seguito di interventi di
meniscectomia, di ricostruzione legamentosa o di pulizia e/o ricostruzione del tessuto
cartilagineo articolare. Inoltre è indicato nel trattamento degli esiti di traumi e/o lesione peri-
articolari o a carico di strutture muscolo-tendinee-legamentose.
43
Quale è il meccanismo d'azione di ChondroGrid®?
ChondroGrid agisce apportando un’alta quantità di collagene idrolizzato biodisponibile al
sito di iniezione. Per via intra-articolare, il collagene idrolizzato a basso peso molecolare si
distribuisce sulla superficie dell’articolazione rinforzando la matrice cartilaginea, deteriorata
dai processi patologici in atto, la cui impalcatura è formata da un fitto intreccio di fibre
collagene. Per via peri-articolare il collagene idrolizzato a basso peso molecolare agisce
quale rinforzo diretto all’impalcatura collagenica danneggiata delle strutture peri-articolari,
quali tendini e/o legamenti, contribuendo ad una riduzione del dolore e ad una più veloce
ripresa funzionale.
Come deve essere utilizzato ChondroGrid?
Secondo uno specifico protocollo terapeutico. Se iniettato per via intra-articolare il
trattamento prevede tre iniezioni: due eseguite a distanza di 15 giorni l’una dall’altra, la terza
dopo un mese dall’ultimo trattamento. Se utilizzato per iniezione peri-articolare, il
trattamento prevede due iniezioni a distanza di 30 giorni l’una dall’altra. Per infiltrazione in
strutture muscolo-tendinee-legamentose, due iniezioni ad un intervallo di circa 10 giorni
l’una dall’altra.
Quali sono i limiti di ChondroGrid?
Sebbene alcuni studi indichino che il collagene idrolizzato di basso peso molecolare possa
anche indurre la rigenerazione delle strutture danneggiate, l’impiego di ChondroGrid deve
essere comunque inteso, allo stato attuale della conoscenza, come coadiuvante nella terapia
sintomatologica e funzionale nelle diverse indicazioni fornite.
ChondroGrid deve essere utilizzato da solo?
Si, ChondroGrid esplica la sua azione da solo, grazie all’azione del collagene idrolizzato che
esso contiene.
ChondroGrid è sicuro?
Si. E' un dispositivo che ha superato tutti i test di sicurezza previsti dalla normativa europea
sui dispositivi medici (marcatura CE).
44
Dove viene fabbricato ChondroGrid ?
ChondroGrid è un dispositivo medico fabbricato in Italia, presso Bioteck, l’azienda leader
italiana nella produzione di dispositivi medici per rigenerazione tessutale.
45
Bibliografia1
Scand J Plast Reconstr Surg Hand Surg Suppl. 1991;23:1-51.
Matrix metabolism and healing in the flexor tendon. Experimental studies on rabbit tendon.
Abrahamsson SO.
I. The rabbit flexor tendon within the synovial sheath contains segments with fibrocartilage-like
areas. These segments have a higher proteoglycan and a lower collagen and non-collagen protein
synthesis compared to the segment with "true" tendon tissue. Cell proliferation is also lower within
the proximal segment than in the intermediate and distal segments. These regional variations should
be considered when interpreting experimental data. They may also be of importance for the variable
healing capacity of different flexor tendon regions. II. Recombinant human insulin-like growth
factor, insulin and fetal calf serum stimulate matrix synthesis and cell proliferation in a dose
dependent manner in flexor tendon explants cultured for three days. rhIGF-I was more potent than
insulin in stimulating cell proliferation and matrix synthesis. rhIGF-I also stimulated matrix
synthesis to a higher degree than FCS. III. In long-term culture of flexor tendon explants, the
addition of rhIGF-I to the culture medium stimulates matrix synthesis, but does not influence turn-
over rates. The total hexosamine and collagen contents in tendons cultured in medium with rhIGF-I
remain at the same level, while non-collagen protein content decreases. There are no major
differences in matrix metabolism between tendons cultured in medium supplemented with FCS or
with rhIGF-I only. rhIGF-I may therefore be used as a growth factor supplement in serum-free
culture of tendon tissue. IV. Dehydration inhibits in vitro matrix synthesis and cell proliferation in
tendon explants. These effects are counteracted by keeping the exposed tendon segments moist with
physiological saline solution during preparation. The sensitivity of tendon tissue to dehydration
should be considered during tendon surgery. V. Tendon explants, cultured in a diffusion chamber,
survive and exhibit an intrinsic capacity for healing. In healing tendon segments incubated for three
weeks, protein synthesis remains unchanged and collagen synthesis decreases, whereas the rate of
cell proliferation increases as compared with native tendons. VI. Endotenon cells of the rabbit
1 La bibliografia riporta gli abstract delle pubblicazioni citate nel documento ordinati in ordine
alfabetico per primo autore. Le pubblicazioni complete sono disponibili su richiesta.
46
flexor tendon can restore the injured tendon surface and bridge the tendon gap. The rabbit flexor
tendon is a morphologically and biochemically heterogeneous tissue with an intrinsic capability for
healing. Tendon tissue is susceptible to dehydration and during exposure quickly looses its viability.
The metabolic and proliferative capacity of the tendon is stimulated by growth factors and rhIGF-I
may be of importance in tendon healing.
47
Cell Mol Life Sci. 2002 Jan;59(1):5-18.
Molecular pathology and pathobiology of osteoarthritic cartilage.
Aigner T, McKenna L.
The biochemical properties of articular cartilage rely on the biochemical composition and integrity
of its extracellular matrix. This matrix consists mainly of a collagen network and the proteoglycan-
rich ground substance. In osteoarthritis, ongoing cartilage matrix destruction takes place, leading to
a progressive loss in joint function. Beside the degradation of molecular matrix components,
destabilization of supramolecular structures such as the collagen network and changes in the
expression profile of matrix molecules also take place. These processes, as well as the pattern of
cellular reaction, explain the pathology of osteoarthritic cartilage degeneration. The loss of
histochemical proteoglycan staining reflects the damage at the molecular level, whereas the
supramolecular matrix destruction leads to fissuring and finally to the loss of the cartilage.
Chondrocytes react by increasing matrix synthesis, proliferating, and changing their cellular
phenotype. Gene expression mapping in situ and gene expression profiling allows characterization
of the osteoarthritic cellular phenotype, a key determinant for understanding and manipulating the
osteoarthritic disease process. Adv Drug Deliv Rev. 2003 Nov 28;55(12):1569-93.
48
Collagens--major component of the physiological cartilage matrix, major target of cartilage
degeneration, major tool in cartilage repair.
Aigner T, Stöve J.
Collagens serve important mechanical functions throughout the body and in particular in the
connective tissues. Additionally, collagens exert important functions as cellular microenvironment
and partly via binding and release of cellular growth mediators. In articular cartilage, fibrillar
collagens are providing most of the biomechanical properties of the extracellular matrix essential
for its functioning. The collagenous matrix is one main target of destructive processes in general
degenerative joint disease and focal matrix lesions. The development of an adequate collagen
framework represents the major aim of therapeutic cartilage repair. In this respect, collagenous
matrices or collagen-imitating scaffolds are more and more emerging as highly suitable vehicles for
cell and (growth) factor transport into cartilage lesion. Thus, collagens are not only major
constituents of connective tissues in terms of integrity and function, they are also major targets of
tissue destruction and regeneration and might become major tools to achieve tissue repair.
49
Eur Urol. 1998;33(2):129-33.
Immunologic aspects of bovine injectable collagen in humans. A review.
Aragona F, D'Urso L, Marcolongo R.
No abstract available
50
Arthroscopy. 2014 Mar;30(3):289-98.
Properties of biologic scaffolds and their response to mesenchymal stem cells.
Beitzel K, McCarthy MB, Cote MP, Russell RP, Apostolakos J, Ramos DM, Kumbar SG, Imhoff
AB, Arciero RA, Mazzocca AD.
PURPOSE: The purpose of this study was to examine, in vitro, the cellular response of human
mesenchymal stem cells (MSCs) to sample types of commercially available scaffolds in comparison
with control, native tendon tissue (fresh-frozen rotator cuff tendon allograft). METHODS: MSCs
were defined by (1) colony-forming potential; (2) ability to differentiate into tendon, cartilage,
bone, and fat tissue; and (3) fluorescence-activated cell sorting analysis (CD73, CD90, CD45).
Samples were taken from fresh-frozen human rotator cuff tendon (allograft), human highly cross-
linked collagen membrane (Arthroflex; LifeNet Health, Virginia Beach, VA), porcine non-cross-
linked collagen membrane (Mucograft; Geistlich Pharma, Lucerne, Switzerland), a human platelet-
rich fibrin matrix (PRF-M), and a fibrin matrix based on platelet-rich plasma (ViscoGel; Arthrex,
Naples, FL). Cells were counted for adhesion (24 hours), thymidine assay for cell proliferation (96
hours), and live/dead stain for viability (168 hours). Histologic analysis was performed after 21
days, and the unloaded scaffolds were scanned with electron microscopy. RESULTS: MSCs were
successfully differentiated into all cell lines. A significantly greater number of cells adhered to both
the non-cross-linked porcine collagen scaffold and PRF-M. Cell activity (proliferation) was
significantly higher in the non-cross-linked porcine collagen scaffold compared with PRF-M and
fibrin matrix based on platelet-rich plasma. There were no significant differences found in the
results of the live/dead assay. CONCLUSIONS: Significant differences in the response of human
MSCs to biologic scaffolds existed. MSC adhesion, proliferation, and scaffold morphology
evaluated by histologic analysis and electron microscopy varied throughout the evaluated types of
scaffolds. Non-cross-linked porcine collagen scaffolds showed superior results for cell adhesion and
proliferation, as well as on histologic evaluation. CLINICAL RELEVANCE: This study enables the
clinician and scientist to choose scaffold materials according to their specific interaction with
MSCs.
51
Curr Med Res Opin. 2006 Nov;22(11):2221-32.
Collagen hydrolysate for the treatment of osteoarthritis and other joint disorders: a review of
the literature.
Bello AE, Oesser S.
BACKGROUND: There is a need for an effective treatment for the millions of people in the United
States with osteoarthritis (OA), a degenerative joint disease. The demand for treatments, both
traditional and non-traditional, will continue to grow as the population ages. SCOPE: This article
reviews the medical literature on the preclinical and clinical research on a unique compound,
collagen hydrolysate. Articles were obtained through searches of the PubMed database
(www.pubmed.gov) through May 2006 using several pairs of key words (collagen hydrolysate and
osteoarthritis; collagen hydrolysate and cartilage; collagen hydrolysate and chondrocytes; collagen
hydrolysate and clinical trial) without date limits. In addition, other sources of information, such as
abstracts presented at scientific congresses and articles in the German medical literature not
available on PubMed, were reviewed and included based on the authors' judgment of their relevance
to the topic of the review. FINDINGS: According to published research, orally administered
collagen hydrolysate has been shown to be absorbed intestinally and to accumulate in cartilage.
Collagen hydrolysate ingestion stimulates a statistically significant increase in synthesis of
extracellular matrix macromolecules by chondrocytes (p < 0.05 compared with untreated controls).
These findings suggest mechanisms that might help patients affected by joint disorders such as OA.
Four open-label and three double-blind studies were identified and reviewed; although many of
these studies did not provide key information--such as the statistical significance of the findings--
they showed collagen hydrolysate to be safe and to provide improvement in some measures of pain
and function in some men and women with OA or other arthritic conditions. CONCLUSION: A
growing body of evidence provides a rationale for the use of collagen hydrolysate for patients with
OA. It is hoped that ongoing and future research will clarify how collagen hydrolysate provides its
clinical effects and determine which populations are most appropriate for treatment with this
supplement.
52
Int J Food Sci Nutr. 2009;60 Suppl 2:99-113.
A randomized controlled trial on the efficacy and safety of a food ingredient, collagen
hydrolysate, for improving joint comfort.
Benito-Ruiz P, Camacho-Zambrano MM, Carrillo-Arcentales JN, Mestanza-Peralta MA, Vallejo-
Flores CA, Vargas-López SV, Villacís-Tamayo RA, Zurita-Gavilanes LA.
INTRODUCTION: Current options to promote joint comfort are limited to medicines that can
reduce pain but can also have adverse effects. Collagen, a major component of joint cartilage, is
found in the diet, particularly in meat. Its hydrolysed form, collagen hydrolysate (CH), is well
absorbed. CH may stimulate the joint matrix cells to synthesize collagen, so helping to maintain the
structure of the joint and potentially to aid joint comfort. METHODS: In a randomized, double-
blind, controlled multicentre trial, 250 subjects with primary osteoarthritis of the knee were given
10 g CH daily for 6 months. RESULTS: There was a significant improvement in knee joint comfort
as assessed by visual analogue scales to assess pain and the Womac pain subscale. Subjects with the
greatest joint deterioration, and with least intake of meat protein in their habitual diets, benefited
most. CONCLUSION: CH is safe and effective and warrants further consideration as a food
ingredient.
53
Biomed Mater. 2009 Jun;4(3):035006.
Early adhesive behavior of bone-marrow-derived mesenchymal stem cells on collagen
electrospun fibers.
Chan CK, Liao S, Li B, Lareu RR, Larrick JW, Ramakrishna S, Raghunath M.
A bioabsorbable nanofibrous scaffold was developed for early adhesion of mesenchymal stem cells
(MSCs). Collagen nanofibers with diameters of 430 +/- 170 nm were fabricated by electrospinning.
Over 45% of the MSC population adhered to this collagen nanofiber after 30 min at room
temperature. Remarkably, collagen-coated P(LLA-CL) electrospun nanofibers were almost as
efficient as collagen nanofibers whereas collagen cast film did not enhance early capture when it
was applied on cover slips. The adhesive efficiency could be further increased to over 20% at 20
min and over 55% at 30 min when collagen nanofibers were grafted with monoclonal antibodies
recognizing CD29 or CD49a. These data demonstrate that the early adhesive behavior is highly
dependent on both the surface texture and the surface chemistry of the substrate. These findings
have potential applications for early capture of MSCs in an ex vivo setting under time constraints
such as in a surgical setting.
54
J Am Acad Dermatol. 1989 Dec;21(6):1203-8.
Reactions to a bovine collagen implant. Clinical and immunologic study in 705 patients.
Charriere G, Bejot M, Schnitzler L, Ville G, Hartmann DJ.
A small percentage of patients treated with bovine collagen implants have adverse reactions
involving both the cellular and humoral types of immune response. We report a clinical follow-up
of 705 subjects treated with a new bovine collagen implant, Atelocollagen (Koken Co. Ltd., Tokyo,
Japan). Sensitization to the implant was evaluated in all subjects by skin testing, and humoral
response was monitored in 166 subjects by measuring the level of circulating antibodies directed
against bovine collagen. Twenty-seven patients (3.8%) exhibited a positive response to a skin test,
and of the remaining 656 patients, an adverse reaction to the implant developed in 2.3%. We found
a strong correlation between the presence of antibodies to collagen and a positive response to skin
testing (92%) or an adverse reaction (100%). In the case of a borderline clinical response to bovine
collagen implantation, anticollagen serologic tests appeared to be a useful tool for the identification
of clinically reactive patients.
55
Biomaterials. 2010 Dec;31(36):9438-51.
Efficacy of hESC-MSCs in knitted silk-collagen scaffold for tendon tissue engineering and
their roles.
Chen JL, Yin Z, Shen WL, Chen X, Heng BC, Zou XH, Ouyang HW.
Human embryonic stem cells (hESC) and their differentiated progenies are an attractive cell source
for transplantation therapy and tissue engineering. Nevertheless, the utility of these cells for tendon
tissue engineering has not yet been adequately explored. This study incorporated hESC-derived
mesenchymal stem cells (hESC-MSCs) within a knitted silk-collagen sponge scaffold, and assessed
the efficacy of this tissue-engineered construct in promoting tendon regeneration. When subjected
to mechanical stimulation in vitro, hESC-MSCs exhibited tenocyte-like morphology and positively
expressed tendon-related gene markers (e.g. Collagen type I & III, Epha4 and Scleraxis), as well as
other mechano-sensory structures and molecules (cilia, integrins and myosin). In ectopic
transplantation, the tissue-engineered tendon under in vivo mechanical stimulus displayed more
regularly aligned cells and larger collagen fibers. This in turn resulted in enhanced tendon
regeneration in situ, as evidenced by better histological scores and superior mechanical performance
characteristics. Furthermore, cell labeling and extracellular matrix expression assays demonstrated
that the transplanted hESC-MSCs not only contributed directly to tendon regeneration, but also
exerted an environment-modifying effect on the implantation site in situ. Hence, tissue-engineered
tendon can be successfully fabricated through seeding of hESC-MSCs within a knitted silk-collagen
sponge scaffold followed by mechanical stimulation.
56
Adv Drug Deliv Rev. 2003 Nov 28;55(12):1595-611.
Brain Res. 2011 Jan 12;1368:71-81.
Synergism of human amnion-derived multipotent progenitor (AMP) cells and a collagen
scaffold in promoting brain wound recovery: pre-clinical studies in an experimental model of
penetrating ballistic-like brain injury.
Chen Z, Lu XC, Shear DA, Dave JR, Davis AR, Evangelista CA, Duffy D, Tortella FC.
One of the histopathological consequences of a penetrating ballistic brain injury is the formation of
a permanent cavity. In a previous study using the penetrating ballistic-like brain injury (PBBI)
model, engrafted human amnion-derived multipotent progenitor (AMP) cells failed to survive when
injected directly in the injury tract, suggesting that the cell survival requires a supportive matrix. In
this study, we seated AMP cells in a collagen-based scaffold, injected into the injury core, and
investigated cell survival and neuroprotection following PBBI. AMP cells suspended in AMP cell
conditioned medium (ACCS) or in a liquefied collagen matrix were injected immediately after a
PBBI along the penetrating injury tract. Injured control rats received only liquefied collagen matrix.
All animals were allowed to survive two weeks. Consistent with our previous results, AMP cells
suspended in ACCS failed to survive; likewise, no collagen was identified at the injury site when
injected alone. In contrast, both AMP cells and the collagen were preserved in the injury cavity
when injected together. In addition, AMP cells/collagen treatment preserved some apparent brain
tissue in the injury cavity, and there was measurable infiltration of endogenous neural progenitor
cells and astrocytes into the preserved brain tissue. AMP cells were also found to have migrated
into the subventricular zone and the corpus callosum. Moreover, the AMP cell/collagen treatment
significantly attenuated the PBBI-induced axonal degeneration in the corpus callosum and
ipsilateral thalamus and improved motor impairment on rotarod performance. Overall, collagen-
based scaffold provided a supportive matrix for AMP cell survival, migration, and neuroprotection.
57
Curr Med Res Opin. 2008 May;24(5):1485-96.
24-Week study on the use of collagen hydrolysate as a dietary supplement in athletes with
activity-related joint pain.
Clark KL, Sebastianelli W, Flechsenhar KR, Aukermann DF, Meza F, Millard RL, Deitch JR,
Sherbondy PS, Albert A.
BACKGROUND: Collagen hydrolysate is a nutritional supplement that has been shown to exert an
anabolic effect on cartilage tissue. Its administration appears beneficial in patients with
osteoarthritis. OBJECTIVE: To investigate the effect of collagen hydrolysate on activity-related
joint pain in athletes who are physically active and have no evidence of joint disease. DESIGN
AND SETTING: A prospective, randomized, placebo-controlled, double-blind study was
conducted at Penn State University in University Park, Pennsylvania. Parameters including joint
pain, mobility, and inflammation were evaluated with the use of a visual analogue scale during a
24-week study phase. STUDY PARTICIPANTS: Between September 2005 and June 2006, 147
subjects who competed on a varsity team or a club sport were recruited. Data from 97 of 147
subjects could be statistically evaluated. INTERVENTION: One hundred and forty-seven subjects
(72 male, 75 female) were randomly assigned to two groups: a group (n = 73) receiving 25 mL of a
liquid formulation that contained 10 g of collagen hydrolysate (CH-Alpha) and a group (n = 74)
receiving a placebo, which consisted of 25 mL of liquid that contained xanthan. MAIN
OUTCOME MEASURES: The primary efficacy parameter was the change in the visual analogue
scales from baseline during the study phase in relation to the parameters referring to pain, mobility,
and inflammation. RESULTS: When data from all subjects (n = 97) were evaluated, six parameters
showed statistically significant changes with the dietary supplement collagen hydrolysate (CH)
compared with placebo: joint pain at rest, assessed by the physician (CH vs. placebo (-1.37 +/- 1.78
vs. -0.90 +/- 1.74 (p = 0.025)) and five parameters assessed by study participants: joint pain when
walking (-1.11 +/- 1.98 vs. -0.46 +/- 1.63, p = 0.007), joint pain when standing (-0.97 +/- 1.92 vs. -
0.43 +/- 1.74, p = 0.011), joint pain at rest (-0.81 +/- 1.77 vs. -0.39 +/- 1.56, p = 0.039), joint pain
when carrying objects (-1.45 +/- 2.11 vs. -0.83 +/- 1.71, p = 0.014) and joint pain when lifting (-
1.79 +/- 2.11 vs. -1.26 +/- 2.09, p = 0.018). When a subgroup analysis of subjects with knee
arthralgia (n = 63) was performed, the difference between the effect of collagen hydrolysate vs.
placebo was more pronounced. The parameter joint pain at rest, assessed by the physician, had a
statistical significance level of p = 0.001 (-1.67 +/- 1.89 vs. -0.86 +/- 1.77), while the other five
parameters based on the participants' assessments were also statistically significant: joint pain when
walking (p = 0.003 (-1.38 +/- 2.12 vs. -0.54 +/- 1.65)), joint pain when standing (p = 0.015 (-1.17
58
+/- 2.06 vs. -0.50 +/- 1.68)), joint pain at rest with (p = 0.021 (-1.01 +/-1.92 vs. -0.47 +/- 1.63)),
joint pain when running a straight line (p = 0.027 (-1.50 +/- 1.97 vs. -0.80 +/- 1.66)) and joint pain
when changing direction (p = 0.026 (-1.87 +/- 2.18 vs. -1.20 +/- 2.10)). CONCLUSION: This was
the first clinical trial of 24-weeks duration to show improvement of joint pain in athletes who were
treated with the dietary supplement collagen hydrolysate. The results of this study have implications
for the use of collagen hydrolysate to support joint health and possibly reduce the risk of joint
deterioration in a high-risk group. Despite the study's size and limitations, the results suggest that
athletes consuming collagen hydrolysate can reduce parameters (such as pain) that have a negative
impact on athletic performance. Future studies are needed to support these findings.
59
PLoS One. 2015 Mar 23;10(3):e0121654.
Curcuminoids extract, hydrolyzed collagen and green tea extract synergically inhibit
inflammatory and catabolic mediator's synthesis by normal bovine and osteoarthritic human
chondrocytes in monolayer.
Comblain F, Sanchez C, Lesponne I, Balligand M, Serisier S, Henrotin Y.
The main objective of this study was to assess the in vitro effects of curcuminoids extract,
hydrolyzed collagen and green tea extract in normal bovine chondrocytes and osteoarthritic human
chondrocytes cultured in monolayer. This study also investigated the synergic or additive effects of
these compounds. Enzymatically isolated primary bovine or human chondrocytes were cultured in
monolayer until confluence and then incubated for 24 hours or 48 hours in the absence or in the
presence of interleukin-1β and with or without curcuminoids extract, hydrolyzed collagen or green
tea extract, added alone or in combination, at different concentrations. Cell viability was neither
affected by these compounds, nor by interleukin 1β. In the absence of interleukin-1β, compounds
did not significantly affect bovine chondrocytes metabolism. In human chondrocytes and in the
absence of interleukin 1β, curcuminoids extract alone or in combination with hydrolyzed collagen
and green tea extract significantly inhibited matrix metalloproteinase-3 production. In interleukin-
1β-stimulated bovine chondrocytes, interleukin-6, inducible nitric oxide synthase, cyclooxygenase2,
matrix metalloproteinase 3, a disintegrin and metalloproteinase with thrombospondin type I motifs
4 and a disintegrin and metalloproteinase with thrombospondin type I motifs 5 expressions were
decreased by curcuminoids extract alone or in combination with hydrolyzed collagen and green tea
extract. The combination of the three compounds was significantly more efficient to inhibit
interleukin-1β stimulated matrix metalloproteinase-3 expression than curcuminoids extract alone. In
interleukin-1β-stimulated human chondrocytes, nitric oxide, interleukin-6 and matrix
metalloproteinase 3 productions were significantly reduced by curcuminoids extract alone or in
combination with hydrolyzed collagen and green tea extract. These findings indicate that a mixture
of curcuminoids extract, hydrolyzed collagen and green tea extract has beneficial effects on
chondrocytes culture in inflammatory conditions and provide a preclinical basis for the in vivo
testing of this mixture.
60
PLoS One. 2016 Jun 8;11(6):e0156902.
Identification of Targets of a New Nutritional Mixture for Osteoarthritis Management
Composed by Curcuminoids Extract, Hydrolyzed Collagen and Green Tea Extract.
Comblain F, Dubuc JE, Lambert C, Sanchez C, Lesponne I, Serisier S, Henrotin Y.
OBJECTIVE: We have previously demonstrated that a mixture of curcuminoids extract, hydrolyzed
collagen and green tea extract (COT) inhibited inflammatory and catabolic mediator's synthesis by
osteoarthritic human chondrocytes. The objective of this study was to identify new targets of COT
using genomic and proteomic approaches. DESIGN: Cartilage specimens were obtained from 12
patients with knee osteoarthritis. Primary human chondrocytes were cultured in monolayer until
confluence and then incubated for 24 or 48 hours in the absence or in the presence of human
interleukin(IL)-1β (10-11M) and with or without COT, each compound at the concentration of 4
μg/ml. Microarray gene expression profiling between control, COT, IL-1β and COT IL-1β
conditions was performed. Immunoassays were used to confirm the effect of COT at the protein
level. RESULTS: More than 4000 genes were differentially expressed between conditions. The key
regulated pathways were related to inflammation, cartilage metabolism and angiogenesis. The IL-1β
stimulated chemokine ligand 6, matrix metalloproteinase-13, bone morphogenetic protein-2 and
stanniocalcin1 gene expressions and protein productions were down-regulated by COT. COT
significantly decreased stanniocalcin1 production in basal condition. Serpin E1 gene expression and
protein production were down-regulated by IL-1β. COT reversed the inhibitory effect of IL-1β.
Serpin E1 gene expression was up-regulated by COT in control condition. CONCLUSION: The
COT mixture has beneficial effect on osteoarthritis physiopathology by regulating the synthesis of
key catabolic, inflammatory and angiogenesis factors. These findings give a scientific rationale for
the use of these natural ingredients in the management of osteoarthritis.
61
J Am Acad Dermatol. 1984 Apr;10(4):638-46.
The immunogenicity of injectable collagen. I. A 1-year prospective study.
Cooperman L, Michaeli D.
With the growing use of collagen-based biomaterials, questions have been raised regarding the
immunogenicity of this protein in humans. Currently a bovine collagen implant is in widespread use
for the correction of dermal contour deficiencies ( Zyderm Collagen Implant (hereafter referred to
as "the implant"); Collagen Corporation, Palo Alto, CA). To investigate potential immunologic
consequences of this material, sixty-one subjects were evaluated in a 1-year prospective study. Two
of the sixty-one subjects (3%) experienced localized, self-limiting inflammatory responses to the
implant material; only in these two subjects could elevated levels of anti-implant collagen
antibodies be measured by radioimmunoassay. These antibodies did not cross-react with human
dermal collagen nor did they result in elevated levels of circulating immune complexes. Routine
blood and urine testing failed to reveal any results of clinical significance. Thus, this protein
displayed only weak antigenic activity in this study population.
62
Cox M, Nelson DR, Lehninger AL (2008).
Lehninger principles of biochemistry.
San Francisco: W.H. Freeman.
No abstract available
63
Ann Intern Med. 1993 Jun 15;118(12):920-8.
Association between bovine collagen dermal implants and a dermatomyositis or a
polymyositis-like syndrome.
Cukier J, Beauchamp RA, Spindler JS, Spindler S, Lorenzo C, Trentham DE.
OBJECTIVE: To determine whether an excess incidence of dermatomyositis or polymyositis or
both exist in patients treated with injectable bovine collagen implants and to characterize the
clinical picture. DESIGN: Historical cohort study (July 1980 through June 1988). PATIENTS:
Patients were identified from personal experience or adverse reaction reports received by the
manufacturer. SETTING: An 8-year period in the United States during which approximately
345,000 patients received implants. RESULTS: Eight patients with dermatomyositis and an
additional patient with polymyositis were identified from approximately 345,000 patients receiving
injectable bovine collagen implants from July 1980 through June 1988. The nine patients with
dermatomyositis or polymyositis were diagnosed an average of 6.4 months (range, 0.7 to 24.9
months) after collagen implant or skin test exposure or both. Eight of the nine patients had a
delayed-type hypersensitivity response at the test or treatment sites or both, and five of six patients
tested were found to have increased serum antibodies to collagen. Compared with the general
population, the incidence of dermatomyositis or polymyositis among collagen-treated patients was
statistically increased (standardized incidence ratio, 5.05; 95% CI, 2.31 to 9.59; P < 0.0001). A
similar analysis of the eight dermatomyositis case patients produced a standardized incidence ratio
of 18.8 (CI, 8.1 to 37.0; P < 0.0001). Using a Monte Carlo simulation, an interval of 6.4 months or
less from exposure to onset of disease was found to be an extremely rare event, occurring less than
72 times per one million simulation trials (CI, 57 to 91). CONCLUSIONS: Because these data
suggest that an immunologic response to bovine type I or type III collagen or both caused this
dermatomyositis or polymyositis-like syndrome, the risks versus benefits for the cosmetic use of
collagen implants should be reassessed.
64
JOM. 2011;April:66-73.
Collagen Scaffolds for Orthopedic Regenerative Medicine.
Cunniffe GM, O'Brien FJ
Collagen and collagen-based scaffolds offer distinct advantages when selected as biomaterials for
use across a broad spectrum of regenerative medicine applications. However, relatively poor
mechanical properties are often perceived to limit their usefulness for orthopedic applications.
These problems can be overcome through enhanced crosslinking mechanisms or through the
addition of a second, stiffer phase such as hydroxyapatite, thus allowing tailored composite
scaffolds to meet specific tissue requirements. This overview will highlight the current state of the
art of these scaffolds, and consider the exciting prospects and future directions of collagen-based
technologies for orthopedic regenerative medicine.
65
Biochim Biophys Acta. 2014 Feb;1838(2):579-88. doi: 10.1016/j.bbamem.2013.07.017. Epub 2013
Jul 24.
Mechanisms of talin-dependent integrin signaling and crosstalk.
Das M, Subbayya Ithychanda S, Qin J, Plow EF.
Cells undergo dynamic remodeling of the cytoskeleton during adhesion and migration on various
extracellular matrix (ECM) substrates in response to physiological and pathological cues. The major
mediators of such cellular responses are the heterodimeric adhesion receptors, the integrins.
Extracellular or intracellular signals emanating from different signaling cascades cause inside-out
signaling of integrins via talin, a cystokeletal protein that links integrins to the actin cytoskeleton.
Various integrin subfamilies communicate with each other and growth factor receptors under
diverse cellular contexts to facilitate or inhibit various integrin-mediated functions. Since talin is an
essential mediator of integrin activation, much of the integrin crosstalk would therefore be
influenced by talin. However, despite the existence of an extensive body of knowledge on the role
of talin in integrin activation and as a stabilizer of ECM-actin linkage, information on its role in
regulating inter-integrin communication is limited. This review will focus on the structure of talin,
its regulation of integrin activation and discuss its potential role in integrin crosstalk. This article is
part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane
channels, receptors and transporters.
66
Acta Biomater. 2010 Oct;6(10):3957-68.
Collagen-hyaluronic acid scaffolds for adipose tissue engineering.
Davidenko N, Campbell JJ, Thian ES, Watson CJ, Cameron RE.
Three-dimensional (3-D) in vitro models of the mammary gland require a scaffold matrix that
supports the development of adipose stroma within a robust freely permeable matrix. 3-D porous
collagen-hyaluronic acid (HA: 7.5% and 15%) scaffolds were produced by controlled freeze-drying
technique and crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride.
All scaffolds displayed uniform, interconnected pore structure (total porosity approximately 85%).
Physical and chemical analysis showed no signs of collagen denaturation during the formation
process. The values of thermal characteristics indicated that crosslinking occurred and that its
efficiency was enhanced by the presence of HA. Although the crosslinking reduced the swelling of
the strut material in water, the collagen-HA matrix as a whole tended to swell more and show
higher dissolution resistance than pure collagen samples. The compressive modulus and elastic
collapse stress were higher for collagen-HA composites. All the scaffolds were shown to support
the proliferation and differentiation 3T3-L1 preadipocytes while collagen-HA samples maintained a
significantly increased proportion of cycling cells (Ki-67+). Furthermore, collagen-HA composites
displayed significantly raised Adipsin gene expression with adipogenic culture supplementation for
8 days vs. control conditions. These results indicate that collagen-HA scaffolds may offer robust,
freely permeable 3-D matrices that enhance mammary stromal tissue development in vitro.
67
J Exp Med. 1967 Aug 1;126(2):331-46.
The serologic specificity of tropocollagen telopeptides.
Davison PF, Levine L, Drake MP, Rubin A, Bump S.
Tropocollagen preparations from carp, buffalo fish, rats, calves, sheep, and humans have been
studied by electron microscopy and serologic methods. Tropocollagens from each species appeared
identical by electron microscopy but they were readily distinguished (except between sheep and
calves) by C'-fixation tests with rabbit antisera against the various tropocollagens. Tests with calf
tropocollagen antiserum showed no distinction between tropocollagen isolated from different
tissues nor between individuals of the same or different strains. The major immunogenic sites in
native tropocollagen are the telopeptides, and these are present on both alpha1- and alpha2-chains.
The C'-fixing activity was lost with heat denaturation of the tropocollagen, but could be recovered
in a concentration-dependent process on cooling. The fact that pure and enzyme-treated collagen
can provoke serologic reaction implies that collagenous sutures and prostheses used in surgery may
lead to sensitization and rejection, a fact which may merit clinical concern.
68
Plast Reconstr Surg. 1987 Apr;79(4):581-94.
Reaction to injectable collagen: results in animal models and clinical use.
DeLustro F, Smith ST, Sundsmo J, Salem G, Kincaid S, Ellingsworth L.
Since its commercial release, Zyderm collagen implant has been used to treat more than 200,000
subjects in the United States for soft-tissue contour defects and more than 250,000 patients
internationally (including the United States). Approximately 3 percent of subjects' skin tested with
Zyderm collagen experience localized hypersensitivity reactions to collagen, whereas
approximately 1 percent of treated patients demonstrate symptoms of hypersensitivity at treatment
sites. Of the latter treatment responses reported since the conclusion of clinical trials with Zyderm,
56 percent occurred following the first treatment, 28 percent following the second, 10 percent
following the third, and 6 percent following subsequent exposures. The data indicate that most
patients receive a median of three treatments (mean = 4.4) with Zyderm collagen, but most patients
who are likely to develop sensitivity to Zyderm collagen appear to respond immunologically to the
test implant or first treatment exposure. Examining these treatment responses, 45 percent of the
patients reported an onset of symptoms within 10 days and 22 percent at more than 30 days
following the last treatment with Zyderm collagen. Erythema was the sole symptom in 24 percent of
cases, whereas erythema plus induration comprised an additional 42 percent. Antibodies against
Zyderm collagen were detected in the sera of 88 percent of these subjects using an ELISA, but no
reactivity was observed against human collagen. Sera from patients reporting only systemic
symptoms were not found to have anticollagen antibodies. These data suggest that the relative risk
of a hypersensitivity reaction to Zyderm collagen does not increase with multiple exposures, since
patients who are going to develop an immune response to bovine collagen react with greatest
frequency to initial injections of collagen. In animal models, Zyderm collagen was shown to be less
immunogenic than other medical devices which are composed of bovine collagen. Specifically,
comparative studies were conducted in which Zyderm collagen and hemostatic agents were
implanted in the guinea pig subcutaneum: sera from animals treated with collagen-derived
hemostatic devices possessed significant levels of anti-implant antibodies (titers greater than 640),
whereas animals treated with Zyderm collagen mounted minimal responses (titers less than 40).
Additional studies were conducted in which implant materials were compared in a guinea pig
parietal (bony defect) model and in a rabbit hemostasis model: in both, Zyderm collagen
demonstrated lower immunogenicity than commercial bovine collagen hemostatic agents.
Histologic results from these studies showed Zyderm
69
Clin Orthop Relat Res. 1990 Nov;(260):263-79.
Immune responses to allogeneic and xenogeneic implants of collagen and collagen derivatives.
DeLustro F, Dasch J, Keefe J, Ellingsworth L.
Whereas xenogeneic collagen has provided a safe and effective biomaterial for numerous medical
applications, there are few instances in which data permit the correlation of the immunologic profile
of well-defined devices with their clinical sequelae. A major exception is the use of injectable
bovine dermal collagen for soft-tissue contour correction. The low incidence of hypersensitivity has
been studied in the context of clinical efficacy and safety with several devices. The findings indicate
that such immunity usually results in the manifestation of local symptoms of dermal inflammation
at sites of treatment that resolve as the implant is resorbed by the host. In contrast, more
immunogenic hemostatic agents may elicit a more frequent or vigorous immune response that is not
clinically visible or relevant in that application. Recent experiences with collagen-based devices for
the repair and regeneration of bone have also demonstrated that the presence of immunity to their
collagenous or non-collagenous components does not necessarily predict adverse clinical sequelae.
Indeed, numerous specific data indicate that this immunity can exist as an epiphenomenon with no
effect on osteogenesis. To get a true composite picture of biocompatibility, significant steps must be
taken to characterize biomaterials properly and to ensure that immunologic, clinical, histologic, and
other pertinent laboratory data are viewed in relation to one another and not in isolation.
70
J Med Dent Sci. 2008 Mar;55(1):71-9.
The axonal regeneration across a honeycomb collagen sponge applied to the transected spinal
cord.
Fukushima K, Enomoto M, Tomizawa S, Takahashi M, Wakabayashi Y, Itoh S, Kuboki Y,
Shinomiya K.
We developed a honeycomb-shaped lyophilized Type I atelocollagen (Honeycomb Collagen: HC)
with different pore sizes, and the effectiveness of the honeycomb shape on nerve regeneration was
examined. We analyzed neurite outgrowth of dorsal root ganglion (DRG) explants on HC, both in
vitro and, with direct implantation of HC into the defects of adult rat spinal cords, in vivo. The
neurites of DRGs on HC extended linearly through the pores. HC with a 400 microm-pore size
enhanced neurite extension, and YIGSR laminin peptide coating to the HC extended more neurites
than fibronectin coating. The HC scaffolds coated with YIGSR were implanted into 2 mm-defects
of spinal cords at the level of T8-9. Four weeks after implantation, the implants had degraded and
been replaced with self-tissues, repairing the injured site. Neurofilament-positive fibers were
observed in the implantation area and passed the borders between the HC and spinal cord stumps.
Functionally, a motor-evoked potential was observed in the quadriceps femoris muscle 10 weeks
after implantation. The electrophysiological examination showed reconstruction of axon tracts over
the implant. This result indicates that our developed honeycomb shape is advantageous for host
spinal cord compared to the random pored sponge shape, and that it promotes axonal regeneration
after spinal cord injury.
71
FEBS Lett. 1971 Feb 9;12(6):341-344.
Different antigenic determinants in the polypeptide chains of human collagen.
Furthmayr H, Beil W, Timpl R.
No abstract available.
72
Clin Exp Rheumatol. 2006 Sep-Oct;24(5):514-20.
Polymerized-type I collagen for the treatment of patients with rheumatoid arthritis. Effect of
intramuscular administration in a double blind placebo-controlled clinical trial.
Furuzawa-Carballeda J, Fenutria-Ausmequet R, Gil-Espinosa V, Lozano-Soto F, Teliz-Meneses
MA, Romero-Trejo C, Alcocer-Varela J.
OBJECTIVE: To determine the efficacy, tolerance and safety of intramuscular injections of
porcine type I collagen-PVP in patients with RA in a long term-therapy. METHODS: The study
was a double blind placebo-controlled and included 30 patients with active RA (ACR). Patients
were treated with intramuscular injections of 2 ml of collagen-PVP (3.4 mg of collagen) or 2 ml of
placebo during 6 months. The follow up was done during the next 6 months. The primary endpoints
included the Ritchie index (RI), swollen joint count, disease activity score (DAS), erythrocyte
sedimentation rate (ESR), and C-reactive protein (CRP). The secondary endpoints included
morning stiffness, pain intensity on a visual analogue scale (VAS), and Spanish-health assessment
questionnaire (HAQ-DI). Improvement was determined using American College of Rheumatology
response criteria (ACR20, 50 and 70). RESULTS: Collagen-PVP was safe and well tolerated. There
were no adverse events. Patients had a statistically significant improvement (p < 0.05) in collagen-
PVP-treated vs. placebo at 6 months of treatment in: swollen joint count (7.1 +/- 0.8 vs. 16.0 +/-
1.6), RI (8.1 +/- 0.8 vs. 15.2 +/- 1.5), morning stiffness (9.2 +/- 3.1 vs. 29.1 +/- 5.9 min), HAQ-DI
(50.0 +/- 10.8 vs. 22.9 +/- 10.3), DAS (3.0 +/- 0.2 vs. 4.9 +/- 0.3), ACR20 (78.6 vs. 71.4%), ACR50
(57.1 vs. 0%) and ACR70 (7.1 vs. 0%) and CRP (1.1 +/- 0.4 vs. 2.5 +/- 0.7). Patients treated with
collagen-PVP required lower doses of methotrexate vs. placebo (12.6 +/- 0.6 vs. 14.2 +/- 0.7 at 6
months and 12.3 +/- 0.8 vs. 15.4 +/- 0.6 at 12 months; p < 0.05). Serological or haematological
parameters remained unchanged. CONCLUSION: Collagen-PVP has been shown to be a safe and
well-tolerated drug for the long-term treatment of RA. Combination of collagen-PVP plus
methotrexate was more efficacious than methotrexate alone. This biodrug can be useful in the
treatment of RA.
73
Eur J Clin Invest. 2009 Jul;39(7):591-7.
Effect of polymerized-type I collagen in knee osteoarthritis. I. In vitro study.
Furuzawa-Carballeda J, Muñoz-Chablé OA, Barrios-Payán J, Hernández-Pando R.
BACKGROUND: Polymerized-type I collagen (polymerized-collagen) is a down-regulator of
inflammation and a tissue regenerator biodrug. The aim of this study was to evaluate its effect in co-
cultures of cartilage and synovial tissue from patients with knee osteoarthritis (OA). MATERIALS
AND METHODS: Cartilage and synovial tissue from five patients with OA were co-cultured for 7
days in the presence or absence of 1% polymerized-collagen. To determine proteoglycans content,
tissues were stained with alcian blue technique. Pro- and anti-inflammatory cytokines [interleukin
(IL)-1beta, IL-8, IL-10, IL-12, tumour necrosis factor (TNF)-alpha, and interferon (IFN)-gamma]
and tissue inhibitor of metalloproteinase (TIMP)-1 were measured in supernatants by ELISA and
results were normalized by total protein concentration. Cartilage oligomeric matrix protein
(COMP), type II collagen, TNF-alpha, IL-10 and Ki-67 expression were determined by
immunohistochemistry. RESULTS: Polymerized-type I collagen induced an increase of 3- to 6fold
cell proliferation (Ki-67), proteoglycans content, and COMP and type II collagen expression,
whereas it inhibited IL-1beta and TNF-alpha production. IL-10 levels were up-regulated in treated
vs. untreated cultures. No differences were found on IL-8 or TIMP-1 levels in supernatants from
polymerized-collagen-treated co-cultures when compared with untreated cultures. IL-12 and IFN-
gamma were undetectable. CONCLUSION: The addition of polymerized-type I collagen to
cartilage and synovial tissue co-cultures induced up-regulation of chondrocytes proliferation and
cartilage extracellular matrix proteins production (COMP, type II collagen and proteoglycans) as
well as an anti-inflammatory cytokine (IL-10) and the down-modulation of pro-inflammatory
cytokines (IL-1beta and TNF-alpha). It is possible that this mechanism might contribute to induce
tissue regeneration and down-regulation of inflammation in OA.
74
Eur J Clin Invest. 2009 Jul;39(7):598-606.
Effect of polymerized-type I collagen in knee osteoarthritis. II. In vivo study.
Furuzawa-Carballeda J, Muñoz-Chablé OA, Macías-Hernández SI, Agualimpia-Janning A.
BACKGROUND: Polymerized-Type I Collagen (Polymerized-Collagen) is an anti-inflammatory
and a tissue regenerator biodrug. The aim of the study was to evaluate the efficacy and safety of
intra-articular injections of Polymerized-Collagen in patients with knee osteoarthritis (OA).
METHODS AND DESIGN: Patients (n=53) were treated with 12 intra-articular injections of 2 mL
of Polymerized-Collagen (n=27) or 2 mL of placebo (n=26) during 6 months. Follow up period was
6 months. The primary endpoints included Western Ontario and McMaster University Osteoarthritis
Index, Lequesne index, and pain intensity on a visual analogue scale (VAS). Secondary outcomes
were patient global score, investigator global score and drug evaluation. Clinical improvement was
determined if the decrease in pain exceeds 20 mm on a VAS and patients achieved at least 20% of
improvement from baseline. Urinary levels of C-terminal crosslinking telopeptide of collagen type
II (CTXII) and serum high-sensitivity C-reactive protein (hsCRP) were determined by enzyme
immunoassays. Statistical analysis was performed by intention to treat. RESULTS: Polymerized-
Collagen was safe and well tolerated. Patients had a statistically significant improvement (P<0.05)
from baseline vs. Polymerized-Collagen and vs. placebo at 6 months in: Lequesne Index (13.1+/-0.5
vs. 7.1+/-0.7 vs. 9.6+/-0.8; P=0.027), WOMAC (9.0+/-0.5 vs. 4.0+/-0.6 vs. 5.80+/-0.8; P=0.032),
patient VAS (60.0+/-2.6 vs. 20.6+/-2.4 vs. 36.1+/-4.5; P=0.003), physician VAS (49.8+/-1.9 vs.
16.8+/-2.9 vs. 29.8+/-2.9; P=0.002), patient global score (1.08+/-0.1 vs. 2.7+/-0.1 vs. 1.9+/-0.2;
P=0.028) and analgesic usage (30.1+/-9.4 vs. 11.0+/-3.4 vs. 17.9+/-4.9; P=0.001). This
improvement was persistent during the follow up. A threefold increase in CTXII was determined in
placebo group. No differences were found on hs CRP and incidence of adverse events between
groups. CONCLUSION: Polymerized-Collagen is safe and effective in the treatment of knee OA.
75
ScientificWorldJournal. 2012;2012:342854.
Polymerized-type I collagen downregulates inflammation and improves clinical outcomes in
patients with symptomatic knee osteoarthritis following arthroscopic lavage: a randomized,
double-blind, and placebo-controlled clinical trial.
Furuzawa-Carballeda J, Lima G, Llorente L, Nuñez-Álvarez C, Ruiz-Ordaz BH, Echevarría-Zuno
S, Hernández-Cuevas V.
OBJECTIVES: Polymerized-type I collagen (polymerized collagen) is a downmodulator of
inflammation and cartilage regenerator biodrug. AIM: To evaluate the effect of intraarticular
injections of polymerized collagen after arthroscopic lavage on inflammation and clinical
improvement in patients with knee osteoarthritis (OA). METHODS: Patients (n = 19) were treated
with 6 intraarticular injections of 2 mL of polymerized collagen (n = 10) or 2 mL of placebo (n = 9)
during 3 months. Followup was 3 months. The primary endpoints included Lequesne index, pain on
a visual analogue scale (VAS), WOMAC, analgesic usage, the number of Tregs and
proinflammatory/anti-inflammatory cytokine-expressing peripheral cells. Secondary outcomes were
Likert score and drug evaluation. Clinical and immunological improvement was determined if the
decrease in pain exceeds 20 mm on a VAS, 20% of clinical outcomes, and inflammatory parameters
from baseline. Urinary levels of C-terminal crosslinking telopeptide of collagen type II (CTXII) and
erythrocyte sedimentation rate (ESR) were determined. RESULTS: Polymerized collagen was safe
and well tolerated. Patients had a statistically significant improvement (P < 0.05) from baseline
versus polymerized collagen and versus placebo at 6 months on Lequesne index, VAS, ESR, Tregs
IL-1β, and IL-10 peripheral-expressing cells. Urinary levels of CTXII were decreased 44% in
polymerized collagen versus placebo. No differences were found on incidence of adverse events
between groups. CONCLUSION: Polymerized collagen is safe and effective on downregulation of
inflammation in patients with knee OA.
76
Plast Reconstr Surg. 1989 May;83(5):875-9.
Identification of the collagen-producing cells in healing flexor tendons.
Garner WL, McDonald JA, Koo M, Kuhn C 3rd, Weeks PM.
A monoclonal antibody to procollagen type I (anti-pC) which specifically stains cells synthesizing
collagen was used to study the healing of chicken flexor tendons in vivo. Healing was assessed
using routine hematoxylin and eosin histology and immunoperoxidase staining with anti-pC.
Studies showed that epitenon cells proliferate 3 days after injury and are producing collagen by 7
days after injury. Tenocytes do not begin producing collagen until 14 to 21 days after injury. From
3 to 5 weeks, the entire substance of the tendon becomes filled with collagen-synthesizing cells.
These cells may have originated from the tendon sheath, the epitenon, or the endotenon; however,
the evidence presented in this study suggests that the epitenon is a major source of these cells.
77
Urol. 1998;33(2):129-33.
Adv Drug Deliv Rev. 2003 Nov 28;55(12):1613-29.
Collagen sponges for bone regeneration with rhBMP-2.
Geiger M, Li RH, Friess W.
In the US alone, approximately 500,000 patients annually undergo surgical procedures to treat bone
fractures, alleviate severe back pain through spinal fusion procedures, or promote healing of non-
unions. Many of these procedures involve the use of bone graft substitutes. An alternative to bone
grafts are the bone morphogenetic proteins (BMPs), which have been shown to induce bone
formation. For optimal effect, BMPs must be combined with an adequate matrix, which serves to
prolong the residence time of the protein and, in some instances, as support for the invading
osteoprogenitor cells. Several factors involved in the preparation of adequate matrices, specifically
collagen sponges, were investigated in order to test the performance in a new role as an implant
providing local delivery of an osteoinductive differentiation factor. Another focus of this review is
the current system consisting of a combination of recombinant human BMP-2 (rhBMP-2) and an
absorbable collagen sponge (ACS). The efficacy and safety of the combination has been clearly
proven in both animal and human trials.
78
Biotechnol Appl Biochem. 2004 Jun;39(Pt 3):329-38.
Inactivation of the bovine-spongiform-encephalopathy (BSE) agent by the acid and alkaline
processes used in the manufacture of bone gelatine.
Grobben AH, Steele PJ, Somerville RA, Taylor DM.
A validation study was carried out to determine the capacity of the traditional acid and alkaline
processes used in the manufacture of bovine bone gelatine to remove and/or inactivate the
transmissible agent that causes BSE (bovine spongiform encephalopathy). Using an accurately
scaled down laboratory process that precisely mimicked the minimum conditions of the industrial
processes, gelatine (gelatin) was manufactured from industrial starting material that had been spiked
with mouse brain infected with the 301V strain of mouse-passaged BSE agent. Clearance factors
were determined by titrating the infectivity levels of the infected mouse brain tissue, the gelatine
extracts, and the final sterilized gelatine solution. The infectivity level of the spiked starting
material was 10(8.4) mouse intracerebral ID(50)/kg (ID(50) is the dose at which half of the
challenged animals were infected). Clearance factors of 10(2.6) and 10(3.7) ID(50) were
demonstrated for the first stages of the acid and alkaline processes respectively during which the
bones are converted to crude gelatine. It was further demonstrated that the complete acid and
alkaline processes both reduced infectivity to undetectable levels, giving clearance factors of
>/=10(4.8) ID(50) for the acid process, and >/=10(4.9) ID(50) for the alkaline process.
79
Dent Traumatol. 2004 Dec;20(6):334-7.
The effect on osteogenesis of type I collagen applied to experimental bone defects.
Gungormus M.
The purpose of the present investigation was to evaluate the effects of type I collagen sponge on the
healing of bone defects. In this study, six adult male rabbits were used. After the induction of
general anesthesia with intraperitoneal kethamine, the anterior surfaces of tibias of the rabbits were
surgically exposed, and two holes with 4 mm in diameter were prepared on each tibia for the
investigation. Only one hole in each tibia was filled with type I collagen, the other unfilled hole was
used as control. During the study, radiopacity changes in the radiographs of the tibias of the rabbits
were evaluated. The animals were killed on the 28th day, and histologic sections of the tibias were
prepared. On the 28th day, it was histopathologically observed that collagen cavities were filled
with new bone. In addition, it was determined that there was an increase in radiopacity of the defect
areas from 14 to 28 days in both groups, and there were statistically a significant difference between
control and collagen groups (P = 0.0001). In this study, consequently, it was determined that type I
collagen sponge in the experimental cavities provides a more rapid regeneration of bone defects
compared with non-filled cavities.
80
JAMA. 1995 Dec 13;274(22):1800-4.
Users' guides to the medical literature. IX. A method for grading health care
recommendations. Evidence-Based Medicine Working Group.
Guyatt GH, Sackett DL, Sinclair JC, Hayward R, Cook DJ, Cook RJ.
No abstract available.
81
Biomaterials. 2008 Feb;29(4):438-47.
An injectable cross-linked scaffold for nucleus pulposus regeneration.
Halloran DO, Grad S, Stoddart M, Dockery P, Alini M, Pandit AS.
Incorporation of scaffolds has long been recognized as a critical element in most tissue engineering
strategies. However with regard to intervertebral disc tissue engineering, the use of a scaffold
containing the principal extracellular matrix components of native disc tissue (i.e. collagen type II,
aggrecan and hyaluronan) has not been investigated. In this study the behavior of bovine nucleus
pulposus cells that were seeded within non-cross-linked and enzymatically cross-linked,
atelocollagen type II based scaffolds containing varying concentrations of aggrecan and hyaluronan
was investigated. Cross-linking atelocollagen type II based scaffolds did not cause any negative
effects on cell viability or cell proliferation over the 7-day culture period. The cross-linked scaffolds
retained the highest proteoglycan synthesis rate and the lowest elution of sulfated
glycosaminoglycan into the surrounding medium. From confined compression testing and volume
reduction measurements, it was seen that the cross-linked scaffolds provided a more stable structure
for the cells compared to the non-cross-linked scaffolds. The results of this study indicate that the
enzymatically cross-linked, composite collagen-hyaluronan scaffold shows the most potential for
developing an injectable cell-seeded scaffold for nucleus pulposus treatment in degenerated
intervertebral discs.
82
J Orthop Sci. 2013 Jul;18(4):627-35.
Treatment of cartilage defects by subchondral drilling combined with covering with
atelocollagen membrane induces osteogenesis in a rat model.
Hamanishi M, Nakasa T, Kamei N, Kazusa H, Kamei G, Ochi M.
BACKGROUND: The coverage of the atelocollagen membrane at the chondral defect after
subchondral drilling might improve the beneficial effects for cartilage repair because of the
prevention of scattering and accumulation of cells and growth factors from bone marrow within the
chondral defect. On the other hand, it might block cells and factors derived from the synovium or
cause high pressure in the chondral defect, resulting in prevention of cells and growth factors
gushing out from the bone marrow, which leads to disadvantages for cartilage repair. METHOD:
We tested this hypothesis in a 2-mm-diameter chondral defect created in the articular cartilage of
the patellar groove in a rat models. Defects were left untreated, or were drilled or drilled and
covered with an atelocollagen membrane; healing was evaluated by histology and gene expression
analysis using real-time polymerase chain reaction and immunohistochemistry. RESULTS:
Membrane coverage induced bone tissue ingrowth into the punched chondral defect. At 1 week,
expression of TGFβ, Sox9, Runx2, osteocalcin, Col1a1, and Col2a1 in the drilling group was
significantly higher than in the covering group. At 4 weeks, expressions of TGFβ, Runx2, and
Col1a1 were all significantly higher in the drilling group, while Sox9, osteocalcin, and Col2a1 were
significantly higher in the covering group. Immunohistochemistry demonstrated Sox9, osteocalcin,
and type II collagen on the bony reparative tissue in the covering group. CONCLUSIONS: These
results suggest that the atelocollagen membrane coverage resulted in inhibition of cartilage repair.
83
Foot Ankle Clin. 2007 Dec;12(4):553-67, v.
Tendon healing.
Hope M, Saxby TS.
An understanding of the processes of tendon healing and tendon-to-bone healing is important for
the intraoperative and postoperative management of patients with tendon ruptures or of patients
requiring tendon transfers in foot and ankle surgery. Knowledge of the normal process allows
clinicians to develop strategies when normal healing fails. This article reviews the important work
behind the identification of the normal phases and control of tendon healing. It outlines the failed
response in tendinopathy and describes tendon-to-bone healing in view of its importance in foot and
ankle surgery.
84
J Reconstr Microsurg. 2001 Feb;17(2):115-23.
A study of induction of nerve regeneration using bioabsorbable tubes.
Itoh S, Takakuda K, Ichinose S, Kikuchi M, Schinomiya K.
A silicone tube (S-tube) was packed with CPLA (copolymer of poly-L-lactide) fibers (S-
tube+CPLA) or collagen fibers (S-tube+fiber). Two types of tube were prepared from a collagen
sheet (Col-tube) and a bioabsorbable atelocollagen membrane for guided tissue regeneration (GTR-
tube). They were packed with collagen fibers or films (Col-tube+fiber, GTR-tube+fiber and GTR-
tube+film). Bridge grafting (15 mm in length) was performed with these tubes in a rat sciatic nerve
model. Specimens were harvested after 8 weeks. Minifascicles were formed in the open space
between the CPLA fibers in the S-tube+CPLA group. Regenerated axons were also formed in the
degenerated collagen fibers in the S-tube+Col group. Immunocytochemistry evaluation revealed
that Schwann cells invaded the space in the absorbing collagen fibers. Histologic analysis of the
regenerated axons in the groups with Col-tubes or GTR-tubes revealed that both the Col-tube and
the GTR-tube packed with collagen fibers were effective in providing a scaffold for regenerating
nerve tissue
85
J Hand Surg Am. 2008 Jan;33(1):102-12.
Tendon: biology, biomechanics, repair, growth factors, and evolving treatment options.
James R, Kesturu G, Balian G, Chhabra AB.
Surgical treatment of tendon ruptures and lacerations is currently the most common therapeutic
modality. Tendon repair in the hand involves a slow repair process, which results in inferior repair
tissue and often a failure to obtain full active range of motion. The initial stages of repair include
the formation of functionally weak tissue that is not capable of supporting tensile forces that allow
early active range of motion. Immobilization of the digit or limb will promote faster healing but
inevitably results in the formation of adhesions between the tendon and tendon sheath, which leads
to friction and reduced gliding. Loading during the healing phase is critical to avoid these adhesions
but involves increased risk of rupture of the repaired tendon. Understanding the biology and
organization of the native tendon and the process of morphogenesis of tendon tissue is necessary to
improve current treatment modalities. Screening the genes expressed during tendon morphogenesis
and determining the growth factors most crucial for tendon development will likely lead to
treatment options that result in superior repair tissue and ultimately improved functional outcomes.
86
Cells Tissues Organs. 2013;198(4):278-88.
Autologous collagen-induced chondrogenesis using fibrin and atelocollagen mixture.
Jeong IH, Shetty AA, Kim SJ, Jang JD, Kim YJ, Chung YG, Choi NY, Liu CH.
For articular cartilage defect treatment, many treatment modalities have been developed. We
evaluate the cartilage repair potential of an atelocollagen and fibrin mixture transplanted to cartilage
defects. A circular, articular cartilage defect 4 mm in diameter was made in the trochlear region in
each of 20 New Zealand white rabbits. The 10 rabbits in the control group were kept without
treatment and the 10 rabbits in the experimental group underwent injection of atelocollagen mixed
with fibrin. At week 12 following surgery the cartilage was observed and histologically compared
in both groups. The surface of the newly generated cartilage was very smooth and even, and we also
noted that the entire area was completely regenerated in the experimental group. The control group
showed incomplete and irregular cartilage formation in the defect. Regarding the histological
scoring, comparison of the two groups differed significantly (p < 0.001). Injection of a mixture of
atelocollagen and fibrin used to treat articular cartilage defects of the knee appears to be an effective
method for cartilage regeneration.
87
Connect Tissue Res. 2013;54(3):218-26.
Cellular and molecular factors in flexor tendon repair and adhesions: a histological and gene
expression analysis.
Juneja SC, Schwarz EM, O'Keefe RJ, Awad HA.
Flexor tendon healing is mediated by cell proliferation, migration, and extracellular matrix synthesis
that contribute to the formation of scar tissue and adhesion. The biological mechanisms of flexor
tendon adhesion formation have been linked to transforming growth factor β (TGF-β). To elucidate
the cellular and molecular events in this pathology, we implanted live flexor digitorum longus grafts
from the reporter mouse Rosa26(LacZ/+) in wild-type recipients, and used histological β-
galactosidase (β-gal) staining to evaluate the intrinsic versus extrinsic cellular origins of scar, and
reverse transcription-polymerase chain reaction to measure gene expression of TGF-β and its
receptors, extracellular matrix proteins, and matrix metalloproteinases (MMPs) and their regulators.
Over the course of healing, graft cellularity and β-gal activity progressively increased, and β-gal-
positive cells migrated out of the Rosa26(LacZ/+) graft. In addition, there was an evidence of influx
of host cells (β-gal-negative) into the gliding space and the graft, suggesting that both graft and host
cells contribute to adhesions. Interestingly, we observed a biphasic pattern in which Tgfb1
expression was the highest in the early phases of healing and gradually decreased thereafter,
whereas Tgfb3 increased and remained upregulated later. The expression of TGF-β receptors was
also upregulated throughout the healing phases. In addition, type III collagen and fibronectin were
upregulated during the proliferative phase of healing, confirming that murine flexor tendon heals by
scar tissue. Furthermore, gene expression of MMPs showed a differential pattern in which
inflammatory MMPs were the highest early and matrix MMPs increased over time. These findings
offer important insights into the complex cellular and molecular factors during flexor tendon
healing.
88
Curr Opin Cell Biol. 2008 Oct;20(5):495-501.
Collagen fibrillogenesis: fibronectin, integrins, and minor collagens as organizers and
nucleators.
Kadler KE, Hill A, Canty-Laird EG.
Collagens are triple helical proteins that occur in the extracellular matrix (ECM) and at the cell-
ECM interface. There are more than 30 collagens and collagen-related proteins but the most
abundant are collagens I and II that exist as D-periodic (where D = 67 nm) fibrils. The fibrils are of
broad biomedical importance and have central roles in embryogenesis, arthritis, tissue repair,
fibrosis, tumor invasion, and cardiovascular disease. Collagens I and II spontaneously form fibrils
in vitro, which shows that collagen fibrillogenesis is a selfassembly process. However, the situation
in vivo is not that simple; collagen I-containing fibrils do not form in the absence of fibronectin,
fibronectin-binding and collagen-binding integrins, and collagen V. Likewise, the thin collagen II-
containing fibrils in cartilage do not form in the absence of collagen XI. Thus, in vivo, cellular
mechanisms are in place to control what is otherwise a protein self-assembly process. This review
puts forward a working hypothesis for how fibronectin and integrins (the organizers) determine the
site of fibril assembly, and collagens V and XI (the nucleators) initiate collagen fibrillogenesis.
89
J Bone Miner Metab. 2012 Nov;30(6):638-50.
Chronological histological changes during bone regeneration on a non-crosslinked
atelocollagen matrix.
Kagawa R, Kishino M, Sato S, Ishida K, Ogawa Y, Ikebe K, Oya K, Ishimoto T, Nakano T, Maeda
Y, Komori T, Toyosawa S.
Cleavage of the antigenic telopeptide region from type I collagen yields atelocollagen, and this is
widely used as a scaffold for bone regeneration combined with cells, growth factors, etc. However,
neither the biological effect of atelocollagen alone or its contribution to bone regeneration has been
well studied. We evaluated the chronological histological changes during bone regeneration
following implantation of non-crosslinked atelocollagen (Koken Co., Ltd.) in rat calvarial defects.
One week after implantation, osteogenic cells positive for runt-related transcription factor 2
(Runx2) and osteoclasts positive for tartrate-resistant acid phosphatase (TRAP) were present in the
atelocollagen implant in the absence of bone formation. The number of Runx2-positive osteogenic
cells and Osterix-positive osteoblasts increased 2 weeks after implantation, and bone matrix
proteins (osteopontin, OPN; osteocalcin, OC; dentin matrix protein 1, DMP1) were distributed in
newly formed bone in a way comparable to normal bone. Some resorption cavities containing
osteoclasts were also present. By 3 weeks after implantation, most of the implanted atelocollagen
was replaced by new bone containing many resorption cavities, and OPN, OC, and DMP1 were
deposited in the residual collagenous matrix. After 4 weeks, nearly all of the atelocollagen implant
was replaced with new bone including hematopoietic marrow. Immunohistochemistry for the
telopeptide region of type I collagen (TeloCOL1) during these processes demonstrated that the
TeloCOL1-negative atelocollagen implant was replaced by TeloCOL1-positive collagenous matrix
and new bone, indicating that new bone was mostly composed of endogenous type I collagen.
These findings suggest that the atelocollagen itself can support bone regeneration by promoting
osteoblast differentiation and type I collagen production.
90
ASAIO J. 2007 Jul-Aug;53(4):506-13.
Regeneration of skeletal muscle using in situ tissue engineering on an acellular collagen
sponge scaffold in a rabbit model.
Kin S, Hagiwara A, Nakase Y, Kuriu Y, Nakashima S, Yoshikawa T, Sakakura C, Otsuji E,
Nakamura T, Yamagishi H.
Because of the limited ability of skeletal muscle to regenerate, resection of a large amount of
muscle mass often results in incomplete recovery due to nonfunctional scar tissue. The aim of this
study was to regenerate skeletal muscle using in situ tissue engineering in a rabbit model. In 18
male rabbits, a muscle defect (1.0 x ~1.0 x ~0.5 cm) was created in the vastus lateralis of both legs.
A piece of cross-linked atelocollagen sponge was then inserted into the defect in one leg, whereas
the defect in the other leg was left untreated. Both defects were finally covered with fascia. Twenty-
four weeks after surgery, the defect that had been filled with the cross-linked atelocollagen sponge
scaffold showed mild concavity and slight adhesion to the fascia, while the control side showed
severe scar formation and shrinkage. Histologically, the regenerating myofibers at the site
containing the collagen sponge were greater in number, diameter, and length than those at the
control site. These results indicate that cross-linked atelocollagen sponge has the potential to act as
a scaffold for muscle tissue regeneration.
91
J Sci Food Agric. 2015 Mar 15;95(4):702-7.
A double-blind, placebo-controlled, randomised, clinical study on the effectiveness of collagen
peptide on osteoarthritis.
Kumar S, Sugihara F, Suzuki K, Inoue N, Venkateswarathirukumara S.
Author information
Abstract
Background: Recent studies show that enzymatically hydrolysed collagen, the collagen peptide, is
absorbed and distributed to joint tissues and has analgesic and anti-inflammatory properties. A
double-blind, placebo-controlled, randomised trial with collagen peptides isolated from pork skin
(PCP) and bovine bone (BCP) sources was carried out to study the effectiveness of orally
supplemented collagen peptide to control the progression of osteoarthritis in patients diagnosed with
knee osteoarthritis. Improvement in treatment was assessed with reduction in Western Ontario
McMaster Universities (WOMAC), visual analogue scale (VAS) and quality of life (QOL) scores
from baseline to 13 weeks (Visit 7). Safety and tolerability were also evaluated. Results: There was
significant reduction from baseline to Visit 7 in the primary end points of WOMAC and VAS
scores and in the secondary end point of QOL score in subjects with PCP and BCP groups, while in
subjects with placebo group the end point indices remained unaltered. Furthermore, all the score
levels of WOMAC, VAS and QOL decreased significantly (P < 0.01) in the study group compared
to placebo group in Visit 7. Conclusion: The study demonstrated that collagen peptides are potential
therapeutic agents as nutritional supplements for the management of osteoarthritis and maintenance
of joint health.
92
Int J Pharm. 2001 Jun 19;221(1-2):1-22.
Biomedical applications of collagen.
Lee CH, Singla A, Lee Y.
Collagen is regarded as one of the most useful biomaterials. The excellent biocompatibility and
safety due to its biological characteristics, such as biodegradability and weak antigenecity, made
collagen the primary resource in medical applications. The main applications of collagen as drug
delivery systems are collagen shields in ophthalmology, sponges for burns/wounds, mini-pellets and
tablets for protein delivery, gel formulation in combination with liposomes for sustained drug
delivery, as controlling material for transdermal delivery, and nanoparticles for gene delivery and
basic matrices for cell culture systems. It was also used for tissue engineering including skin
replacement, bone substitutes, and artificial blood vessels and valves. This article reviews
biomedical applications of collagen including the collagen film, which we have developed as a
matrix system for evaluation of tissue calcification and for the embedding of a single cell
suspension for tumorigenic study. The advantages and disadvantages of each system are also
discussed.
93
Spine (Phila Pa 1976). 2012 Mar 15;37(6):452-8.
Tissue engineering of the intervertebral disc with cultured nucleus pulposus cells using
atelocollagen scaffold and growth factors.
Lee KI, Moon SH, Kim H, Kwon UH, Kim HJ, Park SN, Suh H, Lee HM, Kim HS, Chun HJ,
Kwon IK, Jang JW.
STUDY DESIGN: In vitro experiment using rabbit nucleus pulposus (NP) cells seeded in
atelocollagen scaffolds under the stimulation of growth factors. OBJECTIVE: To demonstrate the
effect of anabolic growth factors in rabbit NP cells cultured in atelocollagen type I and type II.
SUMMARY OF BACKGROUND DATA: Atelocollagen provides intervertebral disc (IVD) cells
for a biocompatible environment to produce extracellular matrix. IVD cells with exogenous
transforming growth factor-beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) also
render an increase in matrix synthesis. However, the effect of anabolic growth factors in NP cells
cultured in atelocollagens was not elucidated before. METHODS: Rabbit NP cell was harvested,
enzymatically digested, and cultured. The NP cells were seeded to atelocollagen type I and type II
scaffolds, and then cultures were exposed to TGF-β1 (10 ng/mL) and/or BMP-2 (100 ng/mL). DNA
synthesis was measured using [4H]-thymidine incorporation. Newly synthesized proteoglycan was
measured using [35S]-sulfate incorporation. Reverse transcription-polymerase chain reactions (RT-
PCRs) for mRNA expression of aggrecan, collagen type I, collagen type II, and osteocalcin were
performed. RESULTS: Rabbit NP cells cultured in atelocollagen type I scaffold showed an increase
(1.7 to 2.4-fold) in DNA synthesis in response to TGF-β1 and/or BMP-2 (P < 0.05), whereas NP
cultures in atelocollagen type II demonstrated a 30% increase in DNA synthesis only with
combination of both growth factors compared with control (P < 0.05). Rabbit NP cells in
atelocollagen type II scaffold with TGF-β1 and combination of both growth factors exhibited robust
5.3- and 5.4-fold increases in proteoglycan synthesis (P < 0.05), whereas any cultures in
atelocollagen type I failed to show any significant increase compared with control. Rabbit NP cells
in atelocollagen type I and type II scaffolds with TGF-β1 and/or BMP-2 demonstrated the
upregulation of aggrecan, collagen type I, and collagen type II mRNA expression compared with
saline control (P < 0.05). The response in transcriptional level was more robust in atelocollagen
type II than in type I. In any event, there is no recognizable expression of osteocalcin (P < 0.05).
CONCLUSION: NP cells in atelocollagens under the stimulation of TGF-β1 and BMP-2 exhibited
anabolic responses in transcriptional and translational levels. Hence, such an approach can provide
a suitable engineered tissue for IVD regeneration with potential for robust refurbishment of matrix.
94
Exp Neurol. 2010 Jun;223(2):645-52.
Bio-printing of collagen and VEGF-releasing fibrin gel scaffolds for neural stem cell culture.
Lee YB, Polio S, Lee W, Dai G, Menon L, Carroll RS, Yoo SS.
Time-released delivery of soluble growth factors (GFs) in engineered hydrogel tissue constructs
promotes the migration and proliferation of embedded cells, which is an important factor for
designing scaffolds that ultimately aim for neural tissue regeneration. We report a tissue
engineering technique to print murine neural stem cells (C17.2), collagen hydrogel, and GF
(vascular endothelial growth factor: VEGF)-releasing fibrin gel to construct an artificial neural
tissue. We examined the morphological changes of the printed C17.2 cells embedded in the
collagen and its migration toward the fibrin gel. The cells showed high viability (92.89+/-2.32%)
after printing, which was equivalent to that of manually-plated cells. C17.2 cells printed within
1mm from the border of VEGF-releasing fibrin gel showed GF-induced changes in their
morphology. The cells printed in this range also migrated toward the fibrin gel, with the total
migration distance of 102.4+/-76.1microm over 3days. The cells in the control samples (fibrin
without the VEGF or VEGF printed directly in collagen) neither proliferated nor migrated. The
results demonstrated that bio-printing of VEGF-containing fibrin gel supported sustained release of
the GF in the collagen scaffold. The presented method can be gainfully used in the development of
three-dimensional (3D) artificial tissue assays and neural tissue regeneration applications.
95
Chin J Traumatol. 2004 Dec;7(6):358-62.
Effect of type I collagen on the adhesion, proliferation, and osteoblastic gene expression of
bone marrow-derived mesenchymal stem cells.
Liu G, Hu YY, Zhao JN, Wu SJ, Xiong Z, Lu R.
OBJECTIVE: To investigate the effects of porous poly lactide-co-glycolide (PLGA) modified by
type I collagen on the adhesion, proliferation, and differentiation of rabbit marrow-derived
mesenchymal stem cells (MSCs). METHODS: The third generation MSCs isolated from mature
rabbits by density gradient centrifugation were cultured at different initial concentrations on 0.3 cm
x 1.2 cm x 2.0 cm 3-D porous PLGA coated by type I collagen in RPMI 1640 containing 10% fetal
calf serum, while cultured on PLGA without type I collagen as control. The cells adhesive and
proliferative behavior at 7, 14, and 21 days after inoculation was assessed by determining the
incorporation rate of [(3)H]-TdR. In order to examine MSCs differentiation, the expression of
osteoblasts marker genes, osteocalcin (OCN), alkaline phosphatase (ALP), osteopontin (OPN)
mRNA, were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), and further
more, the cell morphology at 21 days was also observed by scanning electron microscope (SEM).
RESULTS: Type I collagen promoted cell adhesion on PLGA. The valve was significantly higher
than controls (6 h, 2144 cpm+/-141 cpm vs. 1797 cpm+/-118 cpm, P=0.017; 8 h, 2311 cpm+/-113
cpm vs. 1891 cpm+/-103 cpm, P=0.01). The cells which cultured on PLGA coated with type I
collagen showed significantly higher cell proliferation than controls on the 7 th day (1021 cpm+/-
159 cpm vs. 451 cpm+/-67 cpm, P=0.002), the 14th day (1472 cpm+/-82 cpm vs. 583 cpm+/-67
cpm, P<0.001) and 21 th day (1728 cpm+/-78 cpm vs. 632 cpm+/-55 cpm, P<0.001). Osteoblasts
markers, OCN, ALP, OPN mRNA, were all detected on PLGA coated by type I collagen on the 21
th day, but OCN, OPN mRNA could not be found in controls. Spindle and polygonal cells well
distributed on the polymer coated by type I collagen while cylindric or round cells in controls.
CONCLUSIONS: Type I collagen is effective in promoting the adhesion, proliferation and
differentiation of MSCs on PLGA.
96
Facial Plast Surg. 2014 Dec;30(6):615-22.
Complications of collagen fillers.
Lucey P, Goldberg DJ.
As the skin ages, a deficiency in collagen occurs, thus injectable collagen products have become a
sensible and popular option for dermal filling and volume enhancement. Several types of collagen
have been developed over the years, including animal sources such as bovine and porcine collagen,
as well as human-based sources derived from pieces of the patient's own skin, cadaver skin, and
later cultured from human dermal fibroblasts. While collagen overall has a relatively safe, side
effect profile, there are several complications, both early and late onset, that practitioners and
patients should be aware of. Early complications, occurring within days of the procedure, can be
divided into non-hypersensitivity and hypersensitivity reactions. The non-hypersensitive reactions
include injection site reactions, discoloration, maldistribution, infection, skin necrosis, and the very
rare but dreaded risk of vision loss, whereas the hypersensitivity reactions present usually as
delayed type IV reactions, but can also rarely present as an immediate type I reaction. Late
complications, occurring within weeks to even years after injection, include granuloma formation,
foreign body reactions, and infection secondary to atypical mycobacteria or biofilms. This review
will give a detailed overview of the complications secondary to cutaneous collagen injections.
97
J Biomed Mater Res B Appl Biomater. 2006 Oct;79(1):25-34.
Tissue engineering of articular cartilage with autologous cultured adipose tissue-derived
stromal cells using atelocollagen honeycomb-shaped scaffold with a membrane sealing in
rabbits.
Masuoka K, Asazuma T, Hattori H, Yoshihara Y, Sato M, Matsumura K, Matsui T, Takase B,
Nemoto K, Ishihara M.
Adipose tissue derived stromal cells (ATSCs), which were isolated from adipose tissue of rabbit,
have shown to possess multipotential, that is, they differentiate into osteoblasts and adipocytes in
plate-culturing and into chondrocytes in an established aggregate culture using defined
differentiation-inductive medium. The aim of this study was to evaluate the utility of ATSCs in
tissue engineering procedures for repair of articular cartilage-defects using the atelocollagen
honeycomb-shaped scaffold with a membrane sealing (ACHMS-scaffold). We intended to repair
full-thickness articular cartilage defects in rabbit knees using autologously cultured ATSCs
embedded in the ACHMS-scaffold. ATSCs were incubated within the ACHMS-scaffold to allow a
high density and three-dimensional culture with control medium. An articular cartilage defect was
created on the patellar groove of the femur, and the defect was filled with the ATSCs-containing
ACHMS-scaffold, ACHMS-scaffold alone, or empty (control). Twelve weeks after the operation,
the histological analyses showed that only the defects treated with the ATSCs-containing ACHMS-
scaffold were filled with reparative hyaline cartilage, highly expressed Type II collagen. These
results indicate that transplantation of autologous ATSCs-containing ACHMS-scaffold is effective
in repairing articular cartilage defects.
98
Science. 1969 Dec 19;166(3912):1522-4.
Localization of antigenic determinants in the polypeptide chains of collagen.
Michaeli D, Martin GR, Kettman J, Benjamini E, Leung DY, Blatt BA.
After being injected with collagen from rat or guinea pig skin, rabbits form high titer, species-
specific antibodies to collagen. Antibody is directed primarily against the alpha2 chain of collagen
with little reaction with the alpha1 chain. The amino terminal peptide of cyanogen bromide
cleavage of the alpha2 chain of guinea pig skin collagen, alpha2-CBl, effectively inhibited the
antigen-antibody reaction, an indication that a major antigenic determinant of collagen is located at
the amino terminal of the alpha2 chain.
99
Arch Orthop Trauma Surg. 1997;116(8):454-62.
Histological and biomechanical observations of the rabbit patellar tendon after removal of its
central one-third.
Miyashita H , Ochi M, Ikuta Y.
Using 35 Japanese white rabbits, a study was made of tissue regeneration and the mechanical
properties of the patellar tendon after removal of its central one-third. After removal of the central
one-third of the patellar tendon on one side, in experiment 1 the strength of the entire patellar
tendon including the regenerated tissue was compared with that of the patellar tendon on the
opposite side with the central one-third removed at the time of killing, and in experiment 2 the
strength of only the regenerated tissue was compared with that of the patellar tendon on the opposite
side with two-thirds of the medial and lateral sides removed at the time of death. In experiment 1,
the maximum load showed no significant difference between the operated side and the control. In
one half of the cases, the strength of the operated side including the regenerated tissue was weak,
suggesting weakening of the patellar tendon on the residual bilateral sides. In experiment 2, the
maximum load of the regenerated tissue was significantly lower than that of the control, the former
being 25% of the latter even at 6 months. Histologically, the characteristics of the cells and collagen
fibers gradually approached those of normal tissue, but the crimp pattern of the collagen fibers and
fibrils was evidently smaller than that of the control. These results indicate that regenerated tissue
was still mechanically weak and immature at 6 months.
100
Semin Arthritis Rheum. 2000 Oct;30(2):87-99.
Role of collagen hydrolysate in bone and joint disease.
Moskowitz RW.
OBJECTIVES: To review the current status of collagen hydrolysate in the treatment of
osteoarthritis and osteoporosis. METHODS: Review of past and current literature relative to
collagen hydrolysate metabolism, and assessment of clinical investigations of therapeutic trials in
osteoarthritis and osteoporosis. RESULTS: Hydrolyzed gelatin products have long been used in
pharmaceuticals and foods; these products are generally recognized as safe food products by
regulatory agencies. Pharmaceutical-grade collagen hydrolysate (PCH) is obtained by hydrolysis of
pharmaceutical gelatin. Clinical studies suggest that the ingestion of 10 g PCH daily reduces pain in
patients with osteoarthritis of the knee or hip; blood concentration of hydroxyproline is increased.
Clinical use is associated with minimal adverse effects, mainly gastrointestinal, characterized by
fullness or unpleasant taste. In a multicenter, randomized, double-blind, placebo-controlled trial
performed in clinics in the United States, United Kingdom, and Germany, results showed no
statistically significant differences for the total study group (all sites) for differences of mean pain
score for pain. There was, however, a significant treatment advantage of PCH over placebo in
German sites. In addition, increased efficacy for PCH as compared to placebo was observed in the
overall study population amongst patients with more severe symptomatology at study onset.
Preferential accumulation of 14C-labeled gelatin hydrolysate in cartilage as compared with
administration of 14C-labeled proline has been reported. This preferential uptake by cartilage
suggests that PCH may have a salutary effect on cartilage metabolism. Given the important role for
collagen in bone structure, the effect of PCH on bone metabolism in osteoporotic persons has been
evaluated. Studies of the effects of calcitonin with and without a collagen hydrolysate-rich diet
suggested that calcitonin plus PCH had a greater effect in inhibiting bone collagen breakdown than
calcitonin alone, as characterized by a fall in levels of urinary pyridinoline cross-links. PCH
appeared to have an additive effect relative to use of calcitonin alone. CONCLUSIONS: Collagen
hydrolysate is of interest as a therapeutic agent of potential utility in the treatment of osteoarthritis
and osteoporosis. Its high level of safety makes it attractive as an agent for long-term use in these
chronic disorders.
101
J Dermatol. 2005 May;32(5):376-80.
Atelocollagen sponge and recombinant basic fibroblast growth factor combination therapy
for resistant wounds with deep cavities.
Nakanishi A, Hakamada A, Isoda K, Mizutani H.
Recent advances in bioengineering have introduced materials that enhance wound healing. Even
with such new tools, some deep ulcers surrounded by avascular tissues, including bone, tendon, and
fascia, are resistant to various therapies and easily form deep cavities with loss of subcutaneous
tissue. Atelocollagen sponges have been used as an artificial dermis to cover full-thickness skin
defects. Topical recombinant human basic fibroblast growth factor has been introduced as a growth
factor to induce fibroblast proliferation in skin ulcers. We applied these materials in combination in
two patients with deep resistant wounds: one with a cavity reaching the mediastinum through a
divided sternum and one with deep necrotic wounds caused by electric burns. These wounds did not
respond to the topical basic fibroblast growth factor alone. In contrast, the combination therapy
closed the wounds rapidly without further surgical treatment. This combination therapy is a potent
treatment for resistant wounds with deep cavities.
102
Osteoarthritis Cartilage. 2009 Dec;17(12):1620-7.
Chondroprotective effect of the bioactive peptide prolyl-hydroxyproline in mouse articular
cartilage in vitro and in vivo.
Nakatani S, Mano H, Sampei C, Shimizu J, Wada M.
OBJECTIVE: To investigate the direct effect of prolyl-hydroxyproline (Pro-Hyp) on chondrocytes
under in vivo and in vitro conditions in an attempt to identify Pro-Hyp as the bioactive peptide in
collagen hydrolysate (CH). METHODS: The in vivo effects of CH and Pro-Hyp intake on articular
cartilage were studied by microscopic examination of sections of dissected articular cartilage from
treated C57BL/6J mice. In this study, mice that were fed diets containing excess phosphorus were
used as an in vivo model. This mouse line showed loss of chondrocytes and reduced thickness of
articular cartilage, with abnormality of the subchondral bone. The in vitro effects of CH, Pro-Hyp,
amino acids and other peptides on proliferation, differentiation, glycosaminoglycan content and
mineralization of chondrocytes were determined by MTT activity and staining with alkaline
phosphatase, alcian blue and alizarin red. Expression of chondrogenesis-specific genes in ATDC5
cells was determined by semiquantitative Reverse Transcription Polymerase Chain Reaction (RT-
PCR). RESULTS: In vivo, CH and Pro-Hyp inhibited the loss of chondrocytes and thinning of the
articular cartilage layer caused by phosphorus-induced degradation. In the in vitro study, CH and
Pro-Hyp did not affect chondrocyte proliferation but inhibited their differentiation into mineralized
chondrocytes. A combination of amino acids such as proline, hydroxyproline and prolyl-
hydroxyprolyl-glycine did not affect chondrocyte proliferation or differentiation. Moreover, CH and
Pro-Hyp caused two and threefold increases, respectively, in the staining area of glycosaminoglycan
in the extracellular matrix of ATDC5 cells. RT-PCR indicated that Pro-Hyp increased the aggrecan
mRNA level approximately twofold and decreased the Runx1 and osteocalcin mRNA levels by
two-thirds and one-tenth, respectively. CONCLUSION: Pro-Hyp is the first bioactive edible
peptide derived from CH to be shown to affect chondrocyte differentiation under pathological
conditions.
103
Arthritis Res Ther. 2013 Feb 22;15(1):R32.
Periodic knee injections of collagen tripeptide delay cartilage degeneration in rabbit
experimental osteoarthritis.
Naraoka T, Ishibashi Y, Tsuda E, Yamamoto Y, Kusumi T, Toh S.
INTRODUCTION: Collagen peptides have been reported to possess various biological activities for
various cell types. The purposes of this study were, first, to examine the therapeutic effects of
collagen tripeptide (Ctp) in rabbit osteoarthritis and, second, to explore a synergetic effect with
hyaluronan (HA). METHODS: Osteoarthritis was induced by anterior cruciate ligament transection
of the right knee in 72 Japanese white rabbits and they were divided into four groups (control, Ctp,
HA and Ctp/HA). Each material was injected weekly into the knee, and knee joint samples were
collected 5, 10 and 15 weeks after surgery. Macroscopic and histomorphological analyses of
cartilage were conducted. Expression of type II collagen and matrix metalloproteinase-13 was also
analyzed immunohistochemically. A Tukey's honestly significant difference test was used to
evaluate the statistical significance of difference in the macroscopic, histological and
immnohistochemical results. RESULTS: All treatment groups exhibited slightly higher resistance to
the progression of osteoarthritis than the control group macroscopically 15 weeks after surgery.
Histologically, intra-articular injection of Ctp significantly reduced cartilage degradation 10 weeks
after surgery, and Ctp/HA significantly reduced it 5 weeks after surgery in comparison with the
control. Immunohistochemically, both Ctp-treated and Ctp/HA-treated groups had significantly
increased type II collagen-positive chondrocytes at the fifth week after the surgery, although the
numbers of matrix metalloproteinase-13-positive chondrocytes were not affected. CONCLUSION:
Periodical injections of Ctp and Ctp/HA delayed progression of cartilage degeneration of early
osteoarthritis induced by anterior cruciate ligament transection in rabbits. This effect appears to be
exerted by promotion of type II collagen synthesis predominantly.
104
J Nutr. 1999 Oct;129(10):1891-5.
Oral administration of (14)C labeled gelatin hydrolysate leads to an accumulation of
radioactivity in cartilage of mice (C57/BL).
Oesser S, Adam M, Babel W, Seifert J.
Several investigations showed a positive influence of orally administered gelatin on degenerative
diseases of the musculo-skeletal system. Both the therapeutic mechanism and the absorption
dynamics, however, remain unclear. Therefore, this study investigated the time course of gelatin
hydrolysate absorption and its subsequent distribution in various tissues in mice (C57/BL).
Absorption of (14)C labeled gelatin hydrolysate was compared to control mice administered (14)C
labeled proline following intragastric application. Plasma and tissue radioactivity was measured
over 192 h. Additional "gut sac" experiments were conducted to quantify the MW distribution of
the absorbed gelatin using SDS-electrophoresis and HPLC. Ninety-five percent of enterally applied
gelatin hydrolysate was absorbed within the first 12 h. The distribution of the labeled gelatin in the
various tissues was similar to that of labeled proline with the exception of cartilage, where a
pronounced and long-lasting accumulation of gelatin hydrolysate was observed. In cartilage,
measured radioactivity was more than twice as high following gelatin administration compared to
the control group. The absorption of gelatin hydrolysate in its high molecular form, with peptides of
2.5-15kD, was detected following intestinal passage. These results demonstrate intestinal absorption
and cartilage tissue accumulation of gelatin hydrolysate and suggest a potential mechanism for
previously observed clinical benefits of orally administered gelatin.
105
Cell Tissue Res. 2003 Mar;311(3):393-9. Epub 2003 Feb 25.
Stimulation of type II collagen biosynthesis and secretion in bovine chondrocytes cultured
with degraded collagen.
Oesser S, Seifert J.
The functional integrity of articular cartilage is dependent on the maintenance of the extracellular
matrix (ECM), a process which is controlled by chondrocytes. The regulation of ECM biosynthesis
is complex and a variety of substances have been found to influence chondrocyte metabolism. In the
present study we have investigated the effect of degraded collagen on the formation of type II
collagen by mature bovine chondrocytes in a cell culture model. The culture medium was
supplemented with collagen hydrolysate (CH) and biosynthesis of type II collagen by chondrocytes
was compared to control cells treated with native type I and type II collagen and a collagen-free
protein hydrolysate. The quantification of type II collagen by means of an ELISA technique was
confirmed by immunocytochemical detection as well as by the incorporation of (14)C-proline in the
ECM after a 48 h incubation. Chondrocytes in the control group were maintained in the basal
medium for 11 days. The presence of extracellular CH led to a dose-dependent increase in type II
collagen secretion. However, native collagens as well as a collagen-free hydrolysate of wheat
proteins failed to stimulate the production of type II collagen in chondrocytes. These results clearly
indicate a stimulatory effect of degraded collagen on the type II collagen biosynthesis of
chondrocytes and suggest a possible feedback mechanism for the regulation of collagen turnover in
cartilage tissue.
106
Biosci Biotechnol Biochem. 2010;74(10):2096-9.
Effects of Pro-Hyp, a collagen hydrolysate-derived peptide, on hyaluronic acid synthesis using
in vitro cultured synovium cells and oral ingestion of collagen hydrolysates in a guinea pig
model of osteoarthritis.
Ohara H, Iida H, Ito K, Takeuchi Y, Nomura Y.
Proline-hydroxyproline (Pro-Hyp) stimulated hyaluronic acid production in cultured synovium
cells. It was detected in guinea pig blood after oral ingestion of collagen hydrolysates. Oral
administration of collagen hydrolysates increased the amount of proteoglycans in the epiphyses. It
also reduced the morphological changes associated with osteoarthritic cartilage destruction of the
knee joint. The results suggest that collagen hydrolysates have therapeutic potential for treatment of
osteoarthritis.
107
J Oral Implantol. 2002;28(5):220-5.
Collagen as an implantable material in medicine and dentistry.
Patino MG, Neiders ME, Andreana S, Noble B, Cohen RE.
Collagen is a highly versatile material, extensively used in the medical, dental, and pharmacological
fields. Collagen is capable of being prepared into cross-linked compacted solids or into lattice-like
gels. Resorbable forms of collagen have been used to dress oral wounds, for closure of graft and
extraction sites, and to promote healing. Collagen-based membranes also have been used in
periodontal and implant therapy as barriers to prevent epithelial migration and allow cells with
regenerative capacity to repopulate the defect area. It has been hypothesized that membrane
regenerative techniques facilitate the natural biological potential by creating a favorable
environment for periodontal and peri-implant regeneration. Due to the enormous potential of
collagen-based regenerative barriers, clinicians may benefit from a review of potential applications
of implantable collagen and knowledge of collagen preparation and membrane types as well as from
as awareness of the functional and degradation properties of those materials.
108
J Cell Biochem. 1998 Dec 1;71(3):313-27.
Collagen in tissue-engineered cartilage: types, structure, and crosslinks.
Riesle J, Hollander AP, Langer R, Freed LE, Vunjak-Novakovic G.
The function of articular cartilage as a weight-bearing tissue depends on the specific arrangement of
collagen types II and IX into a three-dimensional organized collagen network that can balance the
swelling pressure of the proteoglycan/water gel. To determine whether cartilage engineered in vitro
contains a functional collagen network, chondrocyte-polymer constructs were cultured for up to 6
weeks and analyzed with respect to the composition and ultrastructure of collagen by using
biochemical and immunochemical methods and scanning electron microscopy. Total collagen
content and the concentration of pyridinium crosslinks were significantly (57% and 70%,
respectively) lower in tissue-engineered cartilage that in bovine calf articular cartilage. However,
the fractions of collagen types II, IX, and X and the collagen network organization, density, and
fibril diameter in engineered cartilage were not significantly different from those in natural articular
cartilage. The implications of these findings for the field of tissue engineering are that differentiated
chondrocytes are capable of forming a complex structure of collagen matrix in vitro, producing a
tissue similar to natural articular cartilage on an ultrastructural scale.
109
Science. 1987 Oct 23;238(4826):491-7.
New perspectives in cell adhesion: RGD and integrins.
Ruoslahti E, Pierschbacher MD.
Rapid progress has been made in the understanding of the molecular interactions that result in cell
adhesion. Many adhesive proteins present in extracellular matrices and in the blood contain the
tripeptide arginine-glycine-aspartic acid (RGD) as their cell recognition site. These proteins include
fibronectin, vitronectin, osteopontin, collagens, thrombospondin, fibrinogen, and von Willebrand
factor. The RGD sequences of each of the adhesive proteins are recognized by at least one member
of a family of structurally related receptors, integrins, which are heterodimeric proteins with two
membrane-spanning subunits. Some of these receptors bind to the RGD sequence of a single
adhesion protein only, whereas others recognize groups of them. The conformation of the RGD
sequence in the individual proteins may be critical to this recognition specificity. On the
cytoplasmic side of the plasma membrane, the receptors connect the extracellular matrix to the
cytoskeleton. More than ten proved or suspected RGD-containing adhesion-promoting proteins
have already been identified, and the integrin family includes at least as many receptors recognizing
these proteins. Together, the adhesion proteins and their receptors constitute a versatile recognition
system providing cells with anchorage, traction for migration, and signals for polarity, position,
differentiation, and possibly growth.
110
Adv Drug Deliv Rev. 2003 Nov 28;55(12):1595-611.
Effect of collagen matrices on dermal wound healing.
Ruszczak Z.
Dermal substitution and wound healing are areas of medicine in which there have been many recent
advances, but neither the commercially available products nor the products currently described in
experimental studies are able to fully substitute for natural living skin. There is an overall consensus
that to heal wounds, the substitution of connective tissue matrix, the main component of each
wound, is necessary. Both artificial and natural polymers have been used to reconstitute dermis.
Nowadays, collagen has been discovered again. Collagen is a natural substrate for cellular
attachment, growth and differentiation, and promotes cellular proliferation and differentiation. Once
dermis reconstruction is done, the covering of the wound surface with both in vitro expanded
epidermis and autologous split-skin transplants is significantly easier and has an improved chance
of success. Nowadays, many commercial and experimental products have been introduced to
improve cutaneous wound healing. This review discusses some of both acellular and cell-containing
products used in the treatment of skin wounds.
111
Biomaterials. 2006 Jan;27(3):335-45.
Regenerative effects of transplanting mesenchymal stem cells embedded in atelocollagen to
the degenerated intervertebral disc.
Sakai D, Mochida J, Iwashina T, Hiyama A, Omi H, Imai M, Nakai T, Ando K, Hotta T.
Intervertebral disc (IVD) degeneration, a common cause of low back pain in humans, is a
relentlessly progressive phenomenon with no currently available effective treatment. In an attempt
to solve this dilemma, we transplanted autologous mesenchymal stem cells (MSCs) from bone
marrow into a rabbit model of disc degeneration to determine if stem cells could repair degenerated
IVDs. LacZ expressing MSCs were transplanted to rabbit L2-L3, L3-L4 and L4-L5 IVDs 2 weeks
after induction of degeneration. Changes in disc height by plain radiograph, T2-weighted signal
intensity in magnetic resonance imaging (MRI), histology, immunohistochemistry and matrix
associated gene expressions were evaluated between normal controls (NC) without operations,
sham operated with only disc degeneration being induced, and MSC-transplanted animals for a 24-
week period. Results showed that after 24 weeks post-MSC transplantation, degenerated discs of
MSC-transplanted group animals regained a disc height value of about 91%, MRI signal intensity of
about 81%, compared to NC group discs. On the other hand, sham-operated group discs
demonstrated the disc height value of about 67% and MRI signal intensity of about 60%.
Macroscopic and histological evaluations confirmed relatively preserved nucleus with circular
annulus structure in MSC-transplanted discs compared to indistinct structure seen in sham.
Restoration of proteoglycan accumulation in MSC-transplanted discs was suggested from
immunohistochemistry and gene expression analysis. These data indicate that transplantation of
MSCs effectively led to regeneration of IVDs in a rabbit model of disc degeneration as suggested in
our previous pilot study. MSCs may serve as a valuable resource in cell transplantation therapy for
degenerative disc disease.
112
Med Biol Eng Comput. 2003 May;41(3):365-71.
Tissue engineering of the intervertebral disc with cultured annulus fibrosus cells using
atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS scaffold).
Sato M, Kikuchi M, Ishihara M, Ishihara M, Asazuma T, Kikuchi T, Masuoka K, Hattori H,
Fujikawa K.
The objective of the study was to investigate the regeneration of intervertebral discs after laser
discectomy using tissue engineering procedures. Annulus fibrosus (AF) cells from the intervertebral
discs of Japanese white rabbits were cultured in an atelocollagen honeycomb-shaped scaffold with a
membrane seal (ACHMS scaffold), to produce a high-density, three-dimensional culture for up to 3
weeks. Although the DNA content in the scaffold increased at a lower rate than that in the
monolayer culture, expression of type II collagen and glycosaminoglycan accumulation in the
scaffold were at higher levels than in the monolayer. The AF cells that had been cultured in the
scaffold for 7 days were allografted into the lacunae of intervertebral discs of recipients (40 rabbits,
14-16 weeks old; average weight, 3.2 kg), whose nucleus pulposus (NP) had been vaporised with an
ICG dye-enhanced laser. The allografted cultured AF cells survived and produced hyaline-like
cartilage. Furthermore, the narrowing of the intervertebral disc space of the cell-containing scaffold
insertion groups was significantly inhibited after 12 post-operative weeks.
113
J Biomed Mater Res B Appl Biomater. 2007 Oct;83(1):181-8.
Effects of growth factors on heparin-carrying polystyrene-coated atelocollagen scaffold for
articular cartilage tissue engineering.
Sato M, Ishihara M, Ishihara M, Kaneshiro N, Mitani G, Nagai T, Kutsuna T, Asazuma T, Kikuchi
M, Mochida J.
The specific aim of our investigation is to study the potential use of a collagen/heparin-carrying
polystyrene (HCPS) composite extracellular matrix for articular cartilage tissue engineering. Here,
we created a high-performance extracellular matrix (HpECM) scaffold to build an optimal
extracellular environment using an HCPS we originally developed, and an atelocollagen
honeycomb-shaped-scaffold (ACHMS-scaffold) with a membrane seal. This scaffold was coated
with HCPS to enable aggregation of heparin-binding growth factors such as FGF-2 and TGF-beta1
within the scaffold. Three-dimensional culture of rabbit articular chondrocytes within the HpECM-
scaffold and subsequent preparation of a tissue-engineered cartilage were investigated. The results
showed remarkably higher cell proliferative activity within the HpECM-pretreated-FGF-2 scaffold
and the sustenance of phenotype within the HpECM-pretreated-TGF-beta1 scaffold. It was thought
that both FGF-2 and TGF-beta1 were stably immobilized in the HpEMC-scaffold since HCPS
generated an extracellular environment similar to that of heparan sulfate proteoglycan within the
scaffold. These results suggest that an ACHMS-scaffold immobilized with HCPS can be a HpECM
for cartilage regeneration to retain the heparin-binding growth factors within the scaffolds.
114
J Agric Food Chem. 2012 Apr 25;60(16):4096-101.
Effect of the novel low molecular weight hydrolyzed chicken sternal cartilage extract, BioCell
Collagen, on improving osteoarthritis-related symptoms: a randomized, double-blind,
placebo-controlled trial.
Schauss AG, Stenehjem J, Park J, Endres JR, Clewell A.
Osteoarthritis (OA) is a significant source of pain and disability. Current medical and surgical
treatments can be costly and have serious side effects. The aim of this randomized, double-blind,
placebo-controlled trial was to investigate the tolerability and efficacy of BioCell Collagen (BCC),
a low molecular weight dietary supplement consisting of hydrolyzed chicken sternal cartilage
extract, in the treatment of OA symptoms. Patients (n = 80) in the study had physician-verified
evidence of progressive OA in their hip and/or knee joint. Joint pain had been present for 3 months
or longer at enrollment, and pain levels were 4 or higher at baseline as assessed by Physician Global
Assessment scores. Subjects were divided into two groups and administered either 2 g of BCC or
placebo for 70 days. Other outcome measurements included visual analogue scale (VAS) for pain
and Western Ontario and McMaster Universities Arthritis Index (WOMAC) scores taken on days 1,
35, and 70. The tolerability profile of the treatment group was comparable to that of the placebo.
Intent-to-treat analysis showed that the treatment group, as compared to placebo, had a significant
reduction of VAS pain on day 70 (p < 0.001) and of WOMAC scores on both days 35 (p = 0.017)
and 70 (p < 0.001). The BCC group experienced a significant improvement in physical activities
compared to the placebo group on days 35 (p = 0.007) and 70 (p < 0.001). BCC was well tolerated
and found to be effective in managing OA-associated symptoms over the study period, thereby
improving patient's activities of daily living. BCC can be considered a potential complement to
current OA therapies.
115
J Musculoskelet Neuronal Interact. 2006 Apr-Jun;6(2):181-90.
Biology of tendon injury: healing, modeling and remodeling.
Sharma P, Maffulli N.
Tendon disorders are frequent, and are responsible for much morbidity both in sport and the
workplace. Although the presence of degenerative changes does not always lead to symptoms, pre-
existing degeneration has been implicated as a risk factor for acute tendon rupture. The term
tendinopathy is a generic descriptor of the clinical conditions in and around tendons arising from
overuse. The terms "tendinosis" and "tendinitis/tendonitis" should only be used after
histopathological examination. Disordered healing is seen in tendinopathy, and inflammation is not
typically seen. In acute injuries, the process of tendon healing is an indivisible process that can be
categorized into three overlapping phases for descriptive purposes. Tendon healing can occur
intrinsically, via proliferation of epitenon and endotenon tenocytes, or extrinsically, by invasion of
cells from the surrounding sheath and synovium. Despite remodeling, the biochemical and
mechanical properties of healed tendon tissue never match those of intact tendon. Tendon injuries
account for considerable morbidity, and often prove disabling for several months, despite what is
considered appropriate management. Chronic problems caused by overuse of tendons probably
account for 30% of all running-related injuries, and the prevalence of elbow tendinopathy in tennis
players can be as high as 40%. The basic cell biology of tendons is still not fully understood, and
the management of tendon injury poses a considerable challenge for clinicians. This article
describes the structure of tendons, and reviews the pathophysiology of tendon injury and healing.
116
Curr Opin Cell Biol. 2012 Oct;24(5):600-6.
Inside-out, outside-in, and inside-outside-in: G protein signaling in integrin-mediated cell
adhesion, spreading, and retraction.
Shen B, Delaney MK, Du X.
The integrin family of cell adhesion receptors mediates bi-directional signaling: 'inside-out'
signaling activates the ligand binding function of integrins and 'outside-in' signaling mediates
cellular responses induced by ligand binding to integrins leading to cell spreading, retraction,
migration, and proliferation. Integrin signaling requires both heterotrimeric G proteins and
monomeric small G proteins. This review focuses on recent development in the roles of G proteins
in integrin outside-in signaling. The finding of direct interaction between the heterotrimeric G
protein subunit Gα13 and integrin β subunits reveals a new mechanism for integrin signaling, and
also uncovers a crosstalk between the signaling pathways initiated by G protein-coupled receptors
(GPCRs) and integrins. This crosstalk, which may be referred to as 'inside-outside-in' signaling,
dynamically regulates contractility and greatly promotes integrin outside-in signaling.
117
J Bone Joint Surg Am. 1997 Dec;79(12):1770-7.
Regeneration of meniscal cartilage with use of a collagen scaffold. Analysis of preliminary
data.
Stone KR, Steadman JR, Rodkey WG, Li ST.
A collagen scaffold was designed for use as a template for the regeneration of meniscal cartilage
and was tested in ten patients in an initial, Food and Drug Administration-approved, clinical
feasibility trial. The goal of the study was to evaluate the implantability and safety of the scaffold as
well as its ability to support tissue ingrowth. The study was based on the findings of in vitro and in
vivo investigations in dogs that had demonstrated cellular ingrowth and tissue regeneration through
the scaffold. Nine patients remained in the study for at least thirty-six months, and one patient
voluntarily withdrew after three months for personal reasons. The collagen scaffold was found to be
implantable and to be safe over the three-year period. Histologically, it supported regeneration of
tissue in meniscal defects of various sizes. No adverse immunological reactions were noted on
sequential serological testing. On second-look arthroscopy, performed either three or six months
after implantation, gross and histological evaluation revealed newly formed tissue replacing the
implant as it was resorbed. At thirty-six months, the nine patients reported a decrease in the
symptoms. According to a scale that assigned 1 point for strenuous activity and 5 points for an
inability to perform sports activity, the average score was 1.5 points before the injury, 3.0 points
after the injury and before the operation, and 2.4 points at six months postoperatively, 2.2 points at
twelve months, 2.0 points at twenty-four months, and 1.9 points at thirty-six months. According to a
scale that assigned 0 points for no pain and 3 points for severe pain, the average pain score was 2.2
points preoperatively and 0.6 point thirty-six months postoperatively. One patient, who had had a
repair of a bucket-handle tear of the medial meniscus and augmentation with the collagen scaffold,
had retearing of the cartilage nineteen months after implantation. Another patient had debridement
because of an irregular area of regeneration at the scaffold-meniscus interface twenty-one months
after implantation. Magnetic resonance imaging scans demonstrated progressive maturation of the
signal within the regenerated meniscus at three, six, twelve, and thirty-six months. These findings
suggest that regeneration of meniscal cartilage through a collagen scaffold is possible. Additional
studies are needed to determine long-term efficacy.
118
Mol Biol Evol. 2011 Jan;28(1):533-42.
Comparative vertebrate evolutionary analyses of type I collagen: potential of COL1a1 gene
structure and intron variation for common bone-related diseases.
Stover DA, Verrelli BC.
Collagen type I alpha 1 (COL1a1), which encodes the primary subunit of type I collagen, the main
structural and most abundant protein in vertebrates, harbors hundreds of mutations linked to human
diseases like osteoporosis and osteogenesis imperfecta. Previous studies have attempted to predict
the phenotypic severity associated with type I collagen mutations, yet an evolutionary analysis that
compares historical and recent selective pressures, including across noncoding regions, has never
been conducted. Here, we use a comparative genomic and species evolutionary analysis
representing ∼450 My of vertebrate history to investigate functional constraints associated with
both exons and introns of the >17-kb COL1a1 gene. We find that although the COL1a1 amino acid
sequence is highly conserved, there are both spatial and temporal signatures of varying selective
constraint across protein domains. Furthermore, sites of high evolutionary constraint significantly
correlate with the location of disease-associated mutations, the latter of which also cluster with
respect to specific severity classes typically categorized in clinical studies. Finally, we find that
COL1a1 introns are significantly short in length with high GC content, patterns that are shared
across highly diverged vertebrates, and which may be a signature of strong stabilizing selection for
high COL1a1 gene expression. In conclusion, although previous studies focused on COL1a1 coding
regions, the current results implicate introns as areas of high selective constraint and targets of
bone-related phenotypic variation. From a broader perspective, our comparative evolutionary
approach provides further resolution to models predicting mutations associated with bone-related
function and disease severity.
119
Biomaterials. 2006 Jun;27(17):3238-48.
Performance of collagen sponge as a 3-D scaffold for tooth-tissue engineering.
Sumita Y, Honda MJ, Ohara T, Tsuchiya S, Sagara H, Kagami H, Ueda M.
Tooth structure can be regenerated by seeding dissociated tooth cells onto polyglycolic acid fiber
mesh, although the success rate of tooth production is low. The present study was designed to
compare the performance of collagen sponge with polyglycolic acid fiber mesh as a 3-D scaffold
for tooth-tissue engineering. Porcine third molar teeth at the early stage of crown formation were
enzymatically dissociated into single cells, and the heterogeneous cells were seeded onto collagen
sponge or the polyglycolic acid fiber mesh scaffolds. Scaffolds were then cultured to evaluate cell
adhesion and ALP activity in vitro. An in vivo analysis was performed by implanting the constructs
into the omentum of immunocompromised rats and evaluating tooth production up to 25 weeks.
After 24h, there were a significantly higher number of cells attached to the collagen sponge scaffold
than the polyglycolic acid fiber mesh scaffold. Similarly, the ALP activity was significantly higher
for the collagen sponge scaffold was than the polyglycolic acid fiber mesh scaffold after 7 days of
culture. The area of calcified tissue formed in the collagen sponge scaffold was also larger than in
the polyglycolic acid fiber mesh scaffold. The results from in vivo experiments show conclusively
that a collagen sponge scaffold allows tooth production with a higher degree of success than
polyglycolic acid fiber mesh. Taken together, the results from this study show that collagen sponge
scaffold is superior to the polyglycolic acid fiber mesh scaffold for tooth-tissue engineering.
120
J Toxicol Sci. 1982 Dec;7 Suppl 2:63-91.
Acute and subacute toxicity studies on collagen wound dressing (CAS) in mice and rats.
Takeda U, Odaki M, Yokota M, Sasaki H, Niizato T, Kawaoto H, Watanabe H, Ito T, Ishiwatari N,
Hayasaka H, Seki M, Koeda T.
Single administration of collagen wound dressing (CAS) made from bovine derm in the form of
finely ground powders was given to mice and rats via i.p., s.c. and p.o. routes and via i.v. route in
the form of physiological saline extracts and it was continuously injected into mice for 28 days via
s.c. route to study its acute and subacute toxicity. Examinations were made of on general
conditions, body weight, food and water consumption, hematology, serum biochemistry, organ
weight, and gross and microscopic findings. Results showed no marked toxicity except for local
irritation which was seen only after parenteral administration. We concluded on the basis of these
animal experiments that there should be no problem in regard to safety after somewhat more
extensive therapeutic application of CAS as a wound dressing in clinical practice.
121
J Biol Chem. 2007 Aug 3;282(31):22437-47.
The size exclusion characteristics of type I collagen: implications for the role of
noncollagenous bone constituents in mineralization.
Toroian D, Lim JE, Price PA.
The mineral in bone is located primarily within the collagen fibril, and during mineralization the
fibril is formed first and then water within the fibril is replaced with mineral. The collagen fibril
therefore provides the aqueous compartment in which mineral grows. Although knowledge of the
size of molecules that can diffuse into the fibril to affect crystal growth is critical to understanding
the mechanism of bone mineralization, there have been as yet no studies on the size exclusion
properties of the collagen fibril. To determine the size exclusion characteristics of collagen, we
developed a gel filtration-like procedure that uses columns containing collagen from tendon and
bone. The elution volumes of test molecules show the volume within the packed column that is
accessible to the test molecules, and therefore reveal the size exclusion characteristics of the
collagen within the column. These experiments show that molecules smaller than a 6-kDa protein
diffuse into all of the water within the collagen fibril, whereas molecules larger than a 40-kDa
protein are excluded from this water. These studies provide an insight into the mechanism of bone
mineralization. Molecules and apatite crystals smaller than a 6-kDa protein can diffuse into all
water within the fibril and so can directly impact mineralization. Although molecules larger than a
40-kDa protein are excluded from the fibril, they can initiate mineralization by forming small
apatite crystal nuclei that diffuse into the fibril, or can favor fibril mineralization by inhibiting
apatite growth everywhere but within the fibril.
122
Annu Rev Biomed Eng. 2012;14:47-71.
Tendon healing: repair and regeneration.
Voleti PB, Buckley MR, Soslowsky LJ.
Injury and degeneration of tendon, the soft tissue that mechanically links muscle and bone, can
cause substantial pain and loss of function. This review discusses the composition and function of
healthy tendon and describes the structural, biological, and mechanical changes initiated during the
process of tendon healing. Biochemical pathways activated during repair, experimental injury
models, and parallels between tendon healing and tendon development are emphasized, and cutting-
edge strategies for the enhancement of tendon healing are discussed.
123
Evol Dev. 2006 Jul-Aug;8(4):370-7.
Molecular evolution of fibrillar collagen in chordates, with implications for the evolution of
vertebrate skeletons and chordate phylogeny.
Wada H, Okuyama M, Satoh N, Zhang S.
Vertebrates have seven types of fibrillar collagens that are encoded by 11 genes. Types I, V, and
XXIV collagens are components of mineralized bone, whereas types II, XI, and XXVII collagens
are components of cartilage. In this study, we traced the molecular evolutionary history of chordate
collagen genes and examined how gene duplications gave rise to the collagen genes used for
skeletons. Our analyses of deuterostome collagen genes, including one amphioxus gene that we
identified in this study, suggest that the common ancestors of deuterostomes possessed three
fibrillar collagen genes. Expression analyses of chordate fibrillar collagen genes suggest that in the
ancestors of chordates, fibrillar collagen was co-opted to the formation of the notochord sheath
independently in three clades. Our results also imply that co-option of collagen genes to cartilage
occurred in clade A (col2A1), clade B (col11A1, 11A2), and clade C (COL27A1). Similarly, some
fibrillar collagen genes have been co-opted for mineralized bone independently from clade A genes
(col1A1, 1A2, 5A2), clade B genes (col5A1), and clade C genes (COL24A1). These frequent co-
options for notochord, cartilage, and mineralized bone must have been accompanied by the rapid
evolution of cis-regulatory elements for transcription. In addition, we found that one of the ascidian
fibrillar collagen genes possesses an amino acid insertion at the identical site of the C-terminal
noncollagenous domain in vertebrate fibrillar collagen genes. This observation raises a suspicion
about the relatively well-accepted phylogeny of the close relationship between amphioxus and
vertebrates.
124
Eur Cell Mater. 2006 Mar 28;11:43-56.
Collagen-hydroxyapatite composites for hard tissue repair.
Wahl DA, Czernuszka JT.
Bone is the most implanted tissue after blood. The major solid components of human bone are
collagen (a natural polymer, also found in skin and tendons) and a substituted hydroxyapatite (a
natural ceramic, also found in teeth). Although these two components when used separately provide
a relatively successful mean of augmenting bone growth, the composite of the two natural materials
exceeds this success. This paper provides a review of the most common routes to the fabrication of
collagen (Col) and hydroxyapatite (HA) composites for bone analogues. The regeneration of
diseased or fractured bones is the challenge faced by current technologies in tissue engineering.
Hydroxyapatite and collagen composites (Col-HA) have the potential in mimicking and replacing
skeletal bones. Both in vivo and in vitro studies show the importance of collagen type,
mineralisation conditions, porosity, manufacturing conditions and crosslinking. The results outlined
on mechanical properties, cell culturing and de-novo bone growth of these devices relate to the
efficiency of these to be used as future bone implants. Solid free form fabrication where a mould
can be built up layer by layer, providing shape and internal vascularisation may provide an
improved method of creating composite structures.
125
J Bone Joint Surg Br. 1989 Jan;71(1):74-80.
Repair of rabbit articular surfaces with allograft chondrocytes embedded in collagen gel.
Wakitani S, Kimura T, Hirooka A, Ochi T, Yoneda M, Yasui N, Owaki H, Ono K.
In an attempt to repair articular cartilage, allograft articular chondrocytes embedded in collagen gel,
were transplanted into full-thickness defects in rabbit articular cartilage. Twenty-four weeks after
the transplantation, the defects were filled with hyaline cartilage, specifically synthesising Type II
collagen. These chondrocytes were autoradiographically proven to have originated from the
transplanted grafts. Assessed histologically the success rate was about 80%, a marked improvement
over the results reported in previous studies on chondrocyte transplantation without collagen gel. By
contrast, the defects without chondrocyte transplantation healed with fibrocartilage. Immunological
enhancement induced by transplanted allogenic chondrocytes or collagen was not significant at
eight weeks after treatment, so far as shown by both direct and indirect blastformation reactions.
Thus, allogenic transplantation of isolated chondrocytes embedded in collagen gel appears to be one
of the most promising methods for the restoration of articular cartilage.
126
J Biomed Mater Res A. 2010 Apr;93(1):123-32.
The application of atelocollagen gel in combination with porous scaffolds for cartilage tissue
engineering and its suitable conditions.
Yamaoka H, Tanaka Y, Nishizawa S, Asawa Y, Takato T, Hoshi K.
For improving the quality of tissue-engineered cartilage, we examined the in vivo usefulness of
porous bodies as scaffolds combined with an atelocollagen hydrogel, and investigated the suitable
conditions for atelocollagen and seeding cells within the engineered tissues. We made tissue-
engineered constructs using a collagen sponge (CS) or porous poly(L-lactide) (PLLA) with human
chondrocytes and 1% hydrogel, the concentration of which maximized the accumulation of
cartilage matrices. The CS was soft with a Young's modulus of less than 1 MPa, whereas the porous
PLLA was very rigid with a Young's modulus of 10 MPa. Although the constructs with the CS
shrank to 50% in size after a 2-month subcutaneous transplantation in nude mice, the PLLA
constructs maintained their original sizes. Both of the porous scaffolds contained some cartilage
regeneration in the presence of the chondrocytes and hydrogel, but the PLLA counterpart
significantly accumulated abundant matrices in vivo. Regarding the conditions of the chondrocytes,
the cartilage regeneration was improved in inverse proportion to the passage numbers among
passages 3-8, and was linear with the cell densities (10(6) to 10(8) cells/mL). Thus, the rigid porous
scaffold can maintain the size of the tissue-engineered cartilage and realize fair cartilage
regeneration in vivo when combined with 1% atelocollagen and some conditioned chondrocytes.
127
Birth Defects Res C Embryo Today. 2013 Sep;99(3):203-22. doi: 10.1002/bdrc.21041.
Tendon and ligament regeneration and repair: clinical relevance and developmental
paradigm.
Yang G, Rothrauff BB, Tuan RS.
As dense connective tissues connecting bone to muscle and bone to bone, respectively, tendon and
ligament (T/L) arise from the somitic mesoderm, originating in a recently discovered somitic
compartment, the syndetome. Inductive signals from the adjacent sclerotome and myotome
upregulate expression of Scleraxis, a key transcription factor for tenogenic and ligamentogenic
differentiation. Understanding T/L development is critical to establishing a knowledge base for
improving the healing and repair of T/L injuries, a high-burden disease due to the intrinsically poor
natural healing response. Current treatment of the three most common tendon injuries-tearing of the
rotator cuff of the shoulder, flexor tendon of the hand, and Achilles tendon-include mostly surgical
repair and/or conservative approaches, including biophysical modalities such as rehabilitation and
cryotherapy. Unfortunately, the fibrovascular scar formed during healing possesses inferior
mechanical and biochemical properties, resulting in compromised tissue functionality. Regenerative
approaches have sought to augment the injured tissue with cells, scaffolds, bioactive agents, and
mechanical stimulation to improve the natural healing response. The key challenges in restoring full
T/L function following injury include optimal combination of these biological agents as well as
their delivery to the injury site. A greater understanding of the molecular mechanisms involved in
T/L development and natural healing, coupled with the capability of producing complex
biomaterials to deliver multiple biofactors with high spatiotemporal resolution and specificity,
should lead to regenerative procedures that more closely recapitulate T/L morphogenesis, thereby
offering future patients the prospect of T/L regeneration, as opposed to simple tissue repair.
128
Mater Sci Eng C Mater Biol Appl. 2015 Dec 1;57:452-63.
Design of biocomposite materials for bone tissue regeneration.
Yunus Basha R, Sampath Kumar TS, Doble M.
Several synthetic scaffolds are being developed using polymers, ceramics and their composites to
overcome the limitations of auto- and allografts. Polymer-ceramic composites appear to be the most
promising bone graft substitute since the natural bone itself is a composite of collagen and
hydroxyapatite. Ceramics provide strength and osteoconductivity to the scaffold while polymers
impart flexibility and resorbability. Natural polymers have an edge over synthetic polymers because
of their biocompatibility and biological recognition property. But, very few natural polymer-
ceramic composites are available as commercial products, and those few are predominantly based
on type I collagen. Disadvantages of using collagen include allergic reactions and pathogen
transmission. The commercial products also lack sufficient mechanical properties. This review
summarizes the recent developments of biocomposite materials as bone scaffolds to overcome these
drawbacks. Their characteristics, in vitro and in vivo performance are discussed with emphasis on
their mechanical properties and ways to improve their performance.
129
Biochem Biophys Res Commun. 2006 May 26;344(1):362-9.
Novel chitosan/collagen scaffold containing transforming growth factor-beta1 DNA for
periodontal tissue engineering.
Zhang Y, Cheng X, Wang J, Wang Y, Shi B, Huang C, Yang X, Liu T.
The current rapid progression in tissue engineering and local gene delivery system has enhanced our
applications to periodontal tissue engineering. In this study, porous chitosan/collagen scaffolds were
prepared through a freeze-drying process, and loaded with plasmid and adenoviral vector encoding
human transforming growth factor-beta1 (TGF-beta1). These scaffolds were evaluated in vitro by
analysis of microscopic structure, porosity, and cytocompatibility. Human periodontal ligament
cells (HPLCs) were seeded in this scaffold, and gene transfection could be traced by green
fluorescent protein (GFP). The expression of type I and type III collagen was detected with RT-
PCR, and then these scaffolds were implanted subcutaneously into athymic mice. Results indicated
that the pore diameter of the gene-combined scaffolds was lower than that of pure chitosan/collagen
scaffold. The scaffold containing Ad-TGF-beta1 exhibited the highest proliferation rate, and the
expression of type I and type III collagen up-regulated in Ad-TGF-beta1 scaffold. After implanted
in vivo, EGFP-transfected HPLCs not only proliferated but also recruited surrounding tissue to
grow in the scaffold. This study demonstrated the potential of chitosan/collagen scaffold combined
Ad-TGF-beta1 as a good substrate candidate in periodontal tissue engineering.
130
Appendici
Appendice A: Foglietto Illustrativo del Dispositivo
CHondroGrid®
Dispositivo sterile monouso Descrizione Collagene idrolizzato a basso peso molecolare, liofilizzato, per iniezione intra- e peri-articolare. Composizione Collagene idrolizzato a basso peso molecolare di origine bovina. Indicazioni CHondroGrid® è indicato per il trattamento della sintomatologia dolorosa e della perdita di funzionalità delle maggiori articolazioni (ginocchio, spalla, anca, polso e caviglia) e delle strutture muscolo-tendinee e legamentose ad esse annesse, causate da patologie degenerative o dovute a esito traumatico o a sovraccarico. Le più comuni indicazioni di utilizzo di CHondroGrid ® sono il trattamento sintomatologico e funzionale di: osteoartrosi, artrosinoviti acute o croniche secondarie ad artrosi o artrite reumatoide, esiti di traumi o lesioni, affaticamento da sovraccarico a carico delle articolazioni sopra indicate. CHondroGrid ® è indicato inoltre in caso di meniscopatie e in preparazione e come terapia di mantenimento a seguito di interventi di meniscectomia, di ricostruzione legamentosa o di pulizia e/o ricostruzione del tessuto cartilagineo articolare. Per queste indicazioni l’applicazione è eseguita per via intra-articolare. Per esiti di traumi e/o lesione peri-articolari o a carico di strutture muscolo-tendinee-ligamentose si procede invece tramite iniezione peri-articolare e infiltrazione delle strutture coinvolte. Meccanismo d’azione Per via intra-articolare: il collagene idrolizzato a basso peso molecolare si distribuisce sulla superficie dell’articolazione rinforzando la matrice cartilaginea, deteriorata dai processi patologici in atto, la cui impalcatura è formata da un fitto intreccio di fibre collagene. CHondroGrid ® svolge quindi un’azione meccanica di rinforzo diretto di strutture collageniche indebolite e/o deteriorate, migliorando la mobilità e contribuendo a ridurre la sintomatologia dolorosa a carico dell’articolazione. Per via peri-articolare: il collagene idrolizzato a basso peso molecolare agisce quale rinforzo diretto all’impalcatura collagenica danneggiata delle strutture peri-articolari, quali tendini e/o legamenti, contribuendo ad una riduzione del dolore e ad una più veloce ripresa funzionale. Protocollo terapeutico Iniezione intra-articolare Il trattamento prevede tre iniezioni: due eseguite a distanza di 15 giorni l’una dall’altra, la terza dopo un mese dall’ultimo trattamento. Iniezione peri-articolare Il trattamento prevede due iniezioni a distanza di 30 giorni l’una dall’altra. Infiltrazione in strutture muscolo-tendinee-ligamentose Due iniezioni ad un intervallo di circa 10 giorni l’una dall’altra. Materiale necessario all’uso del dispositivo (non compreso nella confezione) Una fiala di acqua sterile per iniezioni (2 ml o più). Una siringa sterile (5 ml o più) Due aghi sterili
Istruzioni per l’uso Attenzione: procedere allo svuotamento del liquido articolare o del versamento prima di iniettare CHondroGrid ®.
1. Aprire la fiala di acqua sterile per iniezioni. 2. Aspirarne 2 ml usando la siringa da 5 ml e relativo ago. 3. Aprire il blister, estrarre flacone di liofilizzato e iniettare attraverso il tappo in gomma l’acqua sterile per iniezioni. 4. Agitare fino a completa solubilizzazione. 5. Aspirare nella siringa la soluzione ottenuta. 6. Sostituire l’ago usato con il secondo ago sterile. 7. Somministrare il prodotto avendo cura di avere eseguito una corretta asepsi.
Iniezione intra-articolare Iniettare all’interno dello spazio articolare, se necessario sotto guida strumentale, ad esempio ecografia, specialmente in caso di trattamento dell’anca e della spalla. Infiltrazione peri-articolare e in strutture muscolo-tendinee-legamentose Individuare il punto o i punti di massima dolorabilità ed eventualmente segnarli con una penna dermografica. L’iniezione può essere eseguita sotto guida strumentale in modo da essere certi di raggiungere il sito di lesione. Avvertenze e precauzioni CHondroGrid ® deve essere utilizzato esclusivamente da personale medico qualificato, seguendo procedure strettamente asettiche. CHondroGrid ® non deve essere utilizzato in associazione ad altri composti iniettabili. E’ tuttavia indicato in pazienti precedentemente trattati con iniezione di acido ialuronico o PRP autologo. Nel caso sia richiesta una ulteriore terapia di supporto alla matrice cartilaginea può essere associato a trattamento per via orale con integratori a base di collagene idrolizzato a basso peso molecolare, quali ad esempio CHONDROVITA® o simili. Si raccomanda di non sottoporre l’articolazione trattata a carichi eccessivi nelle ore immediatamente successive all’infiltrazione. Non usare CHondroGrid ® se la confezione risulta aperta o danneggiata. Non iniettare CHondroGrid ® per via endovascolare. Non utilizzare dopo la data di scadenza. La data di scadenza si riferisce al prodotto in confezione integra e conservato in modo corretto. Tenere fuori dalla portata dei bambini. Controindicazioni ed effetti indesiderati Pazienti con accertata ipersensibilità al collagene. Infezione articolare o peri-articolare, emartro, eritema nella zona da infiltrare, chiazze psoriasiche nella zona da infiltrare. L’iniezione intra- e peri-articolare può causare occasionalmente dolore transitorio, leggero gonfiore, reazione cutanea superficiale e arrossamento dovuto all’azione meccanica dell’ago. Queste reazioni sono generalmente di breve durata e si risolvono spontaneamente entro pochi giorni ponendo l’arto a riposo e con l’applicazione di ghiaccio. Contenuto della confezione ed etichette paziente Flacone di vetro in singolo blister di PETG. Foglietto illustrativo. L'etichetta apposta sul blister consiste di sei etichette, staccabili singolarmente, che fungono anche da etichetta paziente per l'apposizione in cartella clinica o altra documentazione. Sterilizzazione e conservazione Il prodotto è sterilizzato tramite irraggiamento a raggi beta a 25 kGy. Conservare al riparo dall’esposizione diretta ai raggi solari, in luogo fresco e asciutto, ad una temperatura compresa tra 4° e 40°C. In condizioni di conservazione corrette l’integrità della confezione e quindi la sterilità del prodotto sono garantite per 2 anni dalla data di produzione (vedi data di scadenza sull’etichetta esterna). Fabbricante Bioteck SpA, Via E. Fermi 49, 36057 Arcugnano (VI), Italia. Fabbricato nello stabilimento di Via G. Agnelli, 3 - 10020 Riva presso Chieri (Torino), Italia. Iscrizione al repertorio dei dispositivi medici e classificazione CND RDM (numero di iscrizione al repertorio): 1412112/R CND (classificazione): P900402 - PRODOTTI RIASSORBIBILI PER RIEMPIMENTO E RICOSTRUZIONE
Rev 20160629 del 29.06.2016
Appendice B: Certificazioni CE di ChondroGrid