Bone structure

July 2013

Ranieri Cancedda, MD University of Genova &

IRCCS AOU San Martino – IST National Cancer Research Institute, Genova

Tissue Repair in Orthopedics: from Tissue Engineering to Regenerative Medicine

Bone defects requiring repair/regeneration

§  Fractures §  Traumatic bone loss §  Bone tumor surgery §  Congenital malformations

The “canonical” tissue engineering approach Crush injury on forearm in

a 30 year old man

Bone Cell Therapy: ���a long way to the clinical application

ü  in vitro studies (cell-biomaterial interactions)

ü  nude mouse model

ü  large animal model

ü  clinical studies

Tortelli et al. Tissue Engineering part A 2009

Bone ECM deposition in vitro in a 3D culture

Implant of MSC seeded scaffolds in nude mice

Mastrogiacomo et al. Tissue Engineering 2006 Giannoni et al. J. Tissue Eng. Regen. Med. 2008 Komlev et al. Tissue Eng. part C 2009

Tibia sheep model

First bone repair by autologous MSC

Quarto R, et al: New Engl J Med 344: 385, 2001

10 months

Number of novel cell therapy treatments for Bone Repair in Europe

Auto Allo Total HSC MSC HSC MSC HSC MSC

2008

Maxillo-facial 2

Orthopedics 10 12

12 12

2009

Maxillo-facial 1 1

Orthopedics 23 43 2

26 44

2010

Maxillo-facial 1 1

Orthopedics 26 19 10

28 29 Modified from Martin et al., Tissue Engineering: Part A; Volumes 16,17,18; 2010-12

Reasons why bone tissue engineering is not making inroads

Regulatory issues (FDA, EMA, National Agencies …..)

Remaining unresolved scientific problems (identification of the “optimal” scaffold, large implant vascularisation ……)

Need of highly sofisticated cell culture facilities High cost of the procedure Difficult logistics Existence of therapeutic alternative

•  Haematoma occurs at the site of tissue injury •  Platelets are activated and release bioactive substances •  Innate immunity cells migrate (Granulocytes and Macrophages) •  Endothelial progenitors move in and angiogenesis begins •  Resident and recruited cells with a differentiation potential increase in number and new tissue formation initiates

Tissue Healing Overview (triggering of the process by platelet released factors)

Bone structure

MSC can engraft in injured tissues and differentiate to replace damaged organs

However, MSC can also act as “factories” of bio-active molecules to limit tissue destruction or enhance repair by a variety of mechanisms

Mesenchymal Stem Cells as “medicinal” cells: induction of endogenous bone repair

Can implanted MSC trigger a cascade of events leading to mobilization of macrophages, angiogenesis and endogenous bone formation?

10.9!3.2!4.7!10.8!Day 7!

20.9!7.6!5.2!13.5!Day 3!

5.6!3.6!3.8!9.6!Day 1!

M2 (CD206+,CD51+)!

(% of total)!

M1 (CD86+,CD40+)!(% of total)!

M2 (CD206+,CD51+) !

(% of total)!

M1 (CD86+,CD40+)!(% of total)!

+ MSC !SKELITE!SKELITE! EMPTY!

0!0.5!

1!1.5!

2!2.5!

3!3.5!

4!

Empty Skelite!Skelite (+MSC)!

M2/

M1

ratio!

Day 1! Day 3! Day 7!

Innate immunity cells migration (Macrophages)

Tasso et al.; submitted

Triggering of the process by platelet released factors

Activated Platelet

Blood  dona(on  

Buffy  coat  isola(on  

Buffy  coat  pool  

Platelet  concentrate  (PRP)  

PLATELET  POOR  PLASMA  (PPP)  

PLATELET  LYSATE  (PL)  

Platelet adjustment to optimal concentration

High speed centrifugation

Freeze-thaw cycles Membrane removal

Validation test at the Transfusional Center

Platelet Derivative Production

RBC  

PRP  

PPP  

PRP  

Leukocytes & Platelets (<1% of total blood)

BM-MSCs

16 hour stimulation with PL or IL-1α or PL + IL-1α

Gene activation analysis

NF-kB activation (binding to specific DNA sequences)

*  *  

*  **  

Fold

incr

ease

control"

Increased synthesis of PGE2 induced by PL in an inflammatory

microenvironment

0

10000

20000

30000

40000

50000

60000

70000

80000

SF IL1 PL PL+IL1

PGE2  

 pg/m

l  *  

*  

*  

**  *  

control"

Hypoxia and cell culture

Research objectives within the IRMI project •  Further investigate the influence of the microenvironment (clot formation, inflammation,

hypoxia) on the paracrine activity of stem cells: bone marrow Mesenchymal Stem Cells (MSCs), Amniotic Fluid Stem Cells (AFSCs)

•  Perform a continuous “basic – translational research” feedback loop to remove identified bottlenecks to the use of allogenic (from donors) Platelet derived sub-fractions.

•  Perform a continuous “basic – translational research” feedback loop to remove identified bottlenecks to the use of conditioned culture medium, and product thereof, from allogenic bone marrow MSCs and Amniotic Fluid Stem Cells (AFSCs)

•  Anticipated bottlenecks include more detailed understanding of the recipient’s body immunoresponse.

•  Develop and optimize products derived from platelets and/or stem cell conditioned media to be used as therapeutic agents either as an injectable solution or combined with biomaterials for the repair of: a) tendon and ligament lesion; b) cartilage and bone lesions and defects (bioactive scaffolds); c) skin lesions such as chronic ulcers, difficult wounds, bedsores, chinks (bioactive membrane).

Biorigen Srl���(Spin off dell’Universita’ degli Studi di Genova)

Biorigen continuerà le ricerche in corso per mantenere ed aumentare il vantaggio competitivo derivante dai brevetti ottenuti e accrescere il potenziale mercato di

riferimento con la brevettazione di prodotti/servizi. Verra’ potenziata l’attivita’ di Ricerca e Sviluppo con particolare attenzione a tematiche relative a:

- Reagenti per colture cellulari basati su derivati piastrinici - Prodotti di medicina rigenerativa per terapia umana e veterinaria nel settori

dermatologico (prevalentemente ulcere e lesioni cutanee), ortopedico e odontoiatrico.