improved cell growth by bio-oss/pla scaffolds for use as a bone substitute

Technology and Health Care 16 (2008) 401–413 401IOS Press

Improved cell growth by Bio-Oss/PLAscaffolds for use as a bone substitute

Annalia Astia,e, Livia Visaib,e, Rossella Doratic, Bice Contic, Enrica Sainob, Sonia Sbarrab,Giulia Gastaldid and Francesco Benazzoa,e

aSMEC Department, Orthopaedic Clinic, University of Pavia, IRCCS Policlinico San Matteo, Pavia,ItalybDepartment of Biochemistry, University of Pavia, Pavia, ItalycDepartment of Pharmaceutical Chemistry, University of Pavia, Pavia, ItalydDepartment of Experimental Medicine, University of Pavia, Pavia, ItalyeCenter of Tissue Engineering (C.I.T.), University of Pavia, Pavia, Italy

Received 23 April 2008

Revised /Accepted 12 July 2008

Abstract. The objective of this study was to investigate the surface modification of a natural bone substitute, Bio-Oss, coatedwith a synthetic polymer poly-D,L-lactide (PLA), in order to improve cell growth. Bio-Oss is a natural bone substitute madeof the mineralized portion of bovine bone. The material is used mainly to fill bone defects in periodontal and maxillofacialsurgery and permit reossification. Poly-α-hydroxyacids such as polylactic acid are receiving an increasing attention due to theirability to retain a great quantity of water, good biocompatibility, low interfacial tension, and minimal mechanical and frictionalirritation. All of these features are appealing from the perspective of bioenvironmental mimicking. The human osteosarcomacell line SAOS-2 was added to the top of scaffolds uncoated or coated with PLA and incubated at 37◦C in 5% CO2 for 15 days.PLA-coated scaffolds improved cell growth. Polymer degradation behaviour, extraction and measurement of the extracellularmatrix proteins of the cultured scaffolds (such as decorin, fibronectin osteocalcin, osteonectin, osteopontin and type-I andtype-III collagen), immunolocalization of bone proteins and morphological analysis of the scaffolds confirmed the bioactiveproperties of Bio-Oss/PLA4M suggesting that it could be a valuable grafting material.

1. Introduction

Bone grafts are commonly required in orthopaedic, oral and maxillofacial surgery for various indi-cations [24,29,35]. Presently surgeons treat hard tissue defects with autogenous bone grafts, the goldstandard for clinical surgery [4,8,29,34], that show optimal skeletal incorporation but often bring aboutcomplications such as rejection.

Tissue engineering represents an alternative approach to the repair and regeneration of damaged humantissue, avoiding the need for a permanent implant [18,31]. In this field a promising approach is to promotetissue regeneration by transplanting tissue engineered constructs made of a biofactor (cells, genes and/orproteins) grown on a porous degradable structure known as scaffold. Far from being a passive componentscaffold material and its porous design play a significant role in tissue regeneration by preserving tissue

∗Address for correspondence: Annalia Asti, Ph.D, SMEC, IRCCS Policlinico San Matteo, Viale Golgi, 5, 27100 Pavia, Italy.Tel.: +39 382 502851; Fax: +39 382 526319; E-mail: [email protected].

0928-7329/08/$17.00 2008 – IOS Press and the authors. All rights reserved

402 A. Asti et al. / Improved cell growth by Bio-Oss/PLA scaffolds for use as a bone substitute

volume, providing temporary mechanical function, and delivering bio factors [2,30]. As in native tissue,cells within a construct respond and adapt to the physical and biological stimuli to which they are exposedin vivo. The cellular component is necessary for the generation of a new tissue through production ofextracellular matrix (ECM); a tissue-engineered construct is ultimately responsible for performing thefunction of the tissue it was designed to replace, that may be mechanical as in the case of structural tissuelike bone [7]. The goal is to replace or repair a sizable defect while only gathering a small tissue sample.Various natural and synthetic bioresorbable materials or a combination of them, are being investigated fordesign and construction of scaffolds for the engineering and regeneration of a wide variety of tissues [16,31].

Bio-Oss is a natural bone substitute made of the mineralized portion of bovine bone. It is obtainedvia a proprietary extraction procedure involving denaturation and elimination of the organic matrixof bone. It is used mainly to fill bone defects in periodontal and maxillofacial surgery and permitreossification. Bio-Oss through its trabecular architecture, pore size (300–500 µm), and high porosity(70–75%) promotes the invasion of blood vessels and bone cells, thereby inducing revitalization andossification of the defect [17]. The presence of interconnected pores of this size is important becausethey increase the surface area available for cell invasion and facilitate the diffusion of organic compoundsduring cell culture as well as the integration of the material by the host organism [3]. Bio-Oss has beenused in clinical applications for more than 15 years and has been scientifically investigated. Resultsshow that this material, thanks to the large hydrophilic inner surface area similar to human bone, presentssuperior handling characteristics. Nevertheless, the cell proliferation rate does not result as well ifcompared to other biomaterials in use [20]. PLA is an aliphatic polyester such as poly (glycolic acid)(PGA) and poly (lactic acid-co-glycolic acid) (PLGA). These synthetic biodegradable polymers donot pose any danger of immunogenicity or possibility of disease transmission. Poly(α-hydroxy acids)are FDA approved, their high biocompatibility originating from the development and clinical use ofsuture materials for surgery [21]. In the physiologic environment these polymers degrade by randomchain scission releasing lactic and glycolic acids which are metabolized by the human body. Theirdegradation rate can be mediated by their composition and molecular weight. Their favorable mechanicalcharacteristics as suture materials have prompted researchers to test their suitability as scaffolds, withpositive results [6,10,11,22,23]. Many practical advantages arise when using synthetic scaffolds sinceit is possible to maintain precise control of material composition and micro-macrostructure, includingporosity. This allows adequate control of scaffold properties, thus creating optimal conditions forcell survival, proliferation and subsequent tissue formation [31]. Chemical composition and surfacetopography influence osteoblast responses [5,25]. In particular regarding bone tissue engineering, thedevelopment of materials comprising a biodegradable polymeric phase and a bioactive inorganic phase,is seen as a promising approach for scaffold production [26]. Therefore investigating methods to studycell-biomaterial interactions is a crucial prerequisite for the development of ideal biomaterials/implantswhich can elicit specific and desirable responses from surrounding tissues [26]. The aim of the presentin vitro study was to evaluate the feasibility of preparing Bio-Oss/PLA scaffolds with characteristicssuitable to cell growth.

2. Materials and methods

2.1. Materials

Bio-Oss scaffolds were provided by the manufacturer Geistlich-Pharma-CH-6110 Wohlusen, Switzer-land, and are commercially available.

A. Asti et al. / Improved cell growth by Bio-Oss/PLA scaffolds for use as a bone substitute 403

Poly-D,L-lactide, Mw 82 kDa, i.v. 0.52 dl/g obtained from Lakeshore Biomaterials, USA. Dimethylcarbonate of analytical grade was obtained from Sigma-Aldrich.

2.2. Preparation of PLA coated scaffolds

A set of 50 Bio-Oss scaffolds was provided by the manufacturer, 21 samples uncoated vs 21 coatedby a casting method with an interpenetrating film of poly D,L-lactide (PLA4M).

The coating solution was prepared as follows: 0.325 mg of PLA 4M was dissolved in 1.6 ml ofdimethyl carbonate (DMC) by magnetic stirring for 15’ at room temperature to give a 5% w/v solution.Bio-Oss samples were soaked in the solution and sonicated for 30’. Subsequently the Bio-Oss/PLA4Msamples were drip-dried under laminar flow hood for 24 hours, and lyophilized overnight at −50◦C toeliminate the residual solvents.

2.3. Cell culture

The human osteosarcoma cell line SAOS-2 was obtained from the American Type Culture Collection(HTB85, ATCC). The cells were cultured in McCoy’s 5A modified medium with L-glutamine andHEPES (Cambrex Bio Science), supplemented with 15% fetal bovine serum, 2% sodium pyruvate, 1%antibiotics, 10−8 M dexamethasone, and 10 mM β-glycerophosphate (Sigma-Aldrich). Ascorbic acid,another osteogenic supplement, is a component of McCoy’s 5A modified medium. The cells werecultured at 37◦C with 5% CO2, routinely trypsinized after confluence, counted, and seeded onto thescaffolds.

2.4. Cell seeding

The scaffolds were sterilized by ethylene oxide at 38◦C for 8 h at 65% relative humidity. After 24 h ofaeration in order to remove the residual ethylene oxide, the scaffolds were placed inside a 24 well-plate(Costar, Corning Inc., NY), washed twice with a physiological solution and incubated with the cellculture medium at 37◦C in the CO2 incubator overnight. A cell suspension of 1 × 107 cells in 200 µlwas added onto the top of each scaffold and after 1 h, 1 ml of culture medium was added to cover thescaffolds. The cells were incubated at 37◦C in 5% CO2 for 15 days. Culture medium was changed everythree days.

2.5. Determination of PLA4M molecular weight

The molecular weights of PLA4M raw material and of PLA4M scaffold coating, were determined byGel Permeation Chromatography (GPC). GPC analysis was performed on six samples of Bio-Oss/PLA4Mscaffolds before incubation with cell cultures and on six samples of Bio-Oss/PLA4M scaffolds afterincubation with cultures for 15 days. Samples of PLA4M raw material were analyzed in triplicatebefore and after incubation with cell cultures. The GPC system consisted of three Ultrastyragel columnsconnected in series (7.7 × 250 mm each, one with 104 Å pores, one with 103 Å pores and one with 500Å), a pump (Varian 9010 (MI), Italy), a detector Prostar 355 RI (Varian (MI), Italy), and a software forcomputing molecular weight distribution (Galaxie Ws, ver. 1.8 Single-Instrument, Varian (MI), Italy).The samples for GPC analysis were prepared by dissolving PLA4M and PLA4M/Bio-Oss scaffoldsin tetrahydrofuran (THF). The sample solutions, at concentrations of about 20 mg/mL, were filteredthrough a 0.45 µm filter (Millipore, USA) before injection into the GPC system, and were eluted with


THF at 1 mL/min. The weight-average molecular weight (Mw) of each sample was calculated usingmonodispersion polystyrene standards, Mw 1,000–150,000 Da. Data are reported as weight averagemolecular weight (Mw), average molecular number (Mn) and polydispersity index (Pi = Mw/Mn).

Results were expressed as the mean ± sd of six scaffolds and as the mean ± sd of three samples ofPLA4M raw material.

2.6. DNA content

Cells attached to the porous scaffolds were lysed by a freeze-thaw method in sterile deionized distilledwater. The released DNA content was evaluated with a fluorometric DNA quantification kit (PicoGreen,Molecular Probes). A DNA standard curve, obtained from a known amount of osteoblasts, was used toexpress the results as cell number per scaffold. The total DNA extracted from the cell cultured scaffoldcoated with PLLA was 102 µg ± 5 µg. The total number of cell obtained from the calibration curve wasapproximately around 17 × 106 ± 2 × 105 cells.

2.7. Set of purified proteins

Decorin [32], fibronectin [33], osteocalcin (immunoenzymatic assay kit, Biomedical Technologies,Inc., Stoughton, MA), osteonectin and osteopontin (immunoenzymatic assay kit, Assay Designs, Inc.,Ann Arbor, MI), type I collagen [28], and type III collagen (Sigma-Aldrich, Inc.).

2.8. Rabbit polyclonal antisera

A set of polyclonal antibodies was selected for ELISA assays. Rabbit polyclonal antisera (anti-type I and III collagen, anti-decorin, anti-osteopontin, anti-osteonectin, and anti-osteocalcin [13,14]were kindly provided by L.W. Fisher (http://csdb.nidcr.nih.gov/csdb/antisera.htm, National Institutesof Health, National Institute of Dental and Craniofacial Research, Craniofacial and Skeletal DiseasesBranch, Matrix Biochemistry Unit, Bethesda, MD).

For polyclonal antibody production against human fibronectin, New Zealand rabbits were injectedintraperitoneally five times at 12-days-intervals with 100 µg of the purified HFn [15]. The antigen wasemulsified with an equal volume of complete Freund’s adjuvant for the first immunization followed byfour injections with incomplete adjuvant. The rabbit was bled, and the sera was tested for reactivity to thepurified HFn using an ELISA assay. The specific IgGs were purified by affinity chromatography on pro-tein G-Sepharose columns according to the manufacturer’s recommendations (Amersham Biosciences).Antibody titers were assayed by ELISA.

2.9. Extraction of the extracellular matrix proteins from the cultured scaffolds

At the end of the culture period, in order to evaluate the amount of the extracellular matrix constituentsover the scaffold surface, the scaffolds were washed extensively with sterile PBS (137 mM NaCl, 2.7 mMKCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH = 7.4) in order to remove the culture medium, and thenincubated for 24 h at 37◦C with 0.5 ml of sterile sample buffer (1.5 M Tris-HCl, 60% [w/v] sucrose,0.8% [w/v] sodium dodecyl sulphate, pH = 8.0). At the end of the incubation period, the sample bufferaliquots were removed, and then the scaffolds were centrifuged at 4000 rpm for 15 min in order to collectthe sample buffer entrapped in the pores.


The total protein concentration was 950 ± 110 µg/0.5 ml as evaluated by the BCA Protein Assay Kit(Pierce Biotechnology). After matrix extraction, the scaffolds were incubated for another 24 h at 37◦Cwith 1 ml of sterile sample buffer, and no protein content was detected.

2.10. ELISA assay

In order to measure the ECM components amount of each protein from the scaffolds, an ELISAassay was performed as follows. At first, calibration curves to measure decorin, fibronectin osteocalcin,osteonectin, osteopontin, and type-I and type-III collagen were performed. Microtiter wells were coatedwith increasing concentrations of each extracted purified protein, from 10 ng to 2 µg, in coating buffer(50 mM Na2CO3, pH = 9.5) overnight at 4◦C. Some of the wells were coated with bovine serumalbumin (BSA) as a negative control. To measure the amount of each extracted protein from thescaffolds, microtiter wells were coated, overnight at 4◦C, with 100 µl of the ECM components (20 µg/mlin coating buffer). After three washes with PBST (PBS containing 0.1% [v/v] Tween 20), the wellswere blocked by incubating with 200 µl of PBS containing 2% (w/v) BSA for 2 h at 22◦C. The wellswere subsequently incubated for 1.5 h at 22◦C with 100 µl of the L.W. Fisher’s anti rabbit polyclonalantisera (1:500 dilution in 1% BSA). After washing, the wells were incubated for 1 h at 22◦C with100 µl of HRP-conjugated goat anti-rabbit IgG (Dako, Glostrup, Denmark) (1:1000 dilution in 1%BSA). The wells were finally incubated with 100 µl of development solution (phosphate-citrate bufferwith o-phenylenediamine dihydrochloride substrate). The colour reaction was stopped with 100 µl of0.5 M H2SO4 and the absorbance values were measured at 490 nm with a microplate reader (Bio-RadLaboratories).

The amount of ECM constituents over the scaffold surface was expressed as fg/(cell×scaffold).

2.11. Confocal laser scanning microscopic (CSLM) studies

To evaluate immunolocalization of bone proteins in the scaffolds, CLSM studies were carried out. Atthe end of the culture period, the scaffolds were fixed with 4% (w/v) paraformaldehyde solution in 0.1 Mphosphate buffer (pH = 7.4) for 8 h at room temperature and washed with PBS (137 mM NaCl, 2.7 mMKCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH = 7.4) three times for 15 min. The scaffolds werethen blocked by incubating with PAT (PBS containing 1% [w/v] bovine serum albumin and 0.02% [v/v]Tween 20) for 2 h at room temperature and washed. L. Fisher’s anti rabbit polyclonal antisera (anti-TypeI collagen, anti-osteopontin and anti-osteocalcin) was used as primary antibody with a dilution equalto 1:1000 in PAT. The incubation with the primary antibodies was performed overnight at 4◦C, whilethe negative controls, instead, were based upon the incubation with PAT alone, overnight at 4 ◦C. Thescaffolds and the negative controls were washed and incubated with Alexa Fluor 488 goat anti-rabbitIgG (H+L) (Molecular Probes) using a dilution of 1:500 in PAT for 1 h at room temperature. At theend of the incubation, the scaffolds were washed in PBS, counterstained with a solution of propidiumiodide (2 µg/ml) to target the cellular nuclei, and then washed. Stained samples were examined underLeica Confocal Laser Scanning Microscope, model TCS SPII (Leica, Heidelberg, Germany) using 63Xoil immersion objective. The excitation and emission wavelengths used for Alexa Fluor 488 were 488and 525 nm, respectively (green fluorescence). Propidium iodide (red fluorescence) was excited at520 nm and emission was monitored at 620 nm. The optical sections of 0.9 µm were collected over a6 µm thickness of scaffold and for each sample, images from three randomly selected positions wereacquired. The resulting stacks of images were analyzed using Leica Confocal Software and subsequentlyprocessed using Image J (National Institute of Health, Wayne Rasband, USA) software. No fluorescencewas observed in the negative controls.


Table 1Results of GPC analyses performed on PLA4M raw materials and PLA4M scaffold coatings

Before incubation After incubationMw ± sd (Da) Mn ± sd (Da) Pi Mw ± sd (Da) Mn ± sd (Da) Pi

PLA4M raw material 59800 ± 3.3 35600 ± 2.8 1.68 48200 ± 4.0 30300 ± 2.6 1.59PLA4M scaffold coating 55670 ± 3.1 32460 ± 3.2 1.71 47700 ± 3.5 31407 ± 3.0 1.52

2.12. Scanning electron microscopy (SEM)

Scanning electron microscopy was performed on Bio-Oss scaffolds and on Bio-Oss/PLA4M scaffoldsbefore and after incubation with SAOS-2 cells. The scaffolds were fixed in glutaraldehyde 2.5% andNa-cacodylate buffer at pH 7.4 for about 2 hrs and then washed with Na-cacodylate buffer for 30 minutes.The dehydration process was performed using an increasing ethanol concentration (from 50 ◦ to 100◦).Samples were then submitted to critical point drying with CO2, mounted on aluminium stubs and goldsputtered (degree of purity 99.9%) under argon atmosphere to allow adequate gold coating of the internalsurface of porous structure (Sputter coater BALZER). Observations and micrographs were performedwith a SEM Cambridge Stereoscan, operating at 20 kV.

2.13. Energy-Dispersive-Spectrometer (EDS)

X-ray microanalysis of the samples was run to detect the presence of elements and their locationwithin the scaffolds The images were obtained with a Cambridge Stereoscan 250, Scanning ElectronMicroscope.

2.14. Standard deviation (sd)

The experiment was repeated six times for each type of scaffold (Bio-Oss and Bio-Oss/PLA4M).Results were expressed as mean ± standard deviation (sd) of six scaffolds, and the mean ± sd of threesamples of PLA4Mraw materials (Table 1).

3. Results

3.1. PLA4M coating molecular weight

The physic-chemical characterization of polymer coatings was performed in order to evaluate: i)if the coating process affects polymer molecular weight (Mw) and molecular number (Mn); ii) thedegradation behaviour of the polymer coating in the time chosen for incubation with cells. Poly-α-hydroxyacids degrade in the physiologic environment by hydrolytic random chain scission. Good scaffoldperformances could be obtained if the polymer keeps its mechanical properties during its incubation withcells, together with its being degraded and reabsorbed in a reasonable time period. Average polymerMw, Mn, and the polydispersity index (Pi), are parameters that indicate polymer integrity. The resultsof GPC analyses reported in Table 1 show the Mw, Mn and Pi, of PLA4M raw material and of PLA4Mscaffold coating, before and after incubation. As shown in Table 1 Mw decreases after incubationboth for polymer raw material and for polymer coating. After 15 days of incubation the average Mwdecreased by 19.39% for PLA4M raw material and by 14.31% for PLA4M coating. Considering that thesensitivity of the GPC technique is about 10% there is no significant difference between the two values.


Table 2Amount of extracellular matrix constituentsover the Bio-Oss/PLA4M scaffold surface af-ter 15 days of SAOS-2 cell culture

fg/cell x scaffold ± sdDecorin 230 ± 3.42Fibronectin 1117 ± 125.08Osteocalcin 180 ± 4.20Osteonectin 200 ± 5.10Osteopontin 294 ± 7.25Type-I collagen 1000 ± 150.25Type-III collagen 350 ± 50.10

Fig. 1. CSLM images of the extracellular matrix proteins produced on the Bio-Oss/PLA4M scaffolds. Immunolocalization oftype-I collagen (panel A), osteopontin (panel B), and osteocalcin (panel C) was performed on Bio-Oss/PLA4M scaffolds whichwere incubated with SAOS-2 cells for 15 days. (Mag. 63x) Nuclei of cells are indicated in red whereas the bone proteins areshown with green fluorescence.

Meanwhile the Mn figures also decreased by comparable values, thus indicating that polymer degradationinvolves loosen of small oligomers soluble in the culture medium. This behaviour is confirmed by thecorresponding decrease in Pi indices. The percentage of the Mw decrease upon incubation is consistentwith the polymer degradation behaviour as reported in the literature [11].

3.2. Characterization of the produced extracellular matrix

In order to evaluate the amount of the ECM constituents over the scaffold surface, an ELISA assay ofthe extracted matrix was performed at the end of the culture period. Table 2 reports the amount of ECMconstituents over the Bio-Oss/PLA4M scaffolds after 15 days of culture, no data have been detected forBio-Oss scaffolds without PLA coating.

As expected, most of the typical bone proteins were detected, with type-1 collagen being the mostrepresentative. However, an underestimation of absolute protein deposition is possible because thesample buffer, used for matrix extraction, contained sodium dodecylsulphate, which may interfere withprotein adsorption during ELISA.

To confirm the presence of bone proteins extracted and detected by ELISA assay, a confocal laserscanning microscopy study of the Bio-Oss/PLA scaffolds was performed. Figure 1 shows the immunolo-calization (indicated in green) of type-I collagen (Panel A), osteopontin (Panel B) and osteocalcin (PanelC) with the associated nuclear counterstaining (coloured in red).

All together these data were in agreement with results of SEM analysis.


A) B)

C) D)

Fig. 2. SEM micrographs of Bio-Oss and Bio-Oss/PLA4M scaffolds before incubation. Panels A and B) Bio-Oss scaffoldwith interconnected pores. For A) bar = 500 µm, B) bar = 200 µm); Panel C) Bio-Oss/PLA4M scaffold. PLA4M coatingresulted to be a continuous layer interpenetrating Bio-Oss pores. (arrow), bar = 1 mm; Panel D) PLA4M polymer at greatermagnification, bar = 20 µm.

3.3. Scanning electron microscopy

Scanning electron microscopy was performed to achieve morphologic characterization of the scaffoldsbefore incubation (Fig. 2A, B), in order to evaluate the suitability of the scaffold preparation method interms of homogeneity and interpenetration of the polymeric film in the scaffold.

Bio-Oss scaffolds (Fig. 2A, B) showed 3D structure with interconnected pores. Porosity rangedbetween 300–500 µm. PLA4M coating resulted in a continuous layer (Fig. 2C) interpenetrating Bio-Osspores (arrow). At higher magnifications (Fig. 2D), PLA4M coating showed pores smaller than 5 µm,probably caused by the evaporation of the solvent during casting. After 15 days of incubation SAOS-2attached and spread well over the surface of porous Bio-Oss/PLA4M scaffolds, as shown in Fig. 3 (PanelA, B, C). SAOS-2 cellular process cover almost the entire material surface indicating the biocompatibilityof the polymer. The presence of fibrous body was observed indicating ECM proteins synthesized by thecells. Cells on the Bio-Oss/PLA4M scaffold show a round morphology with short microvilli on the cellsurface (Fig. 3E). The cavity was also filled with PLA4M, as shown in Fig. 2C. On the contrary the cellsattached sporadically onto the Bio-Oss scaffold without PLA layer, and they appeared to be less spreadout over the surface (Fig. 3D).

3.4. Energy-Dispersive Spectrometer (EDS)

EDS was performed to identify elements and their location within the samples. In presence of cells,on the coated scaffold, x-ray microanalysis (Fig. 4B) showed the presence of calcium and phosphorus,


A) B) C)

E) D)

Fig. 3. SEM micrographs of cells on Bio-Oss and Bio-Oss /PLA4M after 15 days incubation. Panel A) Bio-Oss/PLA4Mscaffold with SAOS-2 cells (arrow), bar = 1 mm. Panels B) insert of Fig. 3A); C) A detail of panel B indicating the presenceof SAOS-cells. Bar = 20 µm. Panel D) Bio-Oss scaffold with few cells on the surface (arrow); bar = 100 µm. Panel E)Bio-Oss/PLA4M scaffold with SAOS-2 cells spread on scaffold surfaces. A plentiful extracellular matrix among cell, can beobserved (arrow) Bar = 10 µm.

inferring that calcium phosphate had formed. We evaluated three samples using x-ray microanalysis (foreach type of scaffold) and in all the samples we obtained the same results; in absence of cells there wereno elements.

4. Discussion

Several cell-substratum interactions are critical for cell attachment growth and function. This meansthe biocompatibility of the biomaterial is very closely related to the cell-behaviour in contact with thebiomaterial, and particularly to cell adhesion on the biomaterial surface [26]. Accordingly, osteoblasticcell behaviour on biomaterials has been shown to be influenced by two different factors: chemicalproperties (composition) and surface energy which determine whether biological molecules will beabsorbed [9]. The composition plays an essential role in osteoblast adhesion on the biomaterial. Themost important characteristic of PLA is that its degradation rate varies depending on its molecular weight,crystallinity, surface area, temperature etc., thereby influencing the subsequent stages of spreading,proliferation and cellular differentiation [19].


A)

B)

Fig. 4. Energy Dispersive Spectrum. Panel A) Analysis of Bio-Oss/PLA4M scaffold without cells; Panel b) Analysis ofBio-Oss/PLA4M incubated with SAOS-2 cells showing the spectrum of calcium and phosphorus.

The initial interaction of host cells with any implant is dominated by the nature of protein fibrin thatis absorbed onto the surface of the material. The material surface can influence cell reaction throughchanges in the cytoskeleton, a network of protein filaments extending through the cell cytoplasm withineukaryotic cells [26]. In cell culture studies with osteoblast-like cells the surface characteristics ofthe material play an important role because osteoblastic cells require a supportive matrix in order tosurvive [1,12,29].

The PLA layer on Bio-Oss scaffold promotes cell adhesion on the substrate as observed by morpho-logical characterisation (SEM) (Fig. 3A, B, C) and immunolocalization of proteins (CLSM) Fig. 1. Itis known that a highly porous scaffold is necessary to allow cell seeding or migration throughout thecavities; pore size is important for tissue growth and determines the internal surface area for cell attach-


ment. A large surface area is required so that a high number of cells sufficient to replace or restore organfunction, can be cultured. PLA is biocompatible, biodegradable, non toxic and has several biomedicalapplications. It is known that cell behaviour and interaction with a biomaterial surface is dependent onproperties such as topography, surface charges and chemistry [17,31].

In this study we observed that SAOS-2 cells adhere to biocompatible polymer layers on the Bio-Osssurface. Porous three-dimensional tissue engineering scaffolds act as temporary ECM for the physicalsupport of cells and to allow manipulation of various cell functions, such as cell adhesion, cell growth, andcell differentiation. In Fig. 3E cells are embedded in the extracellular matrix (ECM). Interaction betweenECM proteins and cells can directly control cell behaviour such as attachment, migration, proliferation,differentiation and apoptosis. A culture of osteoblasts in a conventional monolayer is thought to bepresent under different conditions even when only a small amount of ECM exists to support them. Thethree dimensional scaffold culture systems provide osteoblast–like cells with an environment closer toin vivo conditions than that of the monolayer culture [26].

Energy-Dispersive Spectrum (EDS) of the samples detected the presence of calcium and phosphorusinferring that calcium phosphate had formed. The presence of calcium phosphate would act as anosteoconductive trellis for new bone formation) [27].

Osteocalcin (Table 2), an extracellular non collagenous matrix protein, is a very specific marker forosteoblasts, it is produced by mature osteoblasts during mineralization and found in fully developedmineralized matrices. Once implanted in a body a porous scaffold should maintain its mechanicalstrength and structural integrity until loaded cells adapt to the environment and excrete a sufficientamount of ECM. The scaffold should be completely degraded and adsorbed by the body, and the threedimensional space it occupies should eventually be replaced by newly formed tissue. The results of GPCanalyses performed on the PLA4M coating of scaffolds show that its Mw decreases only by 19.39% in15 days of incubation, thus the polymer maintains its integrity. The mechanical properties of the scaffolddepends mainly on Bio-Oss structure that is uncharged after 15 days of incubation. The polymer mustkeep its integrity for the time needed to improve cell attachment and proliferation. Since polymer coatingis shown to behave similarly to the polymer raw material (Table 1), we can foresee a decrease of 60% inMw in about 60 days of incubation, and complete reabsorption after 90 days of incubation as reported inour previous paper [11]. These times periods are compatible with good scaffold performances showingthat Bio-Oss/PLA4M has an important advantage over allograft and autograft; due to the fact that thePLA coating completely degrades in 3 months. Moreover, since the properties of poly-α-hydroxyacidsin terms of degradation rates, greatly vary depending on their initial molecular weight, it would bepossible to modulate the PLA coating behaviour according to tissue engineering requirements. Thedegradation behavior affects cell vitality, cell growth, and even host response, and plays a critical role inthe engineering process of new tissue. An ideal in vivo degradation rate should be similar to or slightlyless then the rate of tissue formation.

This study was conducted to characterize in vitro a novel scaffold consisting of Bio-Oss and PLA;analysis of the construct morphology, degradation behaviour and characterization of the produced ex-tracellular matrix, osteocalcin, osteopontin and collagen I, III measurement confirm the good bioactiveproperties of the scaffolds.

A complete study would evaluate the possibility of using a Bio-Oss/PLA construct in an animal modelfor an in vivo study, in hopes of introducing tissue engineering to clinical application. The ultimate goalwould be to regenerate damaged tissue and to restore the function of the tissue lost through disease,malformation or accident.


Acknowledgments

This work was supported by Fondazione Cariplo Grant (2004.1424/10.8485 and 2006.0581/10.8485)to F.B., by PRIN (Progetti di Ricerca di Interesse Nazionale) Grant (2006) to L.V., by FAR (Fondodi Ateneo per la Ricerca, Pavia, Italy) Grant (2007) to L.V. and F.B. L.V. thanks the Italian Ministerodell’Istruzione, dell’Universita e della Ricerca, IDEE PROGETTUALI (Grandi Programmi Strategici,D.M. n◦ 24695, Prot. RBIPO6FH7J) and Sovvenzione Globale INGENIO, Fondo Sociale Europeo,Ministero del Lavoro e della Previdenza Sociale and Regione Lombardia.

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