A PVA_PCL_Bioglass Composite with Potential Implications for Osteochondral Tissue Engineering.

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1 A PVA_PCL_Bioglass Composite with Potential Implications for Osteochondral Tissue Engineering. Journal: 2009 MRS Fall Meeting Manuscript ID: Draft Symposium: Symposium RR Date Submitted by the Author: Complete List of Authors: Nair, Prabha Mukundan, Lakshmi; SCTIMST, DTERT Nirmal, Remya; SCTIMST, DTERT Mohan, Neethu; SCTIMST, DTERT Keywords: biomimetic (chemical reaction), bone, tissue

2 Page 1 of 6 A PVA-PCL Bioglass composite with potential implications for osteochondral tissue engineering Prabha D Nair 1,Lakshmi M Mukundan 1, Remya Nirmal 1, Neethu Mohan 1 1. Division of Tissue Engineering &Regeneration Technologies, Sree Chitra Tirunal Institute for Medical Sciences & Technology Trivandrum, Kerala, India ABSTRACT A bioglass of composition SiO 2 (67.12 mol%), CaO (28.5 mol%), and P 2 O 5 (4.38%) was synthesized and stabilized by a novel technique using ethanol. Bioactive glasses have a wide range of application in the field of biomaterials promoting bone bonding as well as bonding to soft tissue. Earlier our lab developed a novel PVA-PCL semi IPN porous and 3D scaffold that was found to favor chondrogenesis. In the present study, a composite of this polymer and bioglass is prepared by an emulsion freeze-drying process, as a porous 3 dimensional scaffold. The scaffolds were characterized for their physiochemical properties and ability to support cartilage tissue regeneration. The composite scaffolds were observed to be non-cytotoxic. The chondrocytes cells cultured in vitro for a month on the composite scaffolds regenerate cartilaginous tissue, secreting GAGs and collagen in amounts nearly comparable to the amounts on the control PVA-PCL scaffold. The composite scaffold is also biomimetic and bioactive and favors mineralization by forming a hydroxycarbonate apatite layer, when immersed in simulated body fluid for a 14 day period. The PVA-PCL-bioglass composite is hence expected to have potential implications as a scaffold for osteochondral tissue engineering. INTRODUCTION Cartilage degeneration caused by congenital abnormalities or disease and trauma, is of great clinical consequence due to the limited intrinsic healing potential of the tissue. The poor repair potential of articular cartilage is attributed to their avascular, aneural and alymphatic nature and lower mitotic activity and turnover rate. Tissue engineering represents an attractive direction to solve the complex problem of cartilage regeneration 1. But the main challenge with this approach is the development of the interface between the artificial cartilage and the underlying bone. Hence research is being carried out towards the repair of cartilage defects with osteochondral tissue engineering. Osteochondral defect which extend through the cartilage to the subchondral bone is repaired with simultaneous regeneration of cartilage and bone and thus provides better anchorage to the newly developed construct. Synthetic biodegradable polymeric scaffolds have gained wide acceptance in cartilage tissue engineering due to their superior mechanical properties and the ability to promote chondrocyte proliferation, maturation and differentiation. Bioactive glasses (BG) and calcium phosphate ceramics are the candidate materials for bone regeneration applications because of their ability to induce specific biological response that leads to the formation of a continuous interface between the implanted material and the tissue 2. Indeed, BG interact well with both hard and soft tissues by the development of the

3 Page 2 of 6 interface called the hydroxycarbonate apatite layer and thus provides a solution to the problem of interfacial attachment at the implant site 3. Thus it is expected that a composite made of synthetic polymer and BG will encourage bonding of the cartilage construct to bone. We have earlier reported a semi IPN scaffold comprised of polyvinyl alcohol (PVA) and polycaprolactone (PCL) to be favorable for chondrogenesis 4. In this study, a composite of two polymers as matrix phase namely PVA-PCL (PP) with bioactive glass as the filler phase (BG) was prepared and investigated for its potential for bioactivity and its ability to support chondrogenesis. Further, the BG used for the study was stabilized by a novel method of ethanol washing. This study particularly involves the physicochemical characterization of the composite scaffold and an analysis of its bioactive potential through the in vitro studies in simulated body fluid (SBF). In addition, studies with chondrocytes culture were also done to confirm its suitability for osteochondral tissue engineering. EXPERIMENTAL Bioactive glass of composition SiO 2 (67.12 mol %), CaO (28.5 mol %), and P 2 O 5 (4.38 mol %) was prepared by the sol gel method as reported elsewhere 5. The synthesized BG powder was stabilized using a novel procedure of ethanol washing. The ethanol washed powder was characterized by FTIR spectroscopy for the removal of residual nitrates and alkoxides. The BG thus stabilized by ethanol washing is used for the preparation of composite scaffolds with PVA (Mw 13,000-23,000) and PCL (Mw 80,000). The preparation of PVA-PCL semi IPN scaffolds is reported else where 8. The physicochemical characterization of the scaffolds was done by FTIR spectroscopic analysis and Thermogravimetric analysis (TGA). The 3D structure, porosity and pore size of the composite scaffolds were evaluated using SEM and micro CT. The cytotoxicity of polymer composite scaffolds was tested in vitro, by direct contact test using L929 cells according to ISO standards 6. The viability of chondrocytes were seeded on PPBG and PP scaffolds was determined using LIVE/DEAD viability/cytotoxicity kit The in vitro bioactivity studies were carried out by soaking the scaffolds for 14 days with a geometric surface area to volume ratio of 0.1cm -1 in SBF. The surfaces of these specimens were analyzed using IR. Chondrocytes at a density of 1 x 10 6 cells was seeded on to PP & PPBG scaffolds each and was cultured in chondrogenic medium for a period of 1 month. The tissue engineered cartilage construct was evaluated for chondrogenic markers and specific extracellular matrix. DISCUSSION Characterization of scaffolds The summary of absorption peaks of raw BG and the stabilized glass are given in Table1.

4 Page 3 of 6 Characteristic groups Raw BG Material Stabiliz -ed BG PP PPBG PPBG reacted with SBF Si-O-Si Strecthing O-Si-O stretching Si-O-Si bending P-O stretching , NO C-O acyclic stretching C=O acyclic stretching OH stretching CH 2 strecthing , C-O bending -2 from CO Table 1. FTIR data of raw BG, stabilized BG, PP scaffold, PPBG composite scaffold and PPBG after immersion in SBF. e d c b a Fig 1: FTIR spectrum of (a) raw BG, (b) stabilized BG, (c) PP scaffold, (d) PPBG composite scaffold, (e) PPBG after immersion in SBF. Composite scaffolds of PP with BG could be prepared with maximum amount of 30 wt % of BG. The spectrum of PP (Fig 1c) reveals its characteristic absorption peaks at cm -1, cm -1 and 3330 cm -1 corresponding to C=O and C O acyclic stretching vibrations of PCL and OH groups of PVA respectively. Similarly, the spectrum of composite with BG (Fig 1d) shows absorption peaks characteristic to both PP and BG % C 94.04% C Instrument: 2960 SDT V2.2B 80 Weight (%) C 61.65% % C Temperature ( C) Universal V3.7A TA Instruments Fig 2: TGA thermogram of PPBG composite scaffold.

5 The TGA thermogram (Fig 2) confirms that the composite contains % bioactive glass, which shows that the composites scaffolds were homogenously mixed having less deviation from the theoretical amount of filler.

6 Page 5 of 6 to the living bone. This apatite layer can be reproduced in vitro using SBF, which has the ion concentration and ph almost equal to the human blood plasma. Hence, it makes possible to analyze the bone bonding capability of the material which in turn relates to the suitability for bone regeneration. The chemical composition of the composite scaffolds after immersion in SBF for 14 days was analyzed by IR and shown in Fig 1(e). The composite sample shows the characteristic peaks of PVA, PCL and BG. Where as in the sample put in SBF, in addition to these peaks, absorption peaks at cm -1 and cm -1 are present and attributed to C-O bending from CO 3-2, which confirms the formation of HCA layer. In addition, the peaks due to Si-O-Si stretching vibrations is absent in the latter sample because with the progress of HCA formation the vibrations due to silica tetrahedral decrease in intensity. The scaffold is expected to be bioactive and hence promote bone formation and bone bonding. Biocompatiblity studies using Chondrocytes The presence of viable chondrocytes was observed after 1-month culture in the polymer composite scaffold by the live dead assay and confocal imaging techniques. The results confirm the biocompatibility of PPBG scaffolds to chondrocytes. (a) (b) Fig 5: Live dead assay showing chondrocytes in the scaffolds after 1 month culture. Dead cells stained red and living cells green (a) PPBG scaffold, (b) PP scaffold Histological analysis of 1 month construct showed that the matrix region was strongly stained by the characteristic red of Safranin O indicating presence of the proteoglycan rich matrix (Fig 6a) corroborated with positive Toludiene blue staining confirming GAG accumulation.(fig 6b). The typical chondrogenic markers collagen type 2 and aggrecan were also expressed in PPBG cartilage construct of 1 month as confirmed by immunohistochemistry (Fig 6 c & d). Quantitative estimation of GAG did not show significant difference between PP as well as PPBG scaffold (Fig 6 e). Hence, the presence of BG has not deteriorated the quality of the cartilage formed.

7 Page 6 of 6 a b GAG Estimation 60 c d C o n c in u g /sc affo ld PP PPBG Fig 6: Histopathological analysis (a) saffranin O stain, (b) Toludiene blue staining, (c) expression of Collagen type 2 (d) Expression of aggrecan, (e) Total GAG content in one month culture. e CONCLUSIONS The physicochemical characterization shows that scaffolds could be produced from a composite comprising of two matrix phase and a filler phase and that the physicochemical properties of the scaffold meets requirement of an ideal scaffold. The in vitro studies of this novel composite scaffold, carried out in the present work, showed that the composite forms a Ca- P rich layer which is indicative of its bioactive potential and potential ability to bond with bone. In addition the cell culture studies also show that the physical and chemical properties of the scaffold provide an optimum condition for chondrocyte survival, proliferation and subsequent tissue formation. Thus the in vitro studies and cell culture studies are also indicative of the suitability of a simple stabilization method for bioactive glass. Hence this study predicts that the novel scaffold made of PVA-PCL and BG would be used in osteochondral tissue engineering where the bioactive potential of BG is utilized for achieving better anchorage of the artificial cartilage construct to bone. The application potential of bioactive glass containing composites therefore could include both hard and soft tissue regeneration and repair. REFERENCES 1. R. G. LeBaron and K. A. Athanasiou, Biomaterals 21, 2575 (2000). 2. L. L. Hench, Bioceramics. J. Am Ceram Soc 81, 1705 (1998). 3. J. Wilson and D. Nolletti. in Handbook on bioactive ceramics: bioactive glasses and glass ceramics edited by T. Yamauro, L. L. Hench and J. Wilson. (CRC publishers, 1990) p N. Mohan and P. D. Nair. J. Biomed. Mater. Res. 84B, 584 (2008). 5. W. Xia and J. Chang. Materials Letters 61, 3251 (2007). 6. ISO : 1999, Biological evaluation of medical products. Part5: Test for in vitro Cytotoxicity.