In this paper we report the differentiating properties of platelet-rich plasma releasates (PRPr) on human chondrocytes within elastomeric polycaprolactone triol-citrate (PCLT-CA) porous scaffold. Human-derived chondrocyte cellular content of glycosaminoglycans (GAGs) and total collagen were determined after seeding into PCLT-CA scaffold enriched with PRPr cells. Immunostaining and real time PCR was applied to evaluate the expression levels of chondrogenic and extracellular gene markers. Seeding of chondrocytes into PCLT-CA scaffold enriched with PRPr showed significant increase in total collagen and GAGs production compared with chondrocytes grown within control scaffold without PRPr cells. The mRNA levels of collagen II and SOX9 increased significantly while the upregulation in Cartilage Oligomeric Matrix Protein (COMP) expression was statistically insignificant. We also report the reduction of the expression levels of collagen I and III in chondrocytes as a consequence of proximity to PRPr cells within the scaffold. Interestingly, the pre-loading of PRPr caused an increase of expression levels of following extracellular matrix (ECM) proteins: fibronectin, laminin and integrin b over the period of 3 days. Overall, our results introduce the PCLT-CA elastomeric scaffold as a new system for cartilage tissue engineering. The method of PRPr cells loading prior to chondrocyte culture could be considered as a potential environment for cartilage tissue engineering as the differentiation and ECM formation is enhanced significantly. Keywords Cartilage tissue engineering Á PCLT-CA scaffolds Á PRPr cells Á Human chondrocytes Á ECM formation 1 Introduction Cartilage healing is a slow and sensitive process that often leads to a permanent cartilage defect. Cartilage repair is commonly suppressed by factors such as avascular condition of articular cartilage, limited chondrocytes in mature and aged tissue and inability of chondrocytes to migrate to the damaged site. In most cases, the fibro-cartilage would replace the damaged cartilage, which does not provide sustained repair and desirable mechanical support & Hussin A. Rothan

Clinical investigations have shown a significant relationship between osteoarthritis (OA) and estrogens levels in menopausal women. Therefore, treatment with exogenous estrogens has been shown to decrease the risk of OA. However, the effect estrogen has not been clearly demonstrated in the chondrocytes using phytoestrogens, which lack the specific side-effects of estrogens, may provide an alternative therapy. This study was designed to examine the possible effects of phytoestrogen (daidzein) on human chondrocyte phenotype and extracellular matrix formation. Phytoestrogens which lack the specific side-effects of estrogens may provide beneficial effect without causing hormone based side effect. Human chondrocytes cells were cultured in 2D (flask) and 3D (PCL-CA scaffold) systems. Daidzein cytotoxic effect was determined by MTT assay. Chondrocyte cellular content of glycosaminoglycans (GAGs), total collagen and chondrogenic gene expression were determined in both culture systems after treatment with daidzein. Daidzein showed time-dependent and dose-independent effects on chondrocyte bioactivity. The compound at low doses showed significant (p \ 0.05) increase in total collagen and GAGs production at similar levels in 2D and 3D culture environment. The mRNA levels of Collagen II and Sox9 were increased significantly (p \ 0.01) after the treatment while the upregulation in COMP expression was statistically insignificant (p [ 0.05). The expression levels of Fibronectin, Laminin and Integrin b1 were significantly increased especially in 3D culture system. This study was illustrated the potential positive effects of daidzein on maintenance of human chondrocyte phenotype and extracellular matrix formation suggesting an attractive and viable alternative therapy for OA. Keywords Phytoestrogens Á Human chondrocyte Á Extracellular matrix Á PCL-CA scaffold Á Osteoarthritis Abbreviations CTX-II C-terminal crosslinked telopeptide type II collagen CA Citric acid COMP Cartilage oligomeric matrix protein COX Cyclooxygenase DMEM Dulbecco's Modification of Eagle's Medium DMMB Dimethylmethylene blue & Thamil Selvee Ramasamy