Polycaprolactone Triol–Citrate Scaffolds Enriched with Human Platelet Releasates Promote Chondrogenic Phenotype and Cartilage Extracellular Matrix Formation

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 β 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.

Cotransfected human chondrocytes: over-expression of IGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans

Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.

Human Chondrocyte Viability against Time and Density in Calcium Alginate In Vitro

One Approach for caraniofacial contouring surgery is to use autogenous cartilage cells localized within a biocompatible polymer. This study investigated varying conditions of polymerization at cell density on the viability of human chondrocytes encapsulated in calcium alginate. Human rib cartilage was dissected, enzymatically dissociated, and the chondrocytes were collected by centrifugation. Alginate discs containig chondrocyte were made from 1.5% sodium alginate and 100 mM CaCI solution. Viability of chondrocytes was measured by quantification of the DNA content per alginate discs at six different time intervals from 0 to 5 weeeks. Significant initial cell loss was observed in the first two weeks after which the survival rate remained stationary. To optimize calcium alginate matrix formation with human chondrocytes, the cell density was varied during manufacture of the alginate discs. Chondrocyte viability was measured after fourteen days of culture as same method mentioned above. Varying the cell density at the time of polymerization from 2.0x10 to 2.0x10' chondrocytes/ml produced a direct relationship between number of cells and chondrocyte viability. The greatest viability(70.2 percent) was observed with cell density of 2.0 x 10' chondrocytes/ml. These data demonstrate that cell density is a critical factor for successful encapsulation of human cartilage cells in a calcium alginate matrix.