Purpose: Current techniques for articular cartilage repair remain suboptimal. The best technique involves the introduction of cultured chondrocytes into the injury site. Experimental results of current chondrocyte culture and expansion techniques (passaging) have shown phenotypic alteration resulting in fibroblast-like cells. Therefore, treatment methods that propose the transplantation of cultured chondrocytes might be transplanting fibroblast-like cells instead of chondrocytes. This experiment explored the difference in genetic expression of chondrocytes left at confluence compared to chondrocytes that were passaged as performed in current culture techniques. It was hypothesized that chondrocytes left at confluence would maintain their collagen I and collagen II gene expression over time.

Method: Fresh normal human articular cartilage was collected from deceased donor patients. The matrix was digested and the chondrocytes were plated in monolayer to create two groups. The first group was cultured and passaged 2? at confluence seven times. The second group was cultured at confluence and left for seven weeks, with medium changes every 3–4 days without passaging. At weekly intervals RNA was extracted from cells in both groups and analyzed with real time PCR, probing specifically for the genes responsible for the production of collagen I, collagen II, aggrecan, and GAPDH. This was done in duplicate.

Results: Collagen II gene expression was maintained over seven weeks in cells left at confluence but was decreased in passaged cells. Collagen I gene expression decreased over seven weeks in cells left at confluence, but remained the same in passaged cells. Aggrecan gene expression remained the same in both groups.

Conclusion: Current culture and expansion techniques that employ passaging (as used in clinical scenarios) result in significant alterations in gene expression that are inconsistent with the current definition of a “chondrocyte”. Culturing chondrocytes at confluence can produce gene expression more similar to native chondrocytes but even these cells have expression of collagen type I that should not be present in chondrocytes. The results of this study suggest that further investigation is required to develop chondrocyte culture and expansion techniques that minimize the de-differentiation of chondrocytes by maintaining collagen II gene expression and eliminating/preventing collagen I gene expression.

Purpose: The introduction of supplementary cells into a region of diseased or damaged tissue is becoming a viable treatment strategy in many areas of medicine. Mesenchymal stem cells (MSCs) are attractive for this purpose because they represent an autologous, multipotent cell source. However, it has been recognized that populations of MSCs represent a heterogenous group of cells with each cell subpopulation possessing unique terminal differential capacity. The CD44 cell surface receptor has previously been identified on some of the cells within the MSC population. It is also present on chondrocytes and is thought to play a critical role in cartilage matrix generation and homeostasis. We hypothesized that a CD44+ purified subpopulation of MSCs will possess enhanced chondrogenic potential and be more suitable for articular cartilage regeneration.

Method: Bone marrow aspirates were collected from orthopaedic patients undergoing iliac crest bone grafting. Human MSCs were isolated and cultured using standard techniques. Flow cytometry was utilized to identify the cell surface antigens characteristic of the MSC population. FACS was utilized to isolate the CD44 positive cells based on antigenic recognition, generating a CD44 positive population and a CD44 negative population. To confirm the multilineage potential of the isolates, defined media and culture conditions were utilized to differentiate both groups into osteocytes, adipocytes and chondrocytes. Real time polymerase chain reaction was utilized to quantify and compare the essential markers, collagen II, collagen I and aggrecan, in the stem cell derived chondrocytes. The CD44 enriched and CD44 depleted populations were compared.

Results: The cells isolated possessed a cell morphology and surface antigen profile consistent with a MSC population. In addition, both experimental groups demonstrated multipotent ability. Real time PCR analysis of the chondrogenic cells demonstrated that the CD44 positive population expressed collagen II and aggrecan at a significantly higher level than the CD44 negative population.

Conclusion: To date no group has successfully identified a relationship between a MSC subpopulation and the multipotent progenitors responsible for generating cartilage. This work demonstrated that there are MSC sub-populations with different potential for chondrogenic expression and represents an important step towards identifying MSC subpopulations with enhanced cartilage formation potential.