Identification of in Situ Progenitor Cell Features Contributing to Cartilage Repair and Strategies for Cartilage Repair Augmentation

Articular cartilage is a thin layer of hyaline cartilaginous tissue covering the ends of long bones. Due to its avascular nature, it possesses very limited regeneration potential. In the event of an injury, the repair is either very limited or not initiated at all. As a result, cartilage lesions degenerate extensively under the influence of damaging cytokines and proinflammatory factors released. An imbalance in anabolic and catabolic activities continues to erode the cartilaginous surface exposing subchondral bone leading to osteoarthritis. Bone marrow stimulation (BMS) initiates repair by fracturing or drilling into subchondral bone. The channels created provide access to the underlying subchondral progenitor cells that are recruited into the defect. Granulation tissue thus formed undergoes a complex cascade of events to generate a repair tissue. BMS is considered a gold standard of cartilage repair strategies since it is easy, relatively less invasive with swift recovery, more economical and appreciable short-mid term relief. However, long term outcome is generally discouraging due to poor durability of repair tissue. The quality of repair tissue is generally fibrocartilagenous with inferior mechanical and biological characteristics compared to hyaline cartilage. The most common drawbacks of the procedure include incomplete regeneration, high inter-individual variability and poor repair outcome especially in older individuals in preclinical as well as clinical studies. Earlier studies have shown that defect location and animal age affect the cartilage repair outcome observed in vivo, suggesting a strong influence of biological characteristics of progenitor cells (Bone Marrow stem cells; BMSCs or mesenchymal stem cells; MSCs) present in subchondral bone. Specifically, rabbit trochlea showed a superior repair and chondrogenic potential compared to the medial femoral condyle. Inspired by these observations, we carried out a study for comprehensive analysis of progenitor cells present in the subchondral bone of condyle and trochlea in young and old rabbits in order to determine the influence of location and age on the properties of progenitor cells. Earlier we had found that drilling to 6 mm improves the repair outcome in rabbit trochlea possibly by providing access to metaphyseal marrow and thus higher progenitor cell population. As a result, MSCs were isolated from epiphyseal (trochlea upper) and metaphyseal (trochlea lower) regions of the trochlea. Collagenase-derived MSCs originated from collagenase digest of bone chips while the digested explants were subsequently cultured to obtain explant-derived MSCs via cell outgrowth. In vitro characterization assays were used to determine the cell yield, clonogenic potential and surface marker expression profile. Chondrogenic differentiation was stimulated using pellets of condylar and trochlear MSCs in the presence of chondrogenic media, rich in chondrogenic stimulating factors such as ascorbate, insulin-transferrin-selenium (ITS) and TGF-βIII. Finally, MSCs were differentiated into osteogenic lineage in presence of osteogenic media containing osteogenic stimulants such as dexamethasone, β-glycerophosphate and ascorbate. The aforementioned MSC characteristics were compared for trochlea vs. condyle, old vs. young and collagenase- vs. explant-derived cultures. MSCs isolated from distal femur epiphyseal bone had stem cell characteristics and fulfilled the stem cell criteria outlined by International Society for Cell Therapy. Location was an important factor influencing the properties of MSCs indicated by low cell yield, clonogenic potential and inferior chondrogenic potential of condylar cells. Results showed that rabbit trochlear vs. condylar subchondral bone yielded a greater number of progenitors with superior cartilaginous matrix expression under chondrogenic conditions suggesting higher intrinsic capacity for cartilage repair compared to condylar subchondral bone. Trochlear MSCs displayed superior chondrogenic differentiation evidenced by higher glycosaminoglycan (GAG) and collagen type II deposition. Age of the donor was an additional factor with significant bearing on the inherent properties of MSCs since growth rate, cell yield, expression of stem cell markers and osteogenic differentiation were found to be significantly superior in younger animals. As expected, no significant difference was observed between collagenase- and explant-derived cultures. Continuing our exploration of the mechanisms underlying variability in BMS procedure, we carried out the next study to verify if the cartilage repair outcome in condyle and trochlea correlated with the pre-existing properties of progenitor cells present in these two sites. A bilateral rabbit model was used to test this hypothesis. For each rabbit, full thickness acute defects were created in one knee and treated by BMS by microdrilling, while the second knee was left intact. Animals were sacrificed 3 weeks later and transverse sections of repair tissue from the treated knee were used to analyze the quality and quantity of repair macroscopically and by safranin- O (Saf-O) and immunostaining. At the time of animal sacrifice, distal femurs were separated and MSCs were isolated from condylar and trochlear regions of the intact contralateral knee for biological characterization of cell yield, clonogenic potential, cell surface marker profile and differentiation potency. BMS induced inferior cartilage repair in condyles of all donors studied further underlining the implication of location in influencing repair outcome. Macroscopic examination revealed extremely poor fill in condylar defects. In contrast, moderate to excellent fill was observed in six out of eight trochlear defects. This led us to identify a donor-dependent variability in repair outcome and the donors were classified as good trochlear responders and poor trochlear responders. In another observation, trochlear MSCs were characterized by increased cell yield, higher clonogenic potential and superior chondrogenic potential. Histopathological analysis indicated better matrix composition, rich in GAG and collagen type II in trochlea both in vivo and in vitro. Donor related variability in repair outcome was also observed in vitro through biological characterization of MSCs. Statistical analysis provided substantial evidence to identify strong, positive location- and donor-dependent correlations between inherent properties of MSCs and cartilage repair outcome in condyle and trochlea and factors with maximum influence on the repair outcome were identified. By this stage, it became imperative to seek after a clinically relevant application of our understanding based on current and past findings. Cartilage lesions are rarely detected in early stages, unfortunately reducing the efficacy of BMS procedure. Unpredictable, highly variable repair of poor quality is observed in degenerative defects, especially in older patients. Platelet-mediated retraction of the blood clot that fills cartilage lesions following BMS is believed to be one underlying cause of inferior quality and durability of repair tissue. Chitosan inhibits this blood clot shrinkage and leads to the formation of a voluminous, adherent and stable clot and thereby enhances cartilage repair by promoting cell recruitment, transient vascularization and subchondral bone remodeling. Platelet-rich-plasma (PRP) contains a 2-10 fold concentration of growth factors and cytokines and has been shown to improve recruitment and chondrogenic potential of subchondral MSCs. Chitosan increases the stability of PRP and promotes the release of platelet-derived growth factors in a more sustained manner. Using a more clinically relevant chronic defect model, we tested the efficacy of implants composed of freeze-dried chitosan solubilized in PRP in improving marrow stimulated cartilage repair outcome compared to BMS augmented with just PRP. Two surgeries were performed in skeletally mature rabbits to create a bilateral trochlear defect model. Defects created in the first surgery were allowed to degenerate over 4 weeks and repaired in a second surgery by BMS. Treatments included BMS augmented with chitosan/PRP implants in one knee and BMS with PRP in the other knee. Repair tissue was harvested 2 months later and transverse sections were used to characterize the quality and quantity of repair by assessing the GAG and coll-II deposition. Subchondral bone repair was analysed by micro-CT. Augmentation of BMS with freeze-dried chitosan/PRP implants in chronic defects improved cartilage repair compared to PRP by increasing quantity and quality of repair tissue and promoting subchondral bone repair. Matrix composition was improved in presence of chitosan/PRP indicated by increased GAG and collagen deposition. MicroCT analysis showed further evidence of increased, albeit incomplete, subchondral bone remodeling induced by chitosan/PRP. In summary, these studies led us to recognize significant donor-, age- and location-dependent variability which might help in understanding the mechanism underneath the unpredictability and inter-individual variability observed with BMS. Needless to say, these observations would be extremely valuable in improving the consistency, durability and quality of BMS repair procedure. Moreover, the innovative chronic model studied here is expected to make significant contributions in progress of cartilage repair field. Finally, chitosan/PRP implants investigated in this research are a promising approach to treat chronic cartilage lesions and could eventually provide relief to osteoarthritic patients with challenging, degenerative defects.