The aim of this study was to evaluate the role of peripheral blood derived mononucleated cells (PBMC) in osteochondral repair. We compared the healing of a critical size osteochondral defect in the medial femoral condyle and lateral trochlear sulcus in an ovine model.
Mesenchymal stem cells (MSCs) have potential for therapeutic repair of cartilage and bone but still require optimization in terms of their capacity to deposit an appropriate extracellular matrix (ECM). Adult human cartilage has a limited capacity for repair and is unusual in that it is one of the few tissues where injury is not followed by an influx of monocytes. We are studying the effects of co-culturing primary monocytes with MSCs differentiating along chondrogenic lineage but in addition we needed to investigate the effects of the monocytes on the mature chondrocytes that will result from the MSCs and will also be present in the host tissue. Human articular cartilage chondrocytes were isolated from human donors undergoing knee replacement surgery for osteoarthritis (OA) with full ethical consent. Cultures were expanded and cells used below passage five for co-culture experiments. Monocytes were prepared from fresh heparinized human blood samples by Ficoll gradient. Co-cultures consisted of either chondrocyte micromasses overlaid with monocytes, or chondrocytes and monocytes seeded together within a collagen/glycosaminoglycan scaffold (Chondromimetic, Tigenix UK). Media, cell pellets and scaffolds were analysed for extracellular matrix (ECM) proteins and proteases by dot blot, western blot, zymography and immunohistochemistry. Human chondrocytes maintained stable micromasses and laid down an ECM for at least 40 days. Human monocytes eventually formed a proliferating cell population with a rounded morphology on top of the chondrocyte micromasses. These cells established an adherent population with a fibroblastic morphology when replated on plastic. Analysis of chondrocyte ECM proteins indicated that monocytes affected deposition of types I and II collagen, decorin and fibronectin and the overall amounts of gelatinases released. RTPCR demonstrated a decrease in type I collagen expression and a concomitant increase in MMP13 expression. The precise interaction between monocytes and and chondrocytes has yet to be established but is thought to involve a mixture of contact and paracrine factors. In this study co-culture of monocytes with chondrocytes resulted in phenotypic changes to the chondrocytes which may warrant the inclusion of monocytes in cartilage/bone repair and also provide information as to the responses of OA chondrocytes to external stimuli.
Meniscal cartilage provides joint stabilisation, load distribution, impact absorption and decreased friction in joints that have a complex movement such as the knee. If the meniscal cartilage degrades or is surgically removed, there is a strong probability, over time, of damage to the articular surface. The ability to regenerate damaged meniscal cartilage with an implanted device that replaces the biological equivalent would allow for joint stabilisation, robust movement and reduce the risk of damage to the articular cartilage. An implant with many of the characteristics of meniscus and with the ability to integrate correctly and firmly with the surrounding tissue, would be advantageous. Inclusion of Platelet Rich Plasma (PRP) into the scaffolds to provide a concentrated source of matrix proteins and autologous growth factors may further enhance the regenerative repair process. To investigate the suitability of the collagen scaffolds, addition of meniscal chondrocytes and or PRP was examined in vitro. Human meniscal chondrocyte cells were isolated, via collagenase digestion, from meniscal cartilage recovered from total knee replacement surgery. Meniscal chondrocytes were cultured in vitro to expand cell numbers. PRP was produced from volunteer's blood using a centrifuge and density based platelet recovery system. Release of Platelet Derived Growth Factor type AB (PDGF-AB) was measured by ELISA as an indicator of the behaviour of the peptide growth factor component. Combinations of scaffold, meniscal chondrocytes and PRP were tested for interaction, suitability and viability. Experiments so far have shown good biocompatibility, in vitro, as meniscal chondrocytes were able to grow within the range of scaffolds produced. Cell retention could be enhanced by addition of PRP to the scaffolds. PDGF-AB was released over 5 days from the scaffold and PRP combination. Further studies are in progress to derive relevant scaffold modifications and combinations for practical, robust, treatment strategies.
