Perlecan in the Natural and Cell Therapy Repair of Human Adult Articular Cartilage: Can Modifications in This Proteoglycan Be a Novel Therapeutic Approach?

Articular cartilage is considered to have limited regenerative capacity, which has led to the search for therapies to limit or halt the progression of its destruction. Perlecan, a multifunctional heparan sulphate (HS) proteoglycan, promotes embryonic cartilage development and stabilises the mature tissue. We investigated the immunolocalisation of perlecan and collagen between donor-matched biopsies of human articular cartilage defects (n = 10 × 2) that were repaired either naturally or using autologous cell therapy, and with age-matched normal cartilage. We explored how the removal of HS from perlecan affects human chondrocytes in vitro. Immunohistochemistry showed both a pericellular and diffuse matrix staining pattern for perlecan in both natural and cell therapy repaired cartilage, which related to whether the morphology of the newly formed tissue was hyaline cartilage or fibrocartilage. Immunostaining for perlecan was significantly greater in both these repair tissues compared to normal age-matched controls. The immunolocalisation of collagens type III and VI was also dependent on tissue morphology. Heparanase treatment of chondrocytes in vitro resulted in significantly increased proliferation, while the expression of key chondrogenic surface and genetic markers was unaffected. Perlecan was more prominent in chondrocyte clusters than in individual cells after heparanase treatment. Heparanase treatment could be a means of increasing chondrocyte responsiveness to cartilage injury and perhaps to improve repair of defects

Human mesenchymal stem cells stimulate EaHy926 endothelial cell migration:combined proteomic and in vitro analysis of the influence of donor-donor variability

Mesenchymal stem cells (MSCs) stimulate angiogenesis within a wound environment and this effect is mediated through paracrine interactions with the endothelial cells present. Here we report that human MSC-conditioned medium (n=3 donors) significantly increased EaHy-926 endothelial cell adhesion and cell migration, but that this stimulatory effect was markedly donor-dependent. MALDI-TOF/TOF mass spectrometry demonstrated that whilst collagen type I and fibronectin were secreted by all of the MSC cultures, the small leucine rich proteoglycan, decorin was secreted only by the MSC culture that was least effective upon EaHy-926 cells. These individual extracellular matrix components were then tested as culture substrata. EaHy-926 cell adherence was greatest on fibronectin-coated surfaces with least adherence on decorin-coated surfaces. Scratch wound assays were used to examine cell migration. EaHy-926 cell scratch wound closure was quickest on substrates of fibronectin and slowest on decorin. However, EaHy-926 cell migration was stimulated by the addition of MSC-conditioned medium irrespective of the types of culture substrates. These data suggest that whilst the MSC secretome may generally be considered angiogenic, the composition of the secretome is variable and this variation probably contributes to donor-donor differences in activity. Hence, screening and optimizing MSC secretomes will improve the clinical effectiveness of pro-angiogenic MSC-based therapies

Mechano-driven regeneration predicts response variations in large animal model based on scaffold implantation site and individual mechano-sensitivity

It is well founded that the mechanical environment may regulate bone regeneration in orthopedic applications. The purpose of this study is to investigate the mechanical contributions of the scaffold and the host to bone regeneration, in terms of subject specificity, implantation site and sensitivity to the mechanical environment. Using a computational approach to model mechano-driven regeneration, bone ingrowth in porous titanium scaffolds was simulated in the distal femur and proximal tibia of three goats and compared to experimental results. The results showed that bone ingrowth shifted from a homogeneous distribution pattern, when scaffolds were in contact with trabecular bone (max local ingrowth 12.47%), to a localized bone ingrowth when scaffolds were implanted in a diaphyseal location (max local ingrowth 20.64%). The bone formation dynamics revealed an apposition rate of 0.37±0.28%/day in the first three weeks after implantation, followed by limited increase in bone ingrowth until the end of the experiment (12 weeks). According to in vivo data, we identified one animal whose sensitivity to mechanical stimulation was higher than the other two. Moreover, we found that the stimulus initiating bone formation was consistently higher in the femur than in the tibia for all the individuals. Overall, the dependence of the osteogenic response on the host biomechanics means that, from a mechanical perspective, the regenerative potential depends on both the scaffold and the host environment. Therefore, this work provides insights on how the mechanical conditions of both the recipient and the scaffold contribute to meet patient and location-specific characteristics

A mathematical model of cartilage regeneration after chondrocyte and stem cell implantation – I: the effects of growth factors

Autologous chondrocyte implantation is a cell-based therapy for treating chondral defects. The procedure begins by inserting chondrocytes into the defect region. The chondrocytes initiate healing by proliferating and depositing extracellular matrix, which allows them to migrate into the defect until it is completely filled with new cartilage. Mesenchymal stem cells can be used instead of chondrocytes with similar long-term results. The main differences are at early times since mesenchymal stem cells must first differentiate into chondrocytes before cartilage is formed. To better understand this repair process, we present a mathematical model of cartilage regeneration after cell therapy. We extend our previous work to include the cell–cell interaction between mesenchymal stem cells and chondrocytes via growth factors. Our results show that matrix formation is enhanced at early times in the presence of growth factors. This study reinforces the importance of mesenchymal stem cell and chondrocyte interaction in the cartilage healing process as hypothesised in experimental studies

A mathematical model of cartilage regeneration after chondrocyte and stem cell implantation – II: the effects of co-implantation

We present a mathematical model of cartilage regeneration after cell therapy, to show how co-implantation of stem cells (mesenchymal stem cells) and chondrocytes into a cartilage defect can impact chondral healing. The key mechanisms involved in the regeneration process are simulated by modelling cell proliferation, migration and differentiation, nutrient diffusion and Extracellular Matrix (ECM) synthesis at the defect site, both spatially and temporally. In addition, we model the interaction between mesenchymal stem cells and chondrocytes by including growth factors. In Part I of this work, we have shown that matrix formation was enhanced at early times when mesenchymal stem cell-to-chondrocyte interactions due to the effects of growth factors were considered. In this article, we show that the additional effect of co-implanting mesenchymal stem cells and chondrocytes further enhances matrix production within the first year in comparison to implanting only chondrocytes or only mesenchymal stem cells. This could potentially reduce healing time allowing the patient to become mobile sooner after surgery

Journal of Theoretical Biology A mathematical model of signalling molecule-mediated processes during regeneration of osteochondral defects after chondrocyte implantation --Manuscript Draft-- Manuscript Number: JTB-D-23-00814R1

Treating bone-cartilage defects is a fundamental clinical problem. The ability of damaged cartilage to self-repair is limited due to its avascularity. Left untreated, these defects can lead to osteoarthritis. Details of osteochondral defect repair are elusive, but animal models indicate healing occurs via an endochondral ossification-like process, similar to that in the growth plate. In the growth plate, the signalling molecules parathyroid hormone-related protein (PTHrP) and Indian Hedgehog (Ihh) form a feedback loop regulating chondrocyte hypertrophy, with Ihh inducing and PTHrP suppressing hypertrophy. To better understand this repair process and to explore the regulatory role of signalling molecules on the regeneration process, we formulate a mathematical model of osteochondral defect regeneration after chondrocyte implantation. The drivers of healing are assumed to be chondrocytes and osteoblasts, and their interaction via signalling molecules. We model cell proliferation, migration and chondrocyte hypertrophy, and matrix production and conversion, spatially and temporally. We further model nutrient and signalling molecule diffusion and their interaction with the cells. We consider the PTHrP-Ihh feedback loop as the backbone mechanisms but the model is flexible to incorporate extra signalling mechanisms if needed. Our mathematical model is able to represent repair of osteochondral defects, starting with cartilage formation throughout the defect. This is followed by chondrocyte hypertrophy, matrix calcification and bone formation deep inside the defect, while cartilage at the surface is maintained and eventually separated from the deeper bone by a thin layer of calcified cartilage. The complete process requires around 48 months. A key highlight of the model demonstrates that the PTHrP-Ihh loop alone is insufficient and an extra mechanism is required to initiate chondrocyte hypertrophy, represented by a critical cartilage density. A parameter sensitivity study reveals that the timing of the repair process crucially depends on parameters, such as the critical cartilage density, and those describing the actions of PTHrP to suppress hypertrophy, such as its diffusion coefficient, threshold concentration and degradation rate. Response to Reviewers: Reply to Reviewers We thank the reviewers for their careful reading of the manuscript and the insightful comments and suggestions they have provided. This has led to the manuscript being thoroughly revised both in the content and structure. The main revisions made are as follows. These are referred to by the corresponding section numbers and page numbers in the revised manuscript