Extracellular vesicles derived from mesenchymal stromal cells mediate endogenous cell growth and migration via the CXCL5 and CXCL6/CXCR2 axes and repair menisci

Background Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are promising candidates for tissue regeneration therapy. However, the therapeutic efficacy of MSC-EVs for meniscus regeneration is uncertain, and the mechanisms underlying MSC-EV-mediated tissue regeneration have not been fully elucidated. The aims of this study were to evaluate the therapeutic efficacy of intra-articular MSC-EV injection in a meniscus defect model and elucidate the mechanism underlying MSC-EV-mediated tissue regeneration via combined bioinformatic analyses. Methods MSC-EVs were isolated from human synovial MSC culture supernatants via ultrafiltration. To evaluate the meniscus regeneration ability, MSC-EVs were injected intra-articularly in the mouse meniscus defect model immediately after meniscus resection and weekly thereafter. After 1 and 3 weeks, their knees were excised for histological and immunohistochemical evaluations. To investigate the mechanisms through which MSC-EVs accelerate meniscus regeneration, cell growth, migration, and chondrogenesis assays were performed using treated and untreated chondrocytes and synovial MSCs with or without MSC-EVs. RNA sequencing assessed the gene expression profile of chondrocytes stimulated by MSC-EVs. Antagonists of the human chemokine CXCR2 receptor (SB265610) were used to determine the role of CXCR2 on chondrocyte cell growth and migration induced by MSC-EVs. Results In the meniscus defect model, MSC-EV injection accelerated meniscus regeneration and normalized the morphology and composition of the repaired tissue. MSC-EVs stimulated chondrocyte and synovial MSC cell growth and migration. RNA sequencing revealed that MSC-EVs induced 168 differentially expressed genes in the chondrocytes and significantly upregulated CXCL5 and CXCL6 in chondrocytes and synovial MSCs. Suppression of CXCL5 and CXCL6 and antagonism of the CXCR2 receptor binding CXCL5 and CXCL6 negated the influence of MSC-EVs on chondrocyte cell growth and migration. Conclusions Intra-articular MSC-EV administration repaired meniscus defects and augmented chondrocyte and synovial MSC cell growth and migration. Comprehensive transcriptome/RNA sequencing data confirmed that MSC-EVs upregulated CXCL5 and CXCL6 in chondrocytes and mediated the cell growth and migration of these cells via the CXCR2 axis.

Characterisation of Regional Human Meniscal Progenitor Cells

Background The surgical treatment of meniscus injury has represented a clinical challenge for decades. Stimulating meniscus regeneration using transplanted meniscal progenitor cells has been suggested as a promising new strategy. However, there is a lack of studies which decisively identify and characterise progenitor cell populations in human meniscus tissues. Methods In this study, donor-matched progenitor cells were isolated via selective fibronectin adhesion from the avascular (PAvas) and vascular (PVas) regions of the meniscus and chondroprogenitors (PChs) from articular cartilage (n=5 donors). In addition, whole mixed populations of cells (MAvas, MVas, MChs) from the same regions were obtained by standard isolation techniques for comparison. The colony formation efficacy of PAvas, PVas and PChs was monitored using Cell-IQ® live cell imaging. Proliferation rates of progenitors were compared with their mixed population counterparts. Cell surface markers indicative of mesenchymal stromal cells (MSCs) profile and progenitor markers were characterised by flow cytometry in all populations. The chondrogenic capacity was assessed via pellet culture assays and measuring chondrogenic gene expression levels, GAG/DNA content and morphology. Results All meniscal progenitor and chondroprogenitor populations showed colony forming capacity in monolayer culture, whereas mixed populations were distributed randomly at passage 0. PVas had significantly lower population doubling times compare to MVas and proliferated faster than PAvas and PChs based on colony forming efficacy. Progenitor populations showed significantly higher positivity for CD49b and CD49c compared to their mixed population counterparts and PChs had a higher positivity level of CD166 compared to mixed chondrocytes. Collagen types II and X expression was significantly downregulated in pellets formed by progenitor populations. GAG/DNA analysis demonstrated that progenitor cells generally produced more GAG than mixed populations. Conclusions Our study demonstrates that the human meniscus contains meniscal progenitor populations in both the avascular and vascular regions. Meniscal progenitors derived from the vascular region exhibit enhanced proliferative and chondrogenic characteristics compared to those from the avascular region; this may associate with the enhanced meniscal healing potential in the vascular region. These findings build on the body of evidence which suggests that meniscal progenitors represent an attractive cell therapy strategy for meniscal regeneration.

Total Meniscus Reconstruction Using a Polymeric Hybrid-Scaffold: Combined with 3D-Printed Biomimetic Framework and Micro-Particle

The meniscus has poor intrinsic regenerative capability, and its injury inevitably leads to articular cartilage degeneration. Although there are commercialized off-the-shelf alternatives to achieve total meniscus regeneration, each has its own shortcomings such as individualized size matching issues and inappropriate mechanical properties. We manufactured a polycaprolactone-based patient-specific designed framework via a Computed Tomography scan images and 3D-printing technique. Then, we completed the hybrid-scaffold by combining the 3D-printed framework and mixture micro-size composite which consists of polycaprolactone and sodium chloride to create a cell-friendly microenvironment. Based on this hybrid-scaffold with an autograft cell source (fibrochondrocyte), we assessed mechanical and histological results using the rabbit total meniscectomy model. At postoperative 12-week, hybrid-scaffold achieved neo-meniscus tissue formation, and its shape was maintained without rupture or break away from the knee joint. Histological and immunohistochemical analysis results showed obvious ingrowth of the fibroblast-like cells and chondrocyte cells as well as mature lacunae that were embedded in the extracellular matrix. Hybrid-scaffolding resulted in superior shape matching as compared to original meniscus tissue. Histological analysis showed evidence of extensive neo-meniscus cell ingrowth. Additionally, the hybrid-scaffold did not induce osteoarthritis on the femoral condyle surface. The 3D-printed hybrid-scaffold may provide a promising approach that can be applied to those who received total meniscal resection, using patient-specific design and autogenous cell source.

3D Printed Poly(ε-Caprolactone)/Meniscus Extracellular Matrix Composite Scaffold Functionalized With Kartogenin-Releasing PLGA Microspheres for Meniscus Tissue Engineering

Meniscus tissue engineering (MTE) aims to fabricate ideal scaffolds to stimulate the microenvironment for recreating the damaged meniscal tissue. Indeed, favorable mechanical properties, suitable biocompatibility, and inherent chondrogenic capability are crucial in MTE. In this study, we present a composite scaffold by 3D printing a poly(ε-caprolactone) (PCL) scaffold as backbone, followed by injection with the meniscus extracellular matrix (MECM), and modification with kartogenin (KGN)-loaded poly(lactic-co-glycolic) acid (PLGA) microsphere (μS), which serves as a drug delivery system. Therefore, we propose a plan to improve meniscus regeneration via KGN released from the 3D porous PCL/MECM scaffold. The final results showed that the hydrophilicity and bioactivity of the resulting PCL/MECM scaffold were remarkably enhanced. In vitro synovium-derived mesenchymal stem cells (SMSCs) experiments suggested that introducing MECM components helped cell adhesion and proliferation and maintained promising ability to induce cell migration. Moreover, KGN-incorporating PLGA microspheres, which were loaded on scaffolds, showed a prolonged release profile and improved the chondrogenic differentiation of SMSCs during the 14-day culture. Particularly, the PCL/MECM-KGN μS seeded by SMSCs showed the highest secretion of total collagen and aggrecan. More importantly, the synergistic effect of the MECM and sustained release of KGN can endow the PCL/MECM-KGN μS scaffolds with not only excellent cell affinity and cell vitality preservation but also chondrogenic activity. Thus, the PCL/MECM-KGN μS scaffolds are expected to have good application prospects in the field of MTE.

Autologous meniscus fragments embedded in atelocollagen gel enhance meniscus repair in a rabbit model

Aims Meniscal injuries are common and often induce knee pain requiring surgical intervention. To develop effective strategies for meniscus regeneration, we hypothesized that a minced meniscus embedded in an atelocollagen gel, a firm gel-like material, may enhance meniscus regeneration through cell migration and proliferation in the gel. Hence, the objective of this study was to investigate cell migration and proliferation in atelocollagen gels seeded with autologous meniscus fragments in vitro and examine the therapeutic potential of this combination in an in vivo rabbit model of massive meniscus defect. Methods A total of 34 Japanese white rabbits (divided into defect and atelocollagen groups) were used to produce the massive meniscus defect model through a medial patellar approach. Cell migration and proliferation were evaluated using immunohistochemistry. Furthermore, histological evaluation of the sections was performed, and a modified Pauli’s scoring system was used for the quantitative evaluation of the regenerated meniscus. Results In vitro immunohistochemistry revealed that the meniscus cells migrated from the minced meniscus and proliferated in the gel. Furthermore, histological analysis suggested that the minced meniscus embedded in the atelocollagen gel produced tissue resembling the native meniscus in vivo. The minced meniscus group also had a higher Pauli’s score compared to the defect and atelocollagen groups. Conclusion Our data show that cells in minced meniscus can proliferate, and that implantation of the minced meniscus within atelocollagen induces meniscus regeneration, thus suggesting a novel therapeutic alternative for meniscus tears. Cite this article: Bone Joint Res 2021;10(4):269–276.

Development of biological meniscus scaffold: Decellularization method and recellularization with meniscal cell population derived from mesenchymal stem cells

Tissue engineering approaches which include a combination of cells and scaffold materials provide an alternative treatment for meniscus regeneration. Decellularization and recellularization techniques are potential treatment options for transplantation. Maintenance of the ultrastructure composition of the extracellular matrix and repopulation with cells are important factors in constructing a biological scaffold and eliminating immunological reactions. The aim of the study is to develop a method to obtain biological functional meniscus scaffolds for meniscus regeneration. For this purpose, meniscus tissue was decellularized by our modified method, a combination of physical, chemical, and enzymatic methods and then recellularized with a meniscal cell population composed of fibroblasts, chondrocytes and fibrochondrocytes that obtained from mesenchymal stem cells. Decellularized and recellularized meniscus scaffolds were analysed biochemically, biomechanically and histologically. Our results revealed that cellular components of the meniscus were successfully removed by preserving collagen and GAG structures without any significant loss in biomechanical properties. Recellularization results showed that the meniscal cells were localized in the empty lacuna on the decellularized meniscus, and also well distributed and proliferated consistently during the cell culture period (p < 0.05). Furthermore, a high amount of DNA, collagen, and GAG contents (p < 0.05) were obtained with the meniscal cell population in recellularized meniscus tissue. The study demonstrates that our decellularization and recellularization methods were effective to develop a biological functional meniscus scaffold and can mimic the meniscus tissue with structural and biochemical features. We predict that the obtained biological meniscus scaffolds may provide avoidance of adverse immune reactions and an appropriate microenvironment for allogeneic or xenogeneic recipients in the transplantation process. Therefore, as a promising candidate, the obtained biological meniscus scaffolds might be verified with a transplantation experiment.