Synergistic Interplay of dECM and Exosomes in Shaping the Cartilage Matrix Microenvironment: A New Paradigm for Regenerative Medicine

CORR Synthesis: What Is the Evidence for the Clinical Use of Stem Cell-based Therapy in the Treatment of Osteoarthritis of the Knee?

Inhibition of BMP signaling with LDN 193189 can influence bone marrow stromal cell fate but does not prevent hypertrophy during chondrogenesis.

The overall goal of my PhD studies was to analyze how the regulation of angiogenesis, a key factor that plays very important roles in tissue repair, could ultimately influence cartilage formation by mesenchymal stromal cells (MSC) and by differentiated nasal chondrocytes (NC) as a proof of concept. Due to their multipotency and great self-renewal capacity, MSC is a highly attractive cell source for cartilage formation and regeneration. MSC have immunomodulatory properties and are in direct contact with inflammatory cells (monocytes) in the cartilage repair environment. By directly regulating the inflammatory response of monocytes and by regulating their phenotype, MSC could significantly benefit from their ability to control inflammatory events during cartilage repair processes. Thus, we also assessed whether MSC could modulate the properties and the phenotype of monocytes in order for monocytes to actively aid MSC in cartilage repair. In chapter 1, we sought a proof of principle to confirm that the blocking of angiogenesis does indeed improve cartilage formation when using genetically-modified nasal chondrocytes (NCs) or antiangiogenic peptides associated with NCs. For this purpose, NCs were genetically-modified to express mouse soluble VEGF receptor-2 (sFlk-1) or were associated with an antiangiogenic peptide in order to have their chondrogenic capacity assessed in vitro and in vivo. Improved cartilage regeneration could be observed after in vivo implantation of NCs in an ectopic nude mouse model. Whereas the anti-angiogenic approaches did not improve chondrogenesis in vitro, frank chondrogenesis occured in vivo only in the constructs generated by NCs expressing sFlk-1 or treated with the peptide. Blood vessel ingrowth was significantly hampered in the anti-angiogenic experimental groups when compared with naive NCs, which correlated with chondrogenis improvement. Strikingly, the anti-angiogenic effect was even more evident when NC donors with low chondrogenic capacity were used, and no-preculture with chondrogenic soluble factors was performed prior to in vitro implantation of the constructs. In chapter 2, we investigated how angiogenesis control by genetically-modified bone marrow-derived MSC could augment their chondrogenic potential in vivo. These MSC were genetically modified to release sFlk-1, the soluble version of VEGF receptor 2 (VEGFR2), which acted as a decoy receptor and could sequester VEGF from the immediate surroundings of MSC. Importantly, no external morphogens were supplemented in order for MSC chondrogenic differentiation to occur. Moreover, the in vivo chondrogenic capacity of sFlk-1-releasing MSC was assessed up to 12 weeks by an ectopic nude mouse model, in which collagen sponges seeded with MSC were implanted subcutaneously. Angiogenesis, as analyzed by blood vessel invasion, was markedly reduced in the constructs seeded with sFlk-1-releasing MSC. Frank and stable cartilage formation was only achieved once VEGF was blocked. In chapter 3, we aimed at investigating whether MSC can instruct monocytes to acquire traits of mesenchymal progenitors or tissue repair macrophages when in direct or indirect contact with the latter. Thus, MSC could instruct monocytes to assist in the tissue-repairing process. MSC and monocytes were cocultured either in 3D collagen sponges or in transwells for a period of five consecutive days. We demonstrated that MSC instruct monocytes into acquiring a hybrid macrophage-mesenchymal phenotype, which varies greatly depending on which kind of contact with MSC the monocytes were exposed to (whether direct cell-to-cell contact or through the release of soluble factors by MSC). However, future studies will be needed to elucidate the mechanism in which MSC could instruct monocytes into differentiating into this hybrid state, and how one could better control this process in order for monocytes to optimally aid MSC in cartilage repair.

Injuries to the articular cartilage and growth plate are significant clinical problems due to their limited ability to regenerate themselves. Despite progress in orthopedic surgery and some success in development of chondrocyte transplantation treatment and in early tissue-engineering work, cartilage regeneration using a biological approach still remains a great challenge. In the last 15 years, researchers have made significant advances and tremendous progress in exploring the potentials of mesenchymal stem cells (MSCs) in cartilage repair. These include (a) identifying readily available sources of and devising appropriate techniques for isolation and culture expansion of MSCs that have good chondrogenic differentiation capability, (b) discovering appropriate growth factors (such as TGF-beta, IGF-I, BMPs, and FGF-2) that promote MSC chondrogenic differentiation, (c) identifying or engineering biological or artificial matrix scaffolds as carriers for MSCs and growth factors for their transplantation and defect filling. In addition, representing another new perspective for cartilage repair is the successful demonstration of gene therapy with chondrogenic growth factors or inflammatory inhibitors (either individually or in combination), either directly to the cartilage tissue or mediated through transducing and transplanting cultured chondrocytes, MSCs or other mesenchymal cells. However, despite these rapid pre-clinical advances and some success in engineering cartilage-like tissue and in repairing articular and growth plate cartilage, challenges of their clinical translation remain. To achieve clinical effectiveness, safety, and practicality of using MSCs for cartilage repair, one critical investigation will be to examine the optimal combination of MSC sources, growth factor cocktails, and supporting carrier matrixes. As more insights are acquired into the critical factors regulating MSC migration, proliferation and chondrogenic differentiation both ex vivo and in vivo, it will be possible clinically to orchestrate desirable repair of injured articular and growth plate cartilage, either by transplanting ex vivo expanded MSCs or MSCs with genetic modifications, or by mobilising endogenous MSCs from adjacent source tissues such as synovium, bone marrow, or trabecular bone.

Synovial Mesenchymal Stem Cell-Derived EV-Packaged miR-31 Downregulates Histone Demethylase KDM2A to Prevent Knee Osteoarthritis

Engineering Stem and Stromal Cell Therapies for Musculoskeletal Tissue Repair.

Chondral defects, generated as a result of trauma and injury or degenerative joint diseases, such as osteoarthritis, represent some of the most challenging orthopedic conditions in terms of natural tissue healing and repair. Damages to the articular cartilage fail to elicit significant reparative activity, due to the acellularity of the tissue. Clonal proliferation of articular chondrocytes is often seen, and results in the production of mechanically inferior fibrocartilage. Extensive degeneration of the articular surface eventually necessitates total joint arthroplasty. There is, therefore, a timely need for the development of biological approaches to cartilage repair. The emerging research discipline of tissue engineering aims to develop functional tissue substitutes by combining experimental approaches in biology and engineering, and represents a particularly attractive technology for the treatment of skeletal diseases, most of which involve tissue degeneration or failure to heal. Functionally, it is instructional to approach cartilage regeneration in the context of developmental chondrogenesis; that is, the formation of cartilage from a progenitor mesenchymal cell population during embryonic development. In particular, adult tissue-derived mesenchymal stem cells (MSCs), which display multilineage differentiation potential, are currently considered a highly promising source of progenitor cells for tissue engineering. Chondrogenesis in the developing vertebrate limb consists of a highly coordinated and orchestrated series of events involving the commitment and differentiation of mesenchymal cells to mature chondrocytes. This process is regulated by the sequential and coordinated expression of genes that encode for specific cell adhesion molecules, growth factors, and extracellular matrix molecules that carry out morphogenetic and signaling activities. Recently, members of the Wnt family of signaling molecules have been functionally implicated in limb development. The Wnts comprise a large family of cysteine-rich glycoproteins that perform a number of inductive and regulatory functions in both normal development and oncogenic transformations. The expression of various Wnts has been identified in the developing limb, and our recent studies showed that Wnts play an important role in mesenchymal chondrogenesis. Interestingly, our recent work with human MSCs indicates the functional involvement of Wnts and the cell adhesion molecular, N-cadherin, as well as members of the transforming growth factor beta superfamily in MSC chondrogenesis, suggesting that regenerative chondrogenesis and developmental chondrogenesis are likely to share common regulatory pathways. By combining MSCs, biodegradable polymeric scaffolds, and the application of growth and differentiation factors, we are currently developing cartilage constructs in vitro that are potentially applicable for cartilage repair in vivo. Specifically, we recently fabricated a nanofibrous scaffold using a synthetic biodegradable polymer, poly(e-caprolactone), and demonstrated its ability to support in vitro chondrogenesis of MSCs. The electrospun porous scaffold consisted of uniform, randomly oriented nanofibers (700 nm diameter); MSCs seeded into the scaffold and cultured in the presence of transforming growth factor beta 1 differentiated to chondrocytes, indicated by gene expression and histological profiles, at a level similar to that observed in high-density pellet cultures. The physical nature and improved mechanical properties of such scaffolds, particularly in comparison with cell pellets, suggest that these constructs may serve as a practical carrier for MSC transplantation in cell-based tissue engineering approaches to cartilage repair, supported by our preliminary animal model study. The future success of cartilage tissue engineering is dependent on a number of requirements, including expansion of progenitor cells, optimization of biomaterial scaffold design, and molecular enhancement of cell differentiation and growth using biologics and gene therapeutic means. We believe that the understanding of developmental chondrogenesis serves as a rational and powerful paradigm for cartilage tissue engineering.

Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self-renewal and multi-lineage differentiation into mesoderm-type of cells, e.g., to osteoblasts, adipocytes, chondrocytes and possibly other cell types including hepatocytes and astrocytes. Due to their ease of culture and multipotentiality, hMSC are increasingly employed as a source for cells suitable for a number of clinical applications, e.g., non-healing bone fractures and defects and also non-skeletal degenerative diseases like heart failure. Currently, the numbers of clinical trials that employ MSC are increasing. However, several biological and biotechnological challenges need to be overcome to benefit from the full potential of hMSC. In this current review, we present some of the most important and recent advances in understanding of the biology of hMSC and their current and potential use in therapy.

Genetic correlations in recombinant inbred mouse strains suggest that cartilage repair is positively correlated with protection from osteoarthritis

Menstrual blood-derived mesenchymal stem cells as a potential tool for cartilage regeneration

<p>Osteoarthritis (OA) is a serious joint inflammation that leads to cartilage degeneration and joint dysfunction. Mesenchymal stem cells (MSCs) are used as a cell-based therapy that showed promising results in promoting cartilage repair. However, recent studies and clinical trials explored unsatisfied outcomes because of slow chondrogenic differentiation and increased calcification without clear reasons. Here, we report that the overexpression of indoleamine 2,3 dioxygenase 1 (IDO1) in the synovial fluid of OA patients impairs chondrogenic differentiation of MSCs in the joint of the OA mice model. The effect of MSCs mixed with IDO1 inhibitor on the cartilage regeneration was tested compared to MSCs mixed with IDO1 in the OA animal model. Further, the mechanism exploring the effect of IDO1 on chondrogenic differentiation was investigated. Subsequently, miRNA transcriptome sequencing was performed for MSCs cocultured with IDO1, and then TargetScan was used to verify the target of miR-122-5p in the SF-MSCs. Interestingly, we found that MSCs mixed with IDO1 inhibitor showed a significant performance to promote cartilage regeneration in the OA animal model, while MSCs mixed with IDO1 failed to stimulate cartilage regeneration. Importantly, the overexpression of IDO1 showed significant inhibition to Sox9 and Collagen type II (COL2A1) through activating the expression of β-catenin, since inhibiting of IDO1 significantly promoted chondrogenic signaling of MSCs (Sox9, COL2A1, Aggrecan). Further, miRNA transcriptome sequencing of SF-MSCs that treated with IDO1 showed significant downregulation of miR-122-5p which perfectly targets Wnt1. The expression of Wnt1 was noticed high when IDO1 was overexpressed. In summary, our results suggest that IDO1 overexpression in the synovial fluid of OA patients impairs chondrogenic differentiation of MSCs and cartilage regeneration through downregulation of miR-122-5p that activates the Wnt1/β-catenin pathway.</p>

Osteoarthritis (OA) is a serious joint inflammation that leads to cartilage degeneration and joint dysfunction. Mesenchymal stem cells (MSCs) are used as a cell-based therapy that showed promising results in promoting cartilage repair. However, recent studies and clinical trials explored unsatisfied outcomes because of slow chondrogenic differentiation and increased calcification without clear reasons. Here, we report that the overexpression of indoleamine 2,3 dioxygenase 1 (IDO1) in the synovial fluid of OA patients impairs chondrogenic differentiation of MSCs in the joint of the OA mice model. The effect of MSCs mixed with IDO1 inhibitor on the cartilage regeneration was tested compared to MSCs mixed with IDO1 in the OA animal model. Further, the mechanism exploring the effect of IDO1 on chondrogenic differentiation was investigated. Subsequently, miRNA transcriptome sequencing was performed for MSCs cocultured with IDO1, and then TargetScan was used to verify the target of miR-122-5p in the SF-MSCs. Interestingly, we found that MSCs mixed with IDO1 inhibitor showed a significant performance to promote cartilage regeneration in the OA animal model, while MSCs mixed with IDO1 failed to stimulate cartilage regeneration. Importantly, the overexpression of IDO1 showed significant inhibition to Sox9 and Collagen type II (COL2A1) through activating the expression of β-catenin, since inhibiting of IDO1 significantly promoted chondrogenic signaling of MSCs (Sox9, COL2A1, Aggrecan). Further, miRNA transcriptome sequencing of SF-MSCs that treated with IDO1 showed significant downregulation of miR-122-5p which perfectly targets Wnt1. The expression of Wnt1 was noticed high when IDO1 was overexpressed. In summary, our results suggest that IDO1 overexpression in the synovial fluid of OA patients impairs chondrogenic differentiation of MSCs and cartilage regeneration through downregulation of miR-122-5p that activates the Wnt1/β-catenin pathway.

Mesenchymal stem cells for cartilage regeneration: Insights into molecular mechanism and therapeutic strategies

Introduction: Cartilage and bone tissues are widely utilized in regenerative medicine, especially in treating injured cartilage and bone. Some attempts were made to produce these tissues from stem cells and scaffolds for a long time, combined with some growth factors and inducible factors. However, owing to the complexity of cartilage and bone tissues, these efforts generated limited results. This chapter explores the production and usage of mesenchymal stem cell (MSC) spheroids in cartilage and bone regeneration. Methods: The data regarding production and applications of mesenchymal stem cell spheroids for cartilage and bone regeneration were searched in the PubMed, Web of Science, and Google scholar databases with the keywords “derived mesenchymal stem cell spheroids”, “cartilage regeneration”, and “bone regeneration”. Results: The formation of cartilage or bone tissue through the use of MSC spheroids mimics the formation of cartilage and bone during embryogenesis. These microtissues can be directly used as materials for transplantation or building blocks to generate the cartilage and bone macrotissues. Conclusion: Based on our studies and recent publications, we support the production and application of microtissues of cartilage and bone from mesenchymal stem cell spheroids for cartilage and bone regeneration.KeywordsBone engineeringCartilage engineeringMicrotissuesMesenchymal stem cellsTissue engineeringRegenerative medicine

Articular cartilage (AC) covers the diarthrodial joints and is responsible for the mechanical distribution of loads across the joints. The majority of its structure and function is controlled by chondrocytes that regulate Extracellular Matrix (ECM) turnover and maintain tissue homeostasis. Imbalance in their function leads to degenerative diseases like Osteoarthritis (OA). OA is characterized by cartilage degradation, osteophyte formation and stiffening of joints. Cartilage degeneration is a consequence of chondrocyte hypertrophy along with the expression of proteolytic enzymes. Matrix Metalloproteinases (MMPs) and A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) are an example of these enzymes that degrade the ECM. Signaling cascades involved in limb patterning and cartilage repair play a role in OA progression. However, the regulation of these remains to be elucidated. Further the role of stem cells and mature chondrocytes in OA progression is unclear. The progress in cell based therapies that utilize Mesenchymal Stem Cell (MSC) infusion for cartilage repair may lead to new therapeutics in the long term. However, many questions are unanswered such as the efficacy of MSCs usage in therapy. This review focuses on the role of chondrocytes in cartilage formation and the progression of OA. Moreover, it summarizes possible alternative therapeutic approaches using MSC infusion for cartilage restoration.