Summary Statement. This study explores the therapeutic use of MSCs to enhance ligament healing from an immuno-modulatory perspective. We report improved healing with MSC treatment, but inconsistent effects on inflammatory markers. Introduction. Mesenchymal stem cell (MSC) use continues to hold untapped potential as a therapeutic agent because: 1) MSCs have the ability to differentiate into several different connective tissues such as cartilage, bone, muscle and fat (1–3), and 2) MSCs can modulate immune and inflammatory responses that affect healing (4, 5). This paradigm shift from differentiation to immune modulation is being studied for different applications (6). Several studies suggest MSCs decrease inflammation by reducing pro-inflammatory cytokines and changing the macrophage phenotype from M1 (classically-activated) to M2 (alternatively-activated) (7–10). However, their immune-modulatory effects within a healing ligament remain unexplored. MSCs can behave differently depending on the tissue and healing environment they encounter, which leads to our interest in MSC immune-modulation in healing ligaments. Methods. Forty-four rats underwent bilateral MCL transection. Days 5 and 14 healing were examined comparing two cell doses (1×10. 6. MSCs or 4×10. 6. MSCs). At the time of surgery, fluorescently-labeled rat MSCs (passage 8–10) were injected into the right MCL, while the left MCL served as a control for normal healing. MCLs were collected at the different time points and processed with immunohistochemistry (n=12). Type 1 macrophages (M1) and type 2 macrophages (M2) were quantified spatially within the healing ligaments. Twelve rats with MSC injections underwent mechanical testing. A multiplex cytokine reader measured 10 different cytokines in the healing ligaments at days 5 and 14. Results. MSCs were detected solely in the healing region and healing region edges at Days 5 and 14 in both dose groups using fluorescence microscopy. At day 5, the higher dose of cells produced significant M2 changes throughout the ligament. There were more M2′s (p=.05) in the distal and proximal healing regions of the normal healing ligament compared to the MSC injection group. There were significant changes in both the low dose and high dose groups at day 14. Fewer M1′s were found in the ends (p=.01) and throughout the MCL (p=.04) in the low dose group. M2′s were decreased in the ends (p=.04), but only in the ligaments that received the higher dose of MSCs. Cytokine analysis showed a greater amount of pro-inflammatory cytokines in the high dose MSC group at Day 5 (IL-1β, IL-2, and Interferon-Y) compared to controls, along with increased IL-12 at Day 14. The low dose MSC injection group demonstrated increased strength with an average failure load of 26.4N compared to 20.9N in the control group (p=.03). Low dose ligaments also exhibited increased stiffness with an average of 12.2 N/mm compared to 10.0 N/mm (p=.01) in control ligaments. Discussion. MSCs improved healing when applied at an appropriate dose as shown by improved mechanical properties at day 14. Interestingly, the smaller dose of 1 million cells proved more successful than the larger dose of 4 million cells. MSCs also affected the cytokine profile and macrophage phenotype at both healing time points, but not always as expected with regard to inflammatory cells and cytokines

Introduction: In this work a bioactive glass-ceramic (GC) in the system SiO2-CaO-P2O5 was evaluated as bone substitute biomaterial. In this sense, the capacity of mesenchymal stem cells (MSCs) to adhere, proliferate and differentiate into osteoblast (OBs) with or without GC was investigated. Two types of culture medium, i.e. growth medium (GM) and osteogenic medium (OM), were evaluated. Materials and Methods: The GC was obtained by heat treatment of a bioactive glass obtained by the sol-gel method. Isolation and culture of MSCs: The adult MSCs were isolated from bone marrow of adult rabbits obtained by direct aspirations of ileac crest. Isolation and culture of OBs: The OBs used as control were obtained by enzymatic digestion. Behavior of MSCs on GC: For the study of the behavior of isolated MSCs on the GC, two series of 96-well plates were seeded, one plate with GM and the other one with OM. The number of cells was evaluated through the XTT assay. OC production and CD90 expression of cells cultured in both media were measured to evaluate the differentiation of MSCs into OBs. Statistical analysis: A variance analysis (ANOVA) was carried out. Results: The number of cells growing in OM and GM, there were no significant differences between them. The MSCs under the conditions of this study expressed an osteoblastic phenotype (OC production, decrease CD90 expression, mineralized extracell matrix). These two effects took place by either the action of exposing the MSCs to a MO and by the effect of the GC

Background. Definitive proof is lacking on mesenchymal stem cell (MSCs) cellular therapy to regenerate bone if biological potential is insufficient. High number of MSCs after GMP expansion may solve the progenitor insufficiency at the injury but clinical trials are pending. Methods. A prospective, multicenter, multinational Phase I/IIa interventional clinical trial was designed under the EU-FP7 REBORNE Project to evaluate safety and early efficacy of autologous expanded MSCs loaded on biomaterial at the fracture site in diaphyseal and/or metaphysodiaphyseal fractures (femur, tibia, humerus) nonunions. The trial included 30 recruited patients among 5 European centres in France, Spain, Germany, and Italy. Safety endpoints (local and general complication rate) and secondary endpoints for early efficacy (number of patients with clinically and radiologically proven bone healing at 12 and 24 weeks) were established. Cultured MSCs from autologous bone marrow, expanded under GMP protocol was the Investigational Medicinal Product, standardised in the participating countries confirming equivalent cell production in all the contributing GMP facilities. Cells were mixed with CE-marked biphasic calcium phosphate biomaterial in the surgical setting, at an implanted dose of 20−106 cells per cc of biomaterial (total 10cc per case) in a single administration, after debridement of the nonunion. Results. Of 30 recruited patients, 28 patients received the treatment and completed the protocol up to 24 weeks (one case pending at submission). No adverse effects related to cells were detected. Two superficial infections associated to musculoskeletal flaps were solved with antibiotics. Preliminary efficacy results at 3 months confirmed 14 consolidations (out of 27 cases, 52%). At 6 months, 20 consolidations (out of 26 cases, 77%) were confirmed. One failure underwent reoperation at 6 months. One case FU was pending at submission. Conclusions. Preliminary results confirm safety, feasibility and efficacy at 3 and 6 months with the described procedure. Level of evidence. II

MiRNAs perform gene regulation that can target approximately 60% of human protein coding genes. Along with many cellular processes, miRNAs have been implicated in stem cell differentiation. Osterix (Osx), which is inhibited by mir-31, is required by MSCs for early osteoblast differentiation resulting in bone formation further downstream. We used antagomir functionalised gold nanoparticles (AuNPs) to block mir-31, which resulted in upregulation of Osx in pre-osteoblastic MG63 cells and human mesenchymal stem cells (MSCs). We used MG63 pre-osteoblastic cell line and human MSCs. Cytotoxicity of AuNPs was assessed by MTT, and cellular uptake of AuNPs was verified by TEM and ICP-MS. Osx RNA levels were determined by Fluidigm analysis and protein expression by In Cell Western analysis. Antagomir-functionalised AuNPs were incubated with cells for an initial 48 hours. (1) No cytotoxic effects were noted in either cell type. (2) Fluidigm analysis identified a varied gene response to antagomir delivery in both cell types, with MSCs recording a reduction of stem cell marker genes nestin, alcam, CD63, and CD44 at day 5 (indicating differentiation). (3) Osx protein levels were increased in both cell types after 48 hour incubation. (4) Downstream MSC analysis demonstrated accelerated osteogenesis at week 3 and 5 (verified by osteocalcin nodule formation) following 48 hour AuNP incubation. RNA analysis in both cell types suggested a shift away from proliferation towards osteoblastic differentiation. This was supported by Osx protein expression, which was increased in both MG63 cells and MSCs. Finally, an increase in the late osteogenic marker (osteocalcin) was verified at weeks 3 and 5 in MSCs after AuNP incubation for 48 hours. These results collectively infer successful delivery of mir-31 antagomirs, which are blocking mir-31-mediated suppression of Osx, resulting in an early increase in Osx, which accelerates MSC osteogenesis downstream

The aim of this study was to examine the therapeutic potential of locally transplanted MSCs or osteoprogenitor cells (OPCs) in delayed unions. Autologous MSCs were cultured in DMEM or osteogenic medium. A femoral osteotomy was created in rats and stabilized with an external fixator. Except for the Control-group (C-group), a delayed union was induced by cauterization of the periosteum and bone marrow removal. After 2 days, these animals received an injection of DMEM in the gap containing MSCs (MSC-group), OPCs (OPC-group) or no cells (Sham-group). Histomorphometrical analysis showed significant differences in the fraction of mineralized bone, cartilage and connective tissue between the C- and the Sham-group after 2 (p=0.001) and 8 weeks (p≤0.009). After 2 weeks, the MSC- and OPC-groups developed a larger cartilage fraction (each p=0.019) compared to the Sham-group. Biomechanical testing after 8 weeks demonstrated a significantly lower torsional stiffness (p=0.001) in the Sham-group compared to the C-group. Both the MSC and OPC groups showed a higher torsional stiffness than the Sham-group with statistically significant differences (p< 0.002) in the OPC-group. Locally applied MSCs and OPCs slightly improved the healing in this model. The MSCs were less effective compared to the OPCs. The less than expected healing improvement of both cell treatments may be related to an unfavourable microenvironment at the application time. An explanation for the superior outcome of the OPCs might be that the OPCs may be protected by macroscopically visible matrix at the transplantation time point

This study was conducted to test the hypothesis that growth factors can reduce the suppressive effect of titanium particles on MSCs. Cultured human MSCs at passage 3 were challenged with prepared cpTi particles at a concentration of 500 particles/cell along with one of the following growth factors: TGF-beta(1) (10 ng/mL), FGF-2 (10 ng/mL), IGF-I (100 ng/mL), and BMP-6 (50 ng/mL). After various periods of time, the treatment effects on cellular proliferation, viability, and osteogenic differentiation were measured. All the four growth factors positively promoted cell proliferation and viability to a varying extent. FGF-2 most effectively enhanced cell proliferation, whereas IGF-I was the most effective growth factor for enhancing cell viability. FGF-2, IGF-I, and BMP-6 reversed the titanium-mediated suppression of osteogenic differentiation, BMP-6 being the most effective one. Various growth factors can mitigate the suppressive effects of titanium particles on MSCs and enhance cell proliferation, viability, and osteogenic differentiation

Purpose. Bone marrow multi-potent stromal cells represent a heterogenous source of cells with great promise in joint cartilage regenerative medicine. However, due to their low numbers upon harvesting, MSCs need to be expanded without compromising their capacity to form chondrocytes (cartilage cells). To date there is no consensus on how to expand MSCs in order to maximize their potential for cartilage repair and nor are there any specific cell signatures of MSCs with chondrogenic propensity. Emerging evidence suggest that marrow stem cells exist in a hypoxic microenvironment. On this basis and in addition to cartilages natural existence in hypoxic environment (1–7% O2), we hypothesized that MSC expansion under hypoxia will result in the enrichment of MSCs with predilection to chondrocytes compared to expansion under the conventional culture conditions of 21% O2. Method. Bone marrow was harvested from the iliac crest of 4 donors (mean age 43.5 years) post informed consent and local ethical approval. Fifteen million mono-nucleated (MNCs) cells were seeded into T150cm2 culture flasks in the presence of alpha MEM plus 10% FBS and 5 ng/ml FGF2. Similarly, 0.25 million MNCs were seeded in 10cm petri dishes for colony forming unit-fibroblastic (CFU-f) assay. The seeded flasks and petri dishes were cultured under normoxia (21% O2) and hypoxia (3% O2). Petri dished cells were cultured for 14 days and those in flasks were cultured until passage 2 (P2). Developed cell colonies per dish were revealed after crystal violet staining. Colony counts and diameters were recorded. P2 cells were treated with a panel of antibodies for cell surface marker analysis by fluorescent activated cell sorting (FACS) flow cytometry. P2 cell pellets were formed and induced towards cartilage in a defined serum free medium containing TGFβ1. Pellets were cultured for 3 weeks under normoxia and were then processed for histological, biochemical and gene expression analyses. Results. The mean number of cell colonies was 1.25-fold higher after hypoxia culture relative to normoxia. There were no differences in colony diameters. A panel of common protein signatures (CD29, CD90, CD105 and CD151) for stem cells declined in expression after expansion in hypoxia. However, other signatures (CD13, CD34 and CD44) expression level increased under hypoxia, whilst CD73 expression was unchanged. Pellets from hypoxia-expanded MSCs showed on average a 1.4-fold increase in chondrogenic capacity as judged by glycosaminoglycan (GAG) matrix per DNA content relative to normoxia pellets. The gene expression of collagen II, SOX9, aggrecan and matrillin-3 increased by 1.2-, 2-, 1.3- and 1.5-fold, respectively, in pellets formed from hypoxia-expanded stem cells relative to their normoxia counterparts. Conclusion. Expansion of stem cells under hypoxia potentiates their capacity to form cartilage with improved cartilage properties. However, there is a need for signatures to identify stem cells with propensity to form cartilage

Cite this article: Bone Joint Res 2023;12(1):5–8.

Aims

Extracellular vesicles (EVs) are nanoparticles secreted by all cells, enriched in proteins, lipids, and nucleic acids related to cell-to-cell communication and vital components of cell-based therapies. Mesenchymal stromal cell (MSC)-derived EVs have been studied as an alternative for osteoarthritis (OA) treatment. However, their clinical translation is hindered by industrial and regulatory challenges. In contrast, platelet-derived EVs might reach clinics faster since platelet concentrates, such as platelet lysates (PL), are already used in therapeutics. Hence, we aimed to test the therapeutic potential of PL-derived extracellular vesicles (pEVs) as a new treatment for OA, which is a degenerative joint disease of articular cartilage and does not have any curative or regenerative treatment, by comparing its effects to those of human umbilical cord MSC-derived EVs (cEVs) on an ex vivo OA-induced model using human cartilage explants.

Introduction

Human Mesenchymal stem cells (hMSCs) are a promising source for articular cartilage repair. Unfortunately, under in vitro conditions, chondrogenically differentiated hMSCs have the tendency to undergo hypertrophy similar to growth plate chondrocytes. Retinoic acid (RA) signalling plays a key role in growth plate hypertrophy. Whilst RA agonists block chondrogenesis and foster hypertrophy during later stages, RAR inverse agonists (IA) enhance chondrogenesis when applied early in culture. Therefore, we hypothesized that treatment with RAR IA will attenuate hypertrophy in chondrogenically differentiated hMSCs. To test this hypothesis, we analysed early (initial chondrogenic differentiation) and late treatment (hypertrophy stage) of hMSCs with an RAR IA.

Summary

Shear stress and hydrostatic effects on the hMSCs early mechano gene response were similar. For the same magnitude gene response, the hydrostatic compression (1.5×10⁵ Pascal) is a 200000 times greater than the force exerted by shear stress (0.7 Pascal).

Impaction allografting is a bone reconstruction technique currently used in lower limb revision arthroplasty. Demineralisation and addition of osteogenic protein-1 (OP-1) can improve the osteoinductivity of the allograft however recent reports indicate significant allograft resorption when it is combined with OP-1 during impaction. Our hypothesis was that hydroxyapatite (HA) and OP-1 could effectively replace demineralised allograft. The objective was to evaluate human mesenchymal stem cell (h-MSC) proliferation (tritiated thymidine incorporation, total DNA Hoechst 33258 and scanning electron microscopy) and osteogenic differentiation (alkaline phosphatase activity) in human demineralised bone matrix (h-DBM) and HA, with or without OP-1. Cell proliferation on HA+OP-1 was significantly higher compared to HA at all time points (p< 0.05) and to DBM alone (day 1, p=0.042; day 14, p< 0.001). Cell proliferation was higher in DBM+OP-1, at all time points compared to HA+OP-1 but only in absolute values. Cell differentiation was significantly higher in HA+OP-1 compared to HA (p< 0.05) but comparable to DBM alone. Differentiation was significantly higher on DBM+OP-1 at all time points compared to HA (p< 0.05) and to HA+OP-1 (p< 0.05). HA is a potential graft expander in impaction allografting. When combined with OP-1 is comparable to DBM alone and being non absorbable may support the impacted graft in the early stages after the administration of OP-1.

Bone regeneration and repair are crucial to ambulation and quality of life. Factors such as poor general health, serious medical comorbidities, chronic inflammation, and ageing can lead to delayed healing and nonunion of fractures, and persistent bone defects. Bioengineering strategies to heal bone often involve grafting of autologous bone marrow aspirate concentrate (BMAC) or mesenchymal stem cells (MSCs) with biocompatible scaffolds. While BMAC shows promise, variability in its efficacy exists due to discrepancies in MSC concentration and robustness, and immune cell composition. Understanding the mechanisms by which macrophages and lymphocytes – the main cellular components in BMAC – interact with MSCs could suggest novel strategies to enhance bone healing. Macrophages are polarized into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, and influence cell metabolism and tissue regeneration via the secretion of cytokines and other factors. T cells, especially helper T1 (Th1) and Th17, promote inflammation and osteoclastogenesis, whereas Th2 and regulatory T (Treg) cells have anti-inflammatory pro-reconstructive effects, thereby supporting osteogenesis. Crosstalk among macrophages, T cells, and MSCs affects the bone microenvironment and regulates the local immune response. Manipulating the proportion and interactions of these cells presents an opportunity to alter the local regenerative capacity of bone, which potentially could enhance clinical outcomes. Cite this article: Bone Joint Res 2024;13(9):462–473

Tissue repair is believed to rely on tissue-resident progenitor cell populations proliferating, migrating, and undergoing differentiation at the site of injury. During these processes, the crosstalk between mesenchymal stromal/stem cells (MSCs) and macrophages has been shown to play a pivotal role. However, the influence of extracellular matrix (ECM) remodelling in this crosstalk, remains elusive. Human MSCs cultured on tissue culture plastic (TCP) and encased within fibrin in vitro were treated with/without TNFα and IFNγ. Human monocytes were cocultured with untreated/pretreated MSCs on TCP or within fibrin. After seven days, the conditioned media (CM) were collected. Human chondrocytes were exposed to CM in a migration assay. The impact of TGFβ was assessed by adding an inhibitor (TGFβRi). Cell activity was assessed using RT-qPCR and XL-protein-profiler-array. Previously, we demonstrated that culturing human MSCs within 3D-environments significantly enhances their immunoregulatory activity in response to pro-inflammatory stimuli. In this study, monocytes were co-cultured with MSCs within fibrin, acquiring a distinct M2-like repair macrophage phenotype in contrast to TCP co-cultures. MSC/macrophage CM characterization using a protein array demonstrated differences in release of several factors, including chemokines, growth factors and ECM components. Chondrocyte migration was significantly reduced in CM from untreated MSC/monocytes co-cultures in fibrin compared to CM of untreated MSCs/monocytes on TCP. This impact on migration was not seen with chondrocytes cultured in CM of monocytes co-cultured with pretreated MSCs in fibrin. The CM of monocytes co-cultured with pretreated MSCs in fibrin up-regulates COL2A1 and SOX9 compared to TCP. Chondrogenesis and migration were TGFβ dependent. MSC/macrophage crosstalk and responsiveness to cytokines are influenced by the ECM environment, which subsequently impacts tissue-resident cell migration and chondrogenesis. The direct effects of ECM on MSC/macrophage secretory phenotype is complemented by the dynamic ECM binding and release of growth factors such as TGFβ

Abstract. Objectives. Tissue repair is believed to rely on tissue-resident progenitor cell populations proliferating, migrating, and undergoing differentiation at the site of injury. During these processes, the crosstalk between mesenchymal stromal/stem cells (MSCs) and macrophages has been shown to play a pivotal role. However, the influence of extracellular matrix (ECM) remodelling in this crosstalk, remains elusive. Methods. Human MSCs cultured on tissue culture plastic (TCP) and encased within fibrin in vitro were treated with/without TNFα and IFNγ. Human monocytes were cocultured with untreated/pretreated MSCs on TCP or within fibrin. After seven days, the conditioned media (CM) were collected. Human chondrocytes were exposed to CM in a migration assay. The impact of TGFβ was assessed by adding an inhibitor (TGFβRi). Cell activity was assessed using RT-qPCR and XL-protein-profiler-array. Results. Previously, we demonstrated that culturing human MSCs within 3D-environments significantly enhances their immunoregulatory activity in response to pro-inflammatory stimuli. In this study, monocytes were co-cultured with MSCs within fibrin, acquiring a distinct M2-like repair macrophage phenotype in contrast to TCP co-cultures. MSC/macrophage CM characterization using a protein array demonstrated differences in release of several factors, including chemokines, growth factors and ECM components. Chondrocyte migration was significantly reduced in CM from untreated MSC/monocytes co-cultures in fibrin compared to CM of untreated MSCs/monocytes on TCP. This impact on migration was not seen with chondrocytes cultured in CM of monocytes co-cultured with pretreated MSCs in fibrin. The CM of monocytes co-cultured with pretreated MSCs in fibrin up-regulates COL2A1 and SOX9 compared to TCP. Chondrogenesis and migration were TGFβ dependent. Conclusion. MSC/macrophage crosstalk and responsiveness to cytokines are influenced by the ECM environment, which subsequently impacts tissue-resident cell migration and chondrogenesis. The direct effects of ECM on MSC/macrophage secretory phenotype is complemented by the dynamic ECM binding and release of growth factors such as TGFβ. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

In relation to regenerative therapies in osteoarthritis and cartilage repair, mesenchymal stromal cells (MSCs) have immunomodulatory functions and influence macrophage behaviour. Macrophages exist as a spectrum of pro-(M1) and anti-(M2) inflammatory phenotypic subsets. In the context of cartilage repair, we investigated MSC-macrophage crosstalk, including specifically the priming of cartilage cells by macrophages to achieve a regenerative rather than fibrotic outcome. Human monocytes were isolated from blood cones and differentiated towards M1 and M2 macrophages. Monocytes (Mo), M1 and M2 macrophages were cultured directly and indirectly (trans-well system) with human bone marrow derived MSCs. MSCs were added during M1 polarisation and separately to already induced M1 cells. Outcomes (M1/M2 markers and ligands/receptors) were evaluated using RT-qPCR and flow cytometry. Influence on chondrogenesis was assessed by applying M1 and M2 macrophage conditioned media (CM) sequentially to cartilage derived cells (recapitulating an acute injury environment). RT-qPCR was used to evaluate chondrogenic/fibrogenic gene transcription. The ratio of M2 markers (CD206 or CD163) to M1 markers (CD38) increased when MSCs were added to Mo/M1 macrophages, regardless of culture system used (direct or indirect). Pro-inflammatory markers (including TNFβ) decreased. CXCR2 expression by both M1 macrophages and MSCs decreased when MSCs were added to differentiated M1 macrophages in transwell. When adding initially M1 CM (for 12 hours) followed by M2 CM (for 12 hours) sequentially to chondrocytes, there was a significant increase of Aggrecan and Collagen type 2 gene expression and decrease in fibroblastic cell surface markers (PDPN/CD90). Mo/M1 macrophages cultured with MSCs, directly or indirectly, are shifted towards a more M2 phenotype. Indirect culture suggests this effect can occur via soluble signaling mediators. Sequential exposure of M1CM followed by M2CM to chondrocytes resulted in increased chondrogenic and reduced fibrotic gene expression, suggesting that an acute pro-inflammatory stimulus may prime chondrocytes before repair

Abstract. Objectives. In relation to regenerative therapies in osteoarthritis and cartilage repair, mesenchymal stromal cells (MSCs) have immunomodulatory functions and influence macrophage behaviour. Macrophages exist as a spectrum of pro-(M1) and anti-(M2) inflammatory phenotypic subsets. In the context of cartilage repair, we investigated MSC-macrophage crosstalk, including specifically the priming of cartilage cells by macrophages to achieve a regenerative rather than fibrotic outcome. Methods. Human monocytes were isolated from blood cones and differentiated towards M1 and M2 macrophages. Monocytes (Mo), M1 and M2 macrophages were cultured directly and indirectly (trans-well system) with human bone marrow derived MSCs. MSCs were added during M1 polarisation and separately to already induced M1 cells. Outcomes (M1/M2 markers and ligands/receptors) were evaluated using RT-qPCR and flow cytometry. Influence on chondrogenesis was assessed by applying M1 and M2 macrophage conditioned media (CM) sequentially to cartilage derived cells (recapitulating an acute injury environment). RT-qPCR was used to evaluate chondrogenic/fibrogenic gene transcription. Results. The ratio of M2 markers (CD206 or CD163) to M1 markers (CD38) increased when MSCs were added to Mo/M1 macrophages, regardless of culture system used (direct or indirect). Pro-inflammatory markers (including TNFa) decreased. CXCR2 expression by both M1 macrophages and MSCs decreased when MSCs were added to differentiated M1 macrophages in transwell. When adding initially M1 CM (for 12 hours) followed by M2 CM (for 12 hours) sequentially to chondrocytes, there was a significant increase of Aggrecan and Collagen type 2 gene expression and decrease in fibroblastic cell surface markers (PDPN/CD90). Conclusions. Mo/M1 macrophages cultured with MSCs, directly or indirectly, are shifted towards a more M2 phenotype. Indirect culture suggests this effect can occur via soluble signaling mediators. Sequential exposure of M1CM followed by M2CM to chondrocytes resulted in increased chondrogenic and reduced fibrotic gene expression, suggesting that an acute pro-inflammatory stimulus may prime chondrocytes before repair. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Osteoarthritis is a common articular cartilage disorder and causes a significant global disease burden. Articular cartilage has a limited capacity of repair and there is increasing interest in the use of cell-based therapies to facilitate repair including the use of Mesenchymal Stromal Cells (MSCs). There is some evidence in the literature that suggests that advancing age is associated with declining MSC function, including reduced proliferation and differentiation potential, and greater cellular apoptosis. In our study, we first performed a systematic review of the literature to determine the effects of chronological age on the in vitro properties of MSCs, and then performed a laboratory study to investigate these properties. We initially conducted a PRISMA systematic review of the literature to review the evidence base for the effects of chronological age on the in vitro properties of MSCs including cell numbers, expansion, cell surface characterization and differentiation potential. This was followed by laboratory based experiments to assess these properties. Tissue from patients undergoing total knee replacement surgery was used to isolate MSCs from the bone fragments using a method developed in our laboratory. The growth kinetics was determined by calculating the population doublings per day. Following expansion in culture, MSCs at P2 were characterised for a panel of cell surface markers using flow cytometry. The cells were positive for CD73, CD90 and CD105, and negative for CD34 and CD45. The differentiation potential of the MSCs was assessed through tri-lineage differentiation assays. Clear differences between the younger and older patients were indicated. Chronological age-related changes in MSC function have important implications on the use of these cells in clinical applications for an ageing population. The results from this study will be used to plan further work looking at the effects of chronological age on cellular senescence and identify pathways that could be targeted to potentially reverse any age-related changes

A promising application of Mesenchymal stem cells (MSCs) is the treatment of non-unions. Substituting bone grafts, MSCs are directly injected into the fracture gap. High cell viability seems to be a prerequisite for therapeutic success. Administration of the MSCs via injection creates shear stresses possibly damaging or destroying the cells. Aim of this study was to investigate the effect of the injection process on cell viability. MSCs were isolated and cultivated from femoral tissue of five subjects undergoing arthroplasty. Prior to injection, the cells were identified as MSCs. After dissolving to a concentration of 1 Million cells/ml, 1 ml of the suspension was injected through a cannula of 200 mm length and 2 mm diameter (14 G) with flow rates of 38 and 100 ml/min. The viability of the MSCs at different flow rates was evaluated by staining to detect the healthy cell fraction. It was analyzed statistically against a control group via the Kruskal-Wallis-test and for equivalence via the TOST procedure. Significance level was set to 5 %, equivalence margin to 20 %. The healthy cell fraction of the control group was 85.88 ± 2.98 %, 86.04 ± 2.53 % at 38 ml/min and 85.48 ± 1.64 % at 100 ml/min. There was no significant difference between the fraction of healthy cells (p = 0.99) for different volume flows, but a significant equivalence between the control group and the two volume flows (38 ml/min: p = 0.002, 100 ml/min: p = 0.001). When injecting MSC solutions, e.g. into a non-union, the viability of the injected cells does not deterioriate significant. The injecting technique is therefore feasible

Varus malalignment increases the susceptibility of cartilage to mechanical overloading, which stimulates catabolic metabolism to break down the extracellular matrix and lead to osteoarthritis (OA). The altered mechanical axis from the hip, knee to ankle leads to knee joint pain and ensuing cartilage wear and deterioration, which impact millions of the aged population. Stabilization of the remaining damaged cartilage, and prevention of further deterioration, could provide immense clinical utility and prolong joint function. Our previous work showed that high tibial osteotomy (HTO) could shift the mechanical stress from an imbalanced status to a neutral alignment. However, the underlying mechanisms of endogenous cartilage stabilization after HTO remain unclear. We hypothesize that cartilage-resident mesenchymal stem cells (MSCs) dampen damaged cartilage injury and promote endogenous repair in a varus malaligned knee. The goal of this study is to further examine whether HTO-mediated off-loading would affect human cartilage-resident MSCs' anabolic and catabolic metabolism. This study was approved by IACUC at Xi'an Jiaotong University. Patients with medial compartment OA (52.75±6.85 yrs, left knee 18, right knee 20) underwent open-wedge HTO by the same surgeons at one single academic sports medicine center. Clinical data was documented by the Epic HIS between the dates of April 2019 and April 2022 and radiographic images were collected with a minimum of 12 months of follow-up. Medial compartment OA with/without medial meniscus injury patients with unilateral Kellgren /Lawrence grade 3–4 was confirmed by X-ray. All incisions of the lower extremity healed well after the HTO operation without incision infection. Joint space width (JSW) was measured by uploading to ImageJ software. The Knee injury and Osteoarthritis Outcome Score (KOOS) toolkit was applied to assess the pain level. Outerbridge scores were obtained from a second-look arthroscopic examination. RNA was extracted to quantify catabolic targets and pro-inflammatory genes (QiaGen). Student's t test for two group comparisons and ANOVA analysis for differences between more than 2 groups were utilized. To understand the role of mechanical loading-induced cartilage repair, we measured the serial changes of joint space width (JSW) after HTO for assessing the state of the cartilage stabilization. Our data showed that HTO increased the JSW, decreased the VAS score and improved the KOOS score significantly. We further scored cartilage lesion severity using the Outerbridge classification under a second-look arthroscopic examination while removing the HTO plate. It showed the cartilage lesion area decreased significantly, the full thickness of cartilage increased and mechanical strength was better compared to the pre-HTO baseline. HTO dampened medial tibiofemoral cartilage degeneration and accelerate cartilage repair from Outerbridge grade 2 to 3 to Outerbridge 0 to 1 compared to untreated varus OA. It suggested that physical loading was involved in HTO-induced cartilage regeneration. Given that HTO surgery increases joint space width and creates a physical loading environment, we hypothesize that HTO could increase cartilage composition and collagen accumulation. Consistent with our observation, a group of cartilage-resident MSCs was identified. Our data further showed decreased expression of RUNX2, COL10 and increased SOX9 in MSCs at the RNA level, indicating that catabolic activities were halted during mechanical off-loading. To understand the role of cartilage-resident MSCs in cartilage repair in a biophysical environment, we investigated the differentiation potential of MSCs under 3-dimensional mechanical loading conditions. The physical loading inhibited catabolic markers (IL-1 and IL-6) and increased anabolic markers (SOX9, COL2). Knee-preserved HTO intervention alleviates varus malalignment-related knee joint pain, improves daily and recreation function, and repairs degenerated cartilage of medial compartment OA. The off-loading effect of HTO may allow the mechanoregulation of cartilage repair through the differentiation of endogenous cartilage-derived MSCs