Bone formation by human paediatric marrow stromal cells in a functional allogeneic immune system.

Allogeneic stem-cell based regenerative medicine is a promising approach for bone defect repair. The use of chondrogenically differentiated human marrow stromal cells (MSCs) has been shown to lead to bone formation by endochondral ossification in immunodeficient pre-clinical models. However, an insight into the interactions between the allogeneic immune system and the human MSC-derived bone grafts has not been fully achieved yet. The choice of a potent source of MSCs isolated from pediatric donors with consistent differentiation and high proliferation abilities, as well as low immunogenicity, could increase the chance of success for bone allografts. In this study, we employed an immunodeficient animal model humanised with allogeneic immune cells to study the immune responses towards chondrogenically differentiated human pediatric MSCs (ch-pMSCs). We show that ch-differentiated pMSCs remained non-immunogenic to allogeneic CD4 and CD8 T cells in an in vitro co-culture model. After subcutaneous implantation in mice, ch-pMSC-derived grafts were able to initiate bone mineralisation in the presence of an allogeneic immune system for 3 weeks without the onset of immune responses. Re-exposing the splenocytes of the humanised animals to pMSCs did not trigger further T cell proliferation, suggesting an absence of secondary immune responses. Moreover, ch-pMSCs generated mature bone after 8 weeks of implantation that persisted for up to 6 more weeks in the presence of an allogeneic immune system. These data collectively show that human allogeneic chondrogenically differentiated pediatric MSCs might be a safe and potent option for bone defect repair in the tissue engineering and regenerative medicine setting.

The response of human macrophages to 3D printed titanium antibacterial implants does not affect the osteogenic differentiation of hMSCs.

Macrophage responses following the implantation of orthopaedic implants are essential for successful implant integration in the body, partly through intimate crosstalk with human marrow stromal cells (hMSCs) in the process of new bone formation. Additive manufacturing (AM) and plasma electrolytic oxidation (PEO) in the presence of silver nanoparticles (AgNPs) are promising techniques to achieve multifunctional titanium implants. Their osteoimmunomodulatory properties are, however, not yet fully investigated. Here, we studied the effects of implants with AgNPs on human macrophages and the crosstalk between hMSCs and human macrophages when co-cultured in vitro with biofunctionalised AM Ti6Al4V implants. A concentration of 0.3 g/L AgNPs in the PEO electrolyte was found to be optimal for both macrophage viability and inhibition of bacteria growth. These specimens also caused a decrease of the macrophage tissue repair related factor C-C Motif Chemokine Ligand 18 (CCL18). Nevertheless, co-cultured hMSCs could osteogenically differentiate without any adverse effects caused by the presence of macrophages that were previously exposed to the PEO (±AgNPs) surfaces. Further evaluation of these promising implants in a bony in vivo environment with and without infection is highly recommended to prove their potential for clinical use.

Intra-articular injection of triamcinolone acetonide sustains macrophage levels and aggravates osteophytosis during degenerative joint disease in mice.

BACKGROUND AND PURPOSE: Corticosteroids such as triamcinolone acetonide (TAA) are potent drugs administered intra-articularly as an anti-inflammatory therapy to relieve pain associated with osteoarthritis (OA). However, the ability of early TAA intervention to mitigate OA progression and modulate immune cell subsets remains unclear. Here, we sought to understand the effect of early intra-articular injection of TAA on OA progression, local macrophages, and peripheral blood monocytes. EXPERIMENTAL APPROACH: Degenerative joint disease was induced by intra-articular injection of collagenase into the knee joint of male C57BL/6 mice. After 1 week, TAA or saline was injected intra-articularly. Blood was taken throughout the study to analyse monocyte subsets. Mice were killed at days 14 and 56 post-induction of collagenase-induced OA (CiOA) to examine synovial macrophages and structural OA features. KEY RESULTS: The percentage of macrophages relative to total live cells present within knee joints was increased in collagenase- compared with saline-injected knees at day 14 and was not altered by TAA treatment. However, at day 56, post-induction of CiOA, TAA-treated knees had increased levels of macrophages compared with the knees of untreated CiOA-mice. The distribution of monocyte subsets present in peripheral blood was not altered by TAA treatment during the development of CiOA. Osteophyte maturation was increased in TAA-injected knees at day 56. CONCLUSION AND IMPLICATIONS: Intra-articular injection of TAA increases long-term synovial macrophage numbers and osteophytosis. Our findings suggest that TAA accentuates the progression of osteoarthritis-associated features when applied to an acutely inflamed knee.

Chondrogenically Primed Human Mesenchymal Stem Cells Persist and Undergo Early Stages of Endochondral Ossification in an Immunocompetent Xenogeneic Model.

Tissue engineering approaches using progenitor cells such as mesenchymal stromal cells (MSCs) represent a promising strategy to regenerate bone. Previous work has demonstrated the potential of chondrogenically primed human MSCs to recapitulate the process of endochondral ossification and form mature bone in vivo, using immunodeficient xenogeneic models. To further the translation of such MSC-based approaches, additional investigation is required to understand the impact of interactions between human MSC constructs and host immune cells upon the success of MSC-mediated bone formation. Although human MSCs are considered hypoimmunogenic, the potential of chondrogenically primed human MSCs to induce immunogenic responses in vivo, as well as the efficacy of MSC-mediated ectopic bone formation in the presence of fully competent immune system, requires further elucidation. Therefore, the aim of this study was to investigate the capacity of chondrogenically primed human MSC constructs to persist and undergo the process of endochondral ossification in an immune competent xenogeneic model. Chondrogenically differentiated human MSC pellets were subcutaneously implanted to wild-type BALB/c mice and retrieved at 2 and 12 weeks post-implantation. The percentages of CD4+ and CD8+ T cells, B cells, and classical/non-classical monocyte subsets were not altered in the peripheral blood of mice that received chondrogenic MSC constructs compared to sham-operated controls at 2 weeks post-surgery. However, MSC-implanted mice had significantly higher levels of serum total IgG compared to sham-operated mice at this timepoint. Flow cytometric analysis of retrieved MSC constructs identified the presence of T cells and macrophages at 2 and 12 weeks post-implantation, with low levels of immune cell infiltration to implanted MSC constructs detected by CD45 and CD3 immunohistochemical staining. Despite the presence of immune cells in the tissue, MSC constructs persisted in vivo and were not degraded/resorbed. Furthermore, constructs became mineralised, with longitudinal micro-computed tomography imaging revealing an increase in mineralised tissue volume from 4 weeks post-implantation until the experimental endpoint at 12 weeks. These findings indicate that chondrogenically differentiated human MSC pellets can persist and undergo early stages of endochondral ossification following subcutaneous implantation in an immunocompetent xenogeneic model. This scaffold-free model may be further extrapolated to provide mechanistic insight to osteoimmunological processes regulating bone regeneration and homeostasis.

Macrophage phenotypes and monocyte subsets after destabilization of the medial meniscus in mice.

Macrophages play an important role in the development and progression of osteoarthritis (OA). The aim of this study was to identify macrophage phenotypes in synovium and monocyte subsets in peripheral blood in C57BL/6 mice by destabilizing the medial meniscus (DMM), and the association of macrophage subsets with OA features. DMM, sham, and non-operated knees were histologically assessed between 1 and 56 days for macrophage polarization states by immunohistochemistry (IHC), cartilage damage, synovial thickening, and osteophytes (n = 9 per timepoint). Naive knees (n = 6) were used as controls. Monocyte and polarized synovial macrophage subsets were evaluated by flow cytometry. CD64 and CD206 levels on IHC were higher at early timepoints in DMM and sham knees compared to naive knees. iNOS labeling intensity was higher in DMM and sham knees than in naive knees from d3 onwards. CD163 expression was unaltered at all timepoints. Even though macrophage polarization profiles were similar in DMM and sham knees, only in DMM knees the presence of iNOS and CD206 associated with synovial thickness, and CD163 staining inversely correlated with osteophyte presence. At day 14, monocyte subset distribution was different in peripheral blood of DMM mice compared with sham mice. In conclusion, monocyte subsets in blood and synovial macrophage phenotypes vary after joint surgery. High levels of iNOS+ , CD163+ , and CD206+ cells are found in both destabilized and sham-operated knees, and coexistence with joint instability may be a requirement to initiate and exacerbate OA progression.

Allogeneic Chondrogenic Mesenchymal Stromal Cells Alter Helper T Cell Subsets in CD4+ Memory T Cells.

Implantation of chondrogenically differentiated mesenchymal stromal cells (MSCs) leads to bone formation in vivo through the process of endochondral ossification. The use of allogeneic MSCs for this purpose may be a promising new approach to replace the current gold standard of bone regeneration. However, the success of using allogeneic cells depends on the interaction between the implanted cells and the host's endogenous immune cells. Th17 T cells and other CD4 helper T cell subtypes have been shown to negatively impact chondrogenesis, however, it is unclear how the interaction between these cells affects bone regeneration mediated by these cells. The aim of the current work was to assess the effect of chondrogenic MSC pellets on Th1, Th2, Th17, and regulatory T cells in vitro. Human MSCs were nonchondrogenic (-TGFß3) and chondrogenically (+TGFß3) differentiated for 7 or 21 days. Memory T cells (sorted from the CD4 population of peripheral blood mononuclear cells [PBMCs]), as well as total PBMCs were cocultured with allogeneic nonchondrogenic and chondrogenic MSC pellets for 3 days. Seven-day differentiated allogeneic nonchondrogenic and chondrogenic MSC pellets that were cocultured with memory T cells resulted in a significant increase in Th2 and a decrease in Th1 T cells. Furthermore, the co-culture of 21-day differentiated nonchondrogenic and chondrogenic MSC pellets with memory T cells resulted in a significant increase in Th2 and Th17 T cells, as well as a decrease in Th1 and regulatory T cells. Interleukin (IL)-6 was identified as a predominant cytokine involved in this interaction between allogeneic chondrogenically differentiated MSC pellets and memory CD4 T cells, with high levels of IL-6 being secreted in the supernatants of this cocultured condition. The findings of this study highlight the potential of chondrogenically differentiated MSC pellets to alter the ratio of Th1 and Th2 as well as Th17 and regulatory T cell subsets. Additional analysis investigating bone formation by chondrogenically differentiated MSCs in an allogeneic setting may identify a novel role of these T cell subsets in bone regeneration processes mediated by chondrogenically differentiated MSCs. Impact statement Allogeneic mesenchymal stromal cells (MSCs) have the potential to be an off-the-shelf treatment for bone repair. However, the lack of knowledge of the immune cells involved in this process has hampered the progression to the clinic. The current study has shown that allogeneic chondrogenic MSCs have the potential to skew the ratio of specific helper CD4 T cell subsets in vitro. This has now provided insight for future in vivo experiments to investigate the role of these T cell subsets in the early stages of bone regeneration mediated by allogeneic chondrogenic MSCs.

Phenotypic Characterization of Bone Marrow Mononuclear Cells and Derived Stromal Cell Populations from Human Iliac Crest, Vertebral Body and Femoral Head.

(1) In vitro, bone marrow-derived stromal cells (BMSCs) demonstrate inter-donor phenotypic variability, which presents challenges for the development of regenerative therapies. Here, we investigated whether the frequency of putative BMSC sub-populations within the freshly isolated mononuclear cell fraction of bone marrow is phenotypically predictive for the in vitro derived stromal cell culture. (2) Vertebral body, iliac crest, and femoral head bone marrow were acquired from 33 patients (10 female and 23 male, age range 14-91). BMSC sub-populations were identified within freshly isolated mononuclear cell fractions based on cell-surface marker profiles. Stromal cells were expanded in monolayer on tissue culture plastic. Phenotypic assessment of in vitro derived cell cultures was performed by examining growth kinetics, chondrogenic, osteogenic, and adipogenic differentiation. (3) Gender, donor age, and anatomical site were neither predictive for the total yield nor the population doubling time of in vitro derived BMSC cultures. The abundance of freshly isolated progenitor sub-populations (CD45-CD34-CD73+, CD45-CD34-CD146+, NG2+CD146+) was not phenotypically predictive of derived stromal cell cultures in terms of growth kinetics nor plasticity. BMSCs derived from iliac crest and vertebral body bone marrow were more responsive to chondrogenic induction, forming superior cartilaginous tissue in vitro, compared to those isolated from femoral head. (4) The identification of discrete progenitor populations in bone marrow by current cell-surface marker profiling is not predictive for subsequently derived in vitro BMSC cultures. Overall, the iliac crest and the vertebral body offer a more reliable tissue source of stromal progenitor cells for cartilage repair strategies compared to femoral head.

Shear and Dynamic Compression Modulates the Inflammatory Phenotype of Human Monocytes in vitro.

Monocytes and their derived macrophages are found at the site of remodeling tissue, such as fracture hematoma, that is exposed to mechanical forces and have been previously implicated in the reparative response. However, the mechanoresponsive of monocytes and macrophages to skeletal tissue-associated mechanical forces and their subsequent contribution to skeletal repair remains unclear. The aim of this study was to investigate the potential of skeletal tissue-associated loading conditions to modulate human monocyte activation and phenotype. Primary human monocytes or the human monocyte reporter cell line, THP1-Blue, were encapsulated in agarose and exposed to a combination of shear and compressive loading for 1 h a day for 3 consecutive days. Exposure of monocytes to mechanical loading conditions increased their pro-inflammatory gene and protein expression. Exposure of undifferentiated monocytes to mechanical loading conditions significantly upregulated gene expression levels of interleukin(IL)-6 and IL-8 compared to free swelling controls. Additionally, multiaxial loading of unstimulated monocytes resulted in increased protein secretion of TNF-α (17.1 ± 8.9 vs. 8 ± 7.4 pg/ml) and MIP-1α (636.8 ± 471.1 vs. 124.1 ± 40.1 pg/ml), as well as IL-13 (42.1 ± 19.8 vs. 21.7 ± 13.6) compared monocytes cultured under free-swelling conditions. This modulatory effect was observed irrespective of previous activation with the M1/pro-inflammatory differentiation stimuli lipopolysaccharide and interferon-γ or the M2/anti-inflammatory differentiation factor interleukin-4. Furthermore, mechanical shear and compression were found to differentially regulate nitric oxide synthase 2 (NOS2) and IL-12B gene expression as well as inflammatory protein production by THP1-Blue monocytes. The findings of this study indicate that human monocytes are responsive to mechanical stimuli, with a modulatory effect of shear and compressive loading observed toward pro-inflammatory mediator production. This may play a role in healing pathways that are mechanically regulated. An in depth understanding of the impact of skeletal tissue-associated mechanical loading on monocyte behavior may identify novel targets to maximize inflammation-mediated repair mechanisms.

Mechanical stimulation of mesenchymal stem cells: Implications for cartilage tissue engineering.

Articular cartilage is a load-bearing tissue playing a crucial mechanical role in diarthrodial joints, facilitating joint articulation, and minimizing wear. The significance of biomechanical stimuli in the development of cartilage and maintenance of chondrocyte phenotype in adult tissues has been well documented. Furthermore, dysregulated loading is associated with cartilage pathology highlighting the importance of mechanical cues in cartilage homeostasis. The repair of damaged articular cartilage resulting from trauma or degenerative joint disease poses a major challenge due to a low intrinsic capacity of cartilage for self-renewal, attributable to its avascular nature. Bone marrow-derived mesenchymal stem cells (MSCs) are considered a promising cell type for cartilage replacement strategies due to their chondrogenic differentiation potential. Chondrogenesis of MSCs is influenced not only by biological factors but also by the environment itself, and various efforts to date have focused on harnessing biomechanics to enhance chondrogenic differentiation of MSCs. Furthermore, recapitulating mechanical cues associated with cartilage development and homeostasis in vivo, may facilitate the development of a cellular phenotype resembling native articular cartilage. The goal of this review is to summarize current literature examining the effect of mechanical cues on cartilage homeostasis, disease, and MSC chondrogenesis. The role of biological factors produced by MSCs in response to mechanical loading will also be examined. An in-depth understanding of the impact of mechanical stimulation on the chondrogenic differentiation of MSCs in terms of endogenous bioactive factor production and signaling pathways involved, may identify therapeutic targets and facilitate the development of more robust strategies for cartilage replacement using MSCs. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:52-63, 2018.

Parathyroid Hormone-Related Protein Gradients Affect the Progression of Mesenchymal Stem Cell Chondrogenesis and Hypertrophy.

INTRODUCTION: Mesenchymal stem cells (MSCs) are considered a promising cell source for cartilage repair strategies due to their chondrogenic differentiation potential. However, their in vitro tendency to progress toward hypertrophy limits their clinical use. This unfavorable result may be due to the fact that MSCs used in tissue engineering approaches are all at the same developmental stage, and have lost crucial spatial and temporal signaling cues. In this study, we sought to investigate the effect of a spatial parathyroid hormone-related protein (PTHrP) signaling gradient on the chondrogenic differentiation of MSCs and progression to hypertrophy. METHODS: Human bone marrow-derived MSCs were transduced with adenoviral vectors overexpressing PTHrP and seeded into fibrin-poly(ester-urethane) scaffolds. To investigate the effect of a spatial PTHrP signaling gradient, scaffolds were seeded with PTHrP-overexpressing MSCs positioned on top of the scaffold, with untransduced MSCs seeded evenly within. Scaffolds were cultured with or without 2 ng/mL transforming growth factor (TGF)-ß1 for 28 days. RESULTS: PTHrP overexpression increased glycosaminoglycan (GAG) production by MSCs irrespective of TGF-ß1 treatment, and exerted differential effects on chondrogenic and hypertrophic gene expression when MSCs were cultured in the presence of a PTHrP signaling gradient. Furthermore, PTHrP-overexpressing MSCs were associated with an increase of endogenous TGF-ß1 production and reduced total MMP-13 secretion compared to controls. CONCLUSION: The presence of a spatial PTHrP signaling gradient may support chondrogenic differentiation of MSCs and promote the formation of a more stable cartilage phenotype in tissue engineering applications.

Joint mimicking mechanical load activates TGFß1 in fibrin-poly(ester-urethane) scaffolds seeded with mesenchymal stem cells.

Transforming growth factor-ß1 (TGF-ß1) is widely used in an active recombinant form to stimulate the chondrogenic differentiation of mesenchymal stem cells (MSCs). Recently, it has been shown that the application of multiaxial load, that mimics the loading within diarthrodial joints, to MSCs seeded in to fibrin-poly(ester-urethane) scaffolds leads to the endogenous production and secretion of TGF-ß1 by the mechanically stimulated cells, which in turn drives the chondrogenic differentiation of the cells within the scaffold. The work presented in this short communication provides further evidence that the application of joint mimicking multiaxial load induces the secretion of TGF-ß1 by mechanically stimulated MSCs. The results of this work also show that joint-like multiaxial mechanical load activates latent TGF-ß1 in response to loading in the presence or absence of cells; this activation was not seen in non-loaded control scaffolds. Despite the application of mechanical load to scaffolds with different distributions/numbers of cells no significant differences were seen in the percentage of active TGF-ß1 quantified in the culture medium of scaffolds from different groups. The similar level of activation in scaffolds containing different numbers of cells, cells at different stages of differentiation or with different distributions of cells suggests that this activation results from the mechanical forces applied to the culture system rather than differences in cellular behaviour. These results are relevant when considering rehabilitation protocols after cell therapy or microfracture, for articular cartilage repair, where increased TGF-ß1 activation in response to joint mobilization may improve the quality of developing cartilaginous repair material. Copyright © 2016 John Wiley & Sons, Ltd.

vIL-10-overexpressing human MSCs modulate naïve and activated T lymphocytes following induction of collagenase-induced osteoarthritis.

BACKGROUND: Recent efforts in osteoarthritis (OA) research have highlighted synovial inflammation and involvement of immune cells in disease onset and progression. We sought to establish the in-vivo immune response in collagenase-induced OA and investigate the ability of human mesenchymal stem cells (hMSCs) overexpressing viral interleukin 10 (vIL-10) to modulate immune populations and delay/prevent disease progression. METHODS: Eight-week-old male C57BL/6 mice were injected with 1 U type VII collagenase over two consecutive days. At day 7, 20,000 hMSCs overexpressing vIL-10 were injected into the affected knee. Control groups comprised of vehicle, 20,000 untransduced or adNull-transduced MSCs or virus alone. Six weeks later knees were harvested for histological analysis and popliteal and inguinal lymph nodes for flow cytometric analysis. RESULTS: At this time there was no significant difference in knee OA scores between any of the groups. A trend toward more damage in animals treated with hMSCs was observed. Interestingly there was a significant reduction in the amount of activated CD4 and CD8 T cells in the vIL-10-expressing hMSC group. CONCLUSIONS: vIL-10-overexpressing hMSCs can induce long-term reduction in activated T cells in draining lymph nodes of mice with collagenase-induced OA. This could lead to reduced OA severity or disease progression over the long term.

Immune modulation to improve tissue engineering outcomes for cartilage repair in the osteoarthritic joint.

Osteoarthritis (OA), the most common form of arthritis, is a disabling degenerative joint disease affecting synovial joints and is associated with cartilage destruction, inflammation of the synovial membrane, and subchondral bone remodeling. Inflammation of the synovial membrane may arise secondary to degenerative processes in articular cartilage (AC), or may be a primary occurrence in OA pathogenesis. However, synovial inflammation plays a key role in the pathogenesis and disease progression of OA through the production of pro-inflammatory mediators, and is associated with cartilage destruction and pain. The triggers that initiate activation of the immune response in OA are unknown, but crosstalk between osteoarthritic chondrocytes, cartilage degradation products, and the synovium may act to perpetuate this response. Increasing evidence has emerged highlighting an important role for pro-inflammatory mediators and infiltrating inflammatory cell populations in the progression of the disease. Tissue engineering strategies hold great potential for the repair of damaged AC in an osteoarthritic joint. However, an in-depth understanding of how OA-associated inflammation impacts chondrocyte and progenitor cell behavior is required to achieve efficient cartilage regeneration in a catabolic osteoarthritic environment. In this review, we will discuss the role of inflammation in OA, and investigate novel immune modulation strategies that may prevent disease progression and facilitate successful cartilage regeneration for the treatment of OA.