Despite the clinical relevance of back pain and intervertebral disc herniation, the lack of reliable models has strained their molecular understanding. We characterized the lumbar spinal phenotype of C57BL/6 and SM/J mice during aging. Interestingly, old SM/J lumbar discs evidenced accelerated degeneration, associated with high rates of disc herniation. SM/J AF's and degenerative human's AF transcriptomic profiles showed altered immune cell, inflammation, and p53 pathways. Old SM/J mice presented increased neuronal markers in herniated discs, thicker subchondral bone, and higher sensitization to pain. Dorsal root ganglia transcriptomic studies and spinal cord analysis exhibited increased pain and neuroinflammatory markers associated with altered extracellular matrix regulation. Immune system single-cell and tissue level analysis showed distinctive T-cell and B-cell modulation and negative correlation between mechanical allodynia and INF-α, IL-1β, IL2, and IL4, respectively. This study underscores the multisystemic network behind back pain and highlights the role of genetic background and the immune system in disc herniation disease. Acknowledgments: This study is supported by grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) R01AR055655, R01AR064733, R01AR074813 to MVR

Bone regeneration is a complex but very well organized process in which the immune system has a decisive role. The adaptive immune system and its experience level (percentage of effector and memory T cells) has been proven to influence the healing cascade especially in the early healing phases. This opens the possibility of an early intervention to enhance bone healing during the primary clinical treatment. Patients stratified for possible delayed bone healing could benefit from immunomodulatory treatment approaches. In pre-clinical studies cells and signaling molecules have been identified that could represent promising candidates to help patients in need

Harnessing the potential of mesenchymal stem cell (MSC) mediated endochondral ossification for the repair of large bone defects represents a promising avenue of investigation as an alternative option to autologous bone transplantation. To date, it has been shown that undifferentiated MSCs are somewhat immune-privileged. In order to induce bone formation from MSCs by endochondral ossification it is usually necessary to first differentiate these cells chondrogenically. However, the status of differentiated cells is less clear than that of undifferentiated MSCs. Furthermore, the fate of implanted bone forming constructs in an allogeneic setting is not known. The potential to use allogeneic MSCs for large bone defect repair would offer opportunities to researchers to develop new therapies using more potent MSC sources and in a more readily available manner with regard to the patient. I will present our research investigating the interactions between chondrogenically primed MSCs and immune cell subsets, namely T cells and dendritic cells. Furthermore, I will discuss the ability of human paediatric MSCs to form bone in the in vivo allogeneic setting.

Macrophages play a critical role in innate immunity by promoting or inhibiting tissue inflammation and repair. Classically, macrophages can differentiate into either pro-inflammatory (M1) or pro-reparative (M2) phenotypes in response to various stimuli. Therefore, this study aimed to address how extracellular vesicles (EVs) derived from polarized macrophages can affect the inflammatory response of tendon cells. For that purpose, human THP-1 cells were stimulated with lipopolysaccharide (LPS), and interleukins -4 and -13 (IL- 4, IL-13), to induce macrophages polarization into M1, M2, and hybrid M1/M2 phenotypes. Subsequently, the EVs were isolated from the culture medium by ultracentrifugation. The impact of these nanovesicles on the inflammation and injury scenarios of human tendon-derived cells (hTDCs), which had previously been stimulated with interleukin- 1 beta (IL-1ß) to mimic an inflammatory scenario, was assessed. We were able to isolate three different nanovesicles populations, showing the typical shape, size and surface markers of EVs. By extensively analyzing the proteomic expression profiles of M1, M2, and M1/M2, distinct proteins that were upregulated in each type of macrophage-derived EVs were identified. Notably, most of the detected pro- inflammatory cytokines and chemokines had higher expression levels in M1-derived EVs and were mostly absent in M2-derived EVs. Hence, by acting as a biological cue, we observed that M2 macrophage-derived EVs increased the expression of the tendon-related marker tenomodulin (TNMD) and tended to reduce the presence of pro-inflammatory markers in hTDCs. Overall, these preliminary results show that EVs derived from polarized macrophages might be a potential tool to modulate the immune system responses becoming a valuable asset in the tendon repair and regeneration fields worthy to be further explored

Anatomically, bone consists of building blocks called osteons, which in turn comprise a central canal that contains nerves and blood vessels. This indicates that bone is a highly innervated and vascularized tissue. The function of vascularization in bone (development) is well-established: providing oxygen and nutrients that are necessary for the formation, maintenance, and healing. As a result, in the field of bone tissue engineering many research efforts take vascularization into account, focusing on engineering vascularized bone. In contrast, while bone anatomy indicates that the role of innervation in bone is equally important, the role of innervation in bone tissue engineering has often been disregarded. For many years, the role of innervation in bone was mostly clear in physiology, where innervation of a skeleton is responsible for sensing pain and other sensory stimuli. Unraveling its role on a cellular level is far more complex, yet more recent research efforts have unveiled that innervation has an influence on osteoblast and osteoclast activity. Such innervation activities have an important role in the regulation of bone homeostasis, stimulating bone formation and inhibiting resorption. Furthermore, due to their anatomical proximity, skeletal nerves and blood vessels interact and influence each other, which is also demonstrated by pathways cross-over and joint responses to stimuli. Besides those closely connected sytems, the immune system plays also a pivotal role in bone regeneration. Certain cytokines are important to attract osteogenic cells and (partially) inhibit bone resorption. Several leukocytes also play a role in the bone regeneration process. Overall, bone interacts with several systems. Aberrations in those systems affect the bone and are important to understand in the context of bone regeneration. This crosstalk has become more evident and is taken more into consideration. This leads to more complex tissue regeneration, but may recapitulate better physiological situations

In orthopedic surgery, implant infections are a serious issue and difficult to treat. The aim of this study was to use superparamagnetic nanoporous silica nanoparticles (MNPSNP) as candidates for directed drug delivery. Currently, short blood circulation half-life due to interactions with the host's immune system hinder nanoparticles in general from being clinically used. PEGylation is an approach to reduce these interactions and to enhance blood circulation time. The effect of PEGylation of the used . 68. Ga-labelled MNPSNP on the distribution and implant accumulation was examined by PET/CT imaging and gamma counting in an implant mouse model. Female Balb/c mice (n=24) received a magnetic implant subcutaneously on the left and a titanium implant on the right hind leg. On day one, 12 of these mice received an additional clodronate®-injection for macrophage depletion. On the second postoperative day, mice were anaesthetized and MNPSNP (native or PEGylated) injected intravenously, followed by a dynamic PET-scan over 60 minutes, a CT- and a static PET-scan at 120 min. As control, 12 mice received only . 68. Ga-MNPSNP (native or PEGylated). Gamma counting of inner organs, urine, blood and implant area was performed as further final analysis. Although PEGylation of the nanoparticles already resulted in lower liver uptakes, both variants of . 68. Ga-labeled MNPSNP accumulated in liver and spleen. Combination of PEGylation with clodronate®-injection led to a highly significant effect whereas clodronate®-injection alone could not reveal significant differences. In gamma counting, a significantly higher %I.D./g was found for the tissue surrounding the magnetic implants compared to the titanium control, although in a low range. PEGylation and/or clodronate®-injection revealed no significant differences regarding nanoparticle accumulation at the implantation site. PEGylation increases circulation time, but MNPSNP accumulation at the implant site was still insufficient for treatment of infections. Additional efforts have to further increase circulation time and local accumulation. Acknowledgements: This work is funded by the German Research Foundation (DFG, project number 280642759)

Biphasic calcium phosphate (BCP) with a characteristic needle-shaped submicron surface topography (MagnetOs) has attracted much attention due to its unique bone-forming ability which is essential for repairing critical-size bone defects such as those found in the posterolateral spine. Previous in vitro and ex-vivo data performed by van Dijk LA and Yuan H demonstrated that these specific surface characteristics drive a favorable response from the innate immune system. This study aimed to evaluate and compare the in vivo performance of three commercially-available synthetic bone grafts, (1) i-FACTOR Putty. ®. , (2) OssDsign. ®. Catalyst Putty and (3) FIBERGRAFT. ®. BG Matrix, with that of a novel synthetic bone graft in a clinically-relevant instrumented sheep posterolateral lumbar spine fusion (PLF) model. The novel synthetic bone graft comprised of BCP granules with a needle-shaped submicron surface topography (MagnetOs) embedded in a highly porous and fibrillar collagen matrix (MagnetOs Flex Matrix). Four synthetic bone grafts were implanted as standalone in an instrumented sheep PLF model for 12 weeks (n=3 bilateral levels per group; levels L2/3 & L4/5), after which spinal fusion was determined by manual palpation, radiograph and µCT imaging (based on the Lenke scale), range-of-motion mechanical testing, and histological and histomorphological evaluation. Radiographic fusion assessment determined bilateral robust bone bridging (Lenke scale A) in 3/3 levels for MagnetOs Flex Matrix compared to 1/3 for all other groups. For µCT, bilateral fusion (Lenke scale A) was found in 2/3 levels for MagnetOs Flex Matrix, compared to 0/3 for i-FACTOR Putty. ®. , 1/3 for OssDsign. ®. Catalyst Putty and 0/3 for FIBERGRAFT. ®. BG Matrix. Fusion assessment for MagnetOs Flex Matrix was further substantiated by histology which revealed significant graft resorption complemented by abundant bone tissue and continuous bony bridging between vertebral transverse processes resulting in bilateral spinal fusion in 3/3 implants. These results show that MagnetOs Flex Matrix achieved better fusion rates compared to three commercially-available synthetic bone grafts when used as a standalone in a clinically-relevant instrumented sheep PLF model

Industrialized countries experience a population aging. Elderly patients, due to the experienced immunity, have a constant pro-inflammatory milieu. Little is known on how adaptive immunity impacts the tissue homeostasis and regeneration. The standardized housing of lab animals is specific pathogen free (SPF). However, this housing condition hinders antigen exposure and thus an aging of the adaptive immune system. We hypothesized that exposure to antigens and a developing adaptive immunity will impact tissue homeostasis and regeneration in mice. Mice kept under SPF housing or non-SPF were examined towards their immune status via flow cytometry, bone structure via microCT and bone competence via biomechanical torsional testing. MSCs from these mice were analyzed regarding their differentiation potential and ECM production under various immune cell signaling. Bone regeneration was analyzed in vivo in a mouse osteotomy model. The memory and effector compartment of the adaptive immunity was significantly increased in mice under non-SPF housing. This housing led to an increased femoral cortical thickness and torsional stiffness (p<0,05), whereas the tissue mineral density was not affected. The differentiation potential of stem cells under the influence of an aged immune milieu was significantly reduced. Bone formation was highly affected by the immune status and availed of a naïve immune cell milieu. Adaptive immunity directly impacts bone tissue formation, by exhibiting a constant stress, leading to structural differences in bone tissue organization as well as mechanical competence. For experimental settings, it appears highly relevant if mouse models have had the chance to develop an experienced immune system

Abstract. Objective. The aim of our systematic review was to report the latest evidence on the effects of CoCr particles on local soft tissue with a focus on its clinical relevance. Methods. PubMed, Embase, and Cochrane Library databases were screened to perform an extensive review. Inclusion criteria were studies of any level of evidence published in peer-reviewed journals reporting clinical and preclinical results written in English. Relative data were extracted and critically analyzed. PRISMA guidelines were applied, and the risk of bias was assessed, as was the methodological quality of the included studies. Results. 30 studies were included after applying the inclusion and exclusion criteria. Of these, 24 were preclinical studies (18 in vitro human studies, 6 animal modal studies, including 3 in vitro and 3 in vivo), 5 were clinical studies and 1 was previous review on similar topic. The presence of metal ions causes cell damage by reducing cell viability, inducing DNA damage, and triggering the secretion of cytokines. Mechanisms of apoptosis, autophagy and necrosis are responsible for the inflammatory reaction observed in ALTR. Conclusion. The available literature on the effects of CoCr particles released from MoM implants shows that metal debris can cause damage to skeletal muscle, the capsule, and provoke osteolysis and inflammation. Therefore, the cytotoxic and genotoxic damages, as well as the interaction with the immune system, affect the success of the arthroplasty and lead to a higher rate of revision surgeries. 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

Abstract. Purpose. It is becoming apparent that mesenchymal stem cells (MSCs) do not directly contribute to mesenchymal tissue regeneration. Pre-clinical attempts to repair large bone defects in big animal models have been hampered by poor MSCs survival after implantation which impedes their direct or indirect effects. Based on previous work, we hypothesized that a venous axial vascularization of the scaffold supporting MSCs or their combination with fresh bone marrow (BM) aspirate would improve their in vivo survival. Methods. Cross-shape profile tubular microporous monetite implants (12mm long, 5mm large) as two longitudinal halves were produced by 3D powder printing. They were implanted around the femoral veins of Wistar rats and loaded with 1mL of BM aspirate either alone or supplemented by 10. 7. MSCs. This was compared with BM-free scaffolds loaded only with 10. 7. MSCs. After 8 weeks bone formation were investigated by micro-CT, scanning electron microscopy, histology and immunohistochemistry. Results. Little bone formation was observed within the scaffold when it was only loaded with MSCs surprisingly. Coupling MSCs, autologous BM and venous perfusion of the scaffold led to a higher volume of new bone than BM alone suggesting that MSCs augmented the bone formation capacity of BM aspirate or enhanced its survival post implantation. Conclusion. Subcutaneous bone formation within 3D-printed implant that mixed of BM with or without MSCs was successfully achieved for the first time by venous perfusion. The inability of MSCs to form differentiated tissues by their own was confirmed in this study; however, contact between MSCs and BM cells and MSCs paracrine secretome (e.g., cytokines, chemokines, extracellular vesicles) may have induced immunomodulatory effects (e.g., macrophages polarization, Treg cells) that triggered bone formation. This approach, if translatable to large animal models, offers immediate clinical value as well as an insight into the role of immune system in tissue regeneration. 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

Introduction. Although osteonecrosis of the femoral head has been observed in young adult patients with autoimmune diseases such as SLE and MCTD that are treated by corticosteroids, the pathogenesis of the osteonecrosis remains unclear. We established a rat model with osteonecrosis of the femoral head by injecting lipopolysaccharide (LPS) and corticosteroid, and assessed consequences of the histopathological alteration of the femoral head, the systemic immune response, and the lipid synthesis. Methods. Male Wistar rats were given 2 mg/kg LPS intravenously on days 0 and 1 and intramuscularly 20 mg/kg methylprednisolone on days 2, 3, and 4. The animals were sacrificed 1, 2, 3, or 4 weeks after the last injection of the methylprednisolone. Histopathological and biochemical analyses were performed every week. The bone samples were then processed for routine hematoxylin and eosin staining to assess the general architecture and injury of the tissue. The triglyceride and the total cholesterol concentrations in the PRP were measured. The levels of various cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, TNF-α) in blood samples were measured. Results. The body weight of the rats over time decreased for 2 weeks but had recovered by week 4. The plasma triglyceride concentrations had decreased significantly by weeks 2 and 3. The total plasma cholesterol concentrations had increased significantly by week 1 but then decreased significantly by week 4. The plasma concentrations of IL-1?α, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ and TNF-α had increased significantly by week 1. These cytokines can all be induced by toll-like receptor 4 (TLR4) signaling. We defined osteonecrosis as the diffuse presence of empty lacunae or pyknotic nuclei of osteocytes in the bone trabeculae, accompanied by surrounding bone marrow cell necrosis. Osteonecrosis of the femoral head was observed only in the epiphysis of the femoral head in sacrificed specimen every week. Histological analysis revealed osteocytic death surrounded by necrotic bone marrow with or without repaired tissue. Conclusion. We established a new rat model of corticosteroid-induced femoral head osteonecrosis. The necrosis that is generated in this model is similar to that seen in patients treated with corticosteroid. In particular, the necrotic lesion was exclusively observed in the proximal epiphysis. LPS is known to activate the immune system via the TLR4 signaling pathway. It has been recognized that the unique immunogenic effects of LPS promote autoimmune disease . LPS and methylprednisolone induced osteonecrosis of the femoral head in rats and this was associated with a disruption of the innate immune system and lipid synthesis. These findings suggest that the TLR4 signaling pathway plays an important role in the pathogenesis for osteonecrosis of the femoral head

Blood transfusion, organ and bone marrow transplantation and allogeneic tissue grafting create the potential for significant immunological challenges through the introduction of non-genetically identical major (HLA) and minor histocompatibility antigens (“allo-antigens”) into the body. Strategies to avoid the complications of immune responses against allo-antigens (transfusion reactions, rejection and graft versus host disease) include HLA matching, immunosuppressive therapies and immune tolerance promoting protocols. In the case of allogeneic mesenchymal stem/stromal cells (allo-MSC), it was initially believed that their combined properties of low HLA expression and inherent immune modulatory functions would render them invisible to the host immune system and, therefore, capable of being permanently accepted without further interventions. For clinical indications such as bone and tendon repair, in which permanent engraftment of allo-MSC or MSC-derived tissue constructs is particularly desirable, this model of “immune privilege” seemed almost too good to be true – and indeed, a decade of experimental research in this area has now convincingly demonstrated that allo-MSC typically elicit cellular (T-cell) and humoral (B-cell/antibody) immune responses in immunocompetent hosts – raising concern about their safety and long-term efficacy in human conditions. However, questions related to the immunogenicity of allo-MSC have evolved beyond a simple yes/no scenario to involve interesting observations and concepts about the potency, diversity, duration, functional characteristics and even potential clinical benfits of immunological responses to allo-MSC. In this presentation, I will summarise and critically evaluate current understanding of allo-MSC immunogenicity under experimental and clinical trial conditions with an emphasis on the implications for orthopaedic therapeutics

Prosthetic Joint Infections (PJIs) are increasing with the use of orthopedic devices on an ageing population. Cutibacterium acnes is a commensal organism that plays an important role in the ecosystem healthy human skin, yet this species is also recognized as a pathogen in foreign body infection: endocarditis, prostatitis and specifically in PJIs. C. acnes is able to escape the immune system. This phenomenon could reflect two bacterial behaviour: the bacterial internalization by host cells and the biofilm formation. In this study, we studied different clinical strains of C. acnes. We noticed that C. acnes isolated from PJIs form 2 fold-more biofilm than the strains isolated from a normal skin in two models (Crystal violet staining and fluorescent microscopy (p=0.04 and p=0.02, respectively, Mann-Whitney test). We did not observe any difference in the internalization rate of those strains by osteoblasts. However, the quantity of biofilm formed by C. acnes before and after the internalization was compared. A significant increase in biofilm formation was observed for the strains isolated from the skin (x2.3±0.07; p=0.008, Mann-Whitney test). However, the hydrophobicity of the skin strains is significantly less important than for the PJIs strains (24.8±13% vs 56.6±12% respectively; p=0.003, Mann-Whitney test) but this did not change after internalization suggesting that there is no cell wall evolution. In conclusion, we studied for the first time the impact of bacterial internalization by osteoblasts on the virulent behaviour of C. acnes, which could explain the hided pathogenicity of this commensal bacterium

Recently, we could illustrate how tightly the bone and the immune system are interconnected during normal homeostasis but even stronger during bone regeneration. Specifically, the patient´s individual ratio of CD8+ effector T cells (TEFF, already identified as potential unfavorable cells for successful healing) to CD4+ regulatory T cells (TREG, one counterpart to CD8+ TEFF in controlling intratissue inflammation) prior to injury/ surgery appears to determine the healing outcome after fracture. We hypothesized that concentrating CD4+ TREG could serve as innovative therapeutic strategy to improve bone healing. We used an adoptive CD4+ TREG transfer in our well-established mouse osteotomy model. Before treatment, we identified the pre-surgery ratio of CD8+ TEFF/ CD4+ TREG by flow cytometry to characterize the healing potential of individual animals. Thereafter, we performed an adoptive CD4+ TREG transfer to reshape inflammation for supporting osteotomy healing. Across all groups, healing outcome was analyzed after 21 days post-surgery by µCT. Whereas TREG were highly supportive in SPF mice, we observed a heterogeneous clustered healing outcome in the non-SPF mice: TREG responder (improved healing outcome; p = 0.038) and TREG non-responder (impaired healing outcome; p = 0.024). Interestingly, the pre-/peri-surgery ratio of CD8+ TEFF/ CD4+ TREG was higher in the TREG non-responder (p=0.057). Thus, the amount of adoptively transferred CD4+ TREG was not sufficient to improve the healing outcome due to initial unfavorable high CD8+ TEFF/CD4+ TREG ratio. These results clearly show the importance of determining the individual immune status of each patient in the clinic before applying an immunotherapeutic approach

Chondrocytes are essential to the maintenance of articular cartilage and it is thought that chondrocyte death occurs early in septic arthritis. Understanding the causes of chondrocyte death will allow the development of chondroprotective strategies to improve long-term outcomes following septic arthritis. We utilised a murine model of septic arthritis using intra-articular injection of 10µL of a 107 concentration of S. aureus suspended in PBS. Seventy-five adult male C57/Bl6 mice were randomised to receive injection of either S. aurues 8325-4 (a wild-type of S. aurues capable of alpha toxin production), DU1090 (an isogenic mutant of 8325-4 that is identical to 8325-4 other than being incapable of producing alpha toxin) or a PBS control. Establishment of septic arthritis was confirmed through gait changes (5 mice/group), limb swelling and histological changes (10 mice/group). 10 animals from each group were sacrificed at 48 hours and the injected knee joints were dissected before being stained with CFMDA (labelling live chondrocytes green) and PI (labelling dead chondrocytes red). The samples were imaged using a confocal laser scanning microscope and the percentage of chondrocyte death was calculated. Mice injected with S. aureus 8325-4 or DU1090 developed septic arthritis with evidence of weight loss, limb swelling and gait changes whereas these were absent in the control group. There was a significantly higher level of chondrocyte death in the group infected with 8325-4 (2.7% chondrocyte viability) when compared to DU1090 (73.9% chondrocyte viability) and PBS injected mice (95% chondrocyte viability). One-Way ANOVA revealed that the difference between each group was statistically different (p < 0.05). Alpha toxin is the major damaging toxin in S. aurues septic arthritis. Any adverse effect of the immune system is negligible in comparison. Development of treatments counteracting the effect of alpha toxin is required

Bone grafts are crucial for the treatment of bone defects caused by tumor excision. The gold standard is autograft but their availability is limited. Allografts are an alternative, but there is a risk of rejection by the immune system. The tissue engineering field is trying to develop vascularized bone grafts, using innovative biomaterials for surgery applications. While the gold standard in bone graft in dentistry is the use of decellularized bovine bone particles (Bio-Oss®), our work has produced a polysaccharide-based composite matrix (composed of PUllulan, DextraNand particles of HydroxyApatite (PUDNHA), as a new scaffold for promoting bone formation and vascularization of the tissue. In the context of bone tissue regeneration, the function of osteoblast and endothelial cells has been extensively studied, while the impact of osteocytes has been regarded as secondary. Nonetheless, the osteocytes represent 90–95% of bone cells and are responsible for orchestration of bone remodeling. Here, we propose an original method to analyze the interaction between bone and biomaterials, after in vivo implantation of the matrix PUDNHA in an experimental sheep model. Our objectives are to analyze the network established by osteocytes in the newly formed tissue induced by the matrix, as well as their interactions with the blood vessels. Sheep have been implanted with the Bio-Oss® or the PUDNHA using the sinus lift technique. After 3 (3M) and 6 months (6M), the animals were euthanazied and the explants were fixed, analyzed by X-ray, embedded in Methylmetacrylate/Buthylmetacrylate and analyzed histologically by Trichrome staining. Thereafter, the samples (n=3/group) were polished using different sand papers. A final polish was realized using a 1µm Diamond polishing compound. The blocks were incubated 10 or 30s with 37% phosphoric acid to remove the mineral on the surface, then dipped in 2.6% sodium hypochlorite to remove the collagen. The samples were air dried overnight, metallized with Gold palladium the following day, before being imaged with a SEM. As expected, PUDNHA activates bone regeneration in this sinus lift model after 3M and 6M. X-ray analysis and histological data revealed more bone regeneration at 6M versus 3M in both groups. With this acid eching technique, we were able to visualize the interface of bone with the biomaterials. This treatment coupled with SEM analysis, confirmed the increase of bone formation with time of implantation in both groups. In addition, SEM images revealed that osteocyte alignment and their network were different in the new regenerated bone compared to the host bone. Moreover, images showed the direct contact of the osteocytes with the blood vessels formed in the new regenerated bone. This acid eching technique can be useful in the field of biomaterials to see the relationship between cells, blood vessels and the material implanted and understand how the new bone is forming around the different biomaterials

Alarmins- also referred to as damage associated molecular patterns (DAMPS)- are endogenous molecules mobilized in response to tissue damage known to activate the innate immune system and regulate tissue repair and remodelling. The molecular mechanisms that regulate inflammatory and remodelling pathways in tendinopathy are largely unknown therefore identifying early immune effectors is essential to understanding the pathology. S100A8 and S100A9 are low molecular weight calcium binding proteins primarily released by activated phagocytes in an inflammatory setting and also secreted as a heterodimeric complex that exhibits cytokine like functions. Based on our previous investigations we sought evidence of S100A8/A9 expression in human tendinopathy and thereafter, to explore mechanisms whereby S100 proteins may regulate inflammatory mediators and matrix regulation in human tenocytes. Torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from patients undergoing arthroscopic stabilisation surgery. S100A8/A9 expression was analysed at transcript and protein level using quantitative RT-PCR and immunohistochemistry, respectively. Primary human tenocytes were cultured from hamstring tendon tissue obtained during hamstring tendon ACL reconstruction. The in vitro effect of recombinant human S100 A8/A9 on primary human tenocytes was measured using quantitative RT-PCR and ELISA. Immunohistochemistry of tendinopathic tissues demonstrated the presence of S100 A8/A9 in diseased tissues compared to control tissue. In addition, early pathological diseased tissue indicated greater S100A9 expression compared with established diseased pathology. These findings were reflected by data obtained at transcript level from diseased tissues. Recombinant human S100A8, A9 and A8/A9 complex led to significant increase in expression of inflammatory mediators, including IL-6 in vitro. Further analysis via quantitative RT-PCR demonstrated recombinant S100A8, A9 and A8/A9 complex treatment on tenocytes, in vitro, had no direct effect on the expression of genes involved in matrix remodelling. The presence of S100A8 and S100A9 in early tendinopathic lesions suggests expression is upregulated in response to cellular damage. S100A8 and S100A9 are endogenous ligands of Toll-like receptors (TLRs) and receptor for advanced glycation end products (RAGE). These receptors have known regulatory effects on immune mediated cytokine production. We propose S100A8 and S100A9 as active alarmins in the early stages of tendinopathy and thus targeting of its downstream signalling may offer novel therapeutic approaches in the management of human tendon disorders

Mesenchymal stem cells (MSCs) are believed to be immune-privileged due to lack of antigen-presenting-cell related markers, however, evidence suggests that MSCs are immunogenic and are attacked by the immune system. Our research investigates the hypothesis that there are differences between MSC clones from the same individual in terms of their morphology, proliferation, differentiation and immune profile. Our goal is to discover immune-privileged stem cells, which can act as a universal allogenic mesenchymal stem cell donor to facilitate bone ingrowth for osteosarcoma patients status post tumor excision and prosthesis implantation. Serial dilutions of bone-marrow derived (BMMSCs) and adipose derived mesenchymal stem cells (ADMSCs) from same animal were carried out in order to isolate single-cell clones. From a single animal we obtained 3 clones from BMMSCs and 3 from ADMSCs. This procedure was repeated for another other 2 animals. The proliferation rate and cell doubling time of each clonal culture was measured. The proliferation rate of mixed clonal cultures was also measured. The tri-differentiation potential of the clonal cultures was compared and a comparison was also made with the original isolates from bone marrow and fat. The immune-privileged properties were measured by flow cytometry and immuno-staining for the major histocompatibility complex (MHC) antigens. To measure the immune response a mixed leucocyte reaction was used but where leucocytes from a different individual were mixed with the clonal MSC cells. All isolates were able to differentiate into osteoblasts, chondrocytes and adipocytes. All clonal cultures revealed significantly different proliferation rates and doubling times when compared with each other and with mixed cultures. All clonal cultures showed different surface marker presentations, which included differences in the expression of MHC antigens. One clone isolated from ADMSCs showed lack of MHCI and MHCII. Our mixed leucocyte reaction and MHC staining showed variety of immune-modulation and this was related to the expression of the MHC antigens. All clones tri-differentiated and therefore show a degree of ‘stemness’. MSCs are generally are believed not to express MHC II and to be immune-privileged. However, this study shows that the expression of these antigens in clones isolated from bone marrow and from fat is variable. A heterogeneous result indicates individual differences between MSCs, even from same origin. The immune response elicited by MSCs is complicated. MSCs have been shown to release interleukin 10, which could inhibit the immune response but on the other hand interferon-gamma could enhance MHCII presentation in some MSCs. Our results confirmed our hypothesis because clonal cultures isolated from different sources of MSCs in the same animal showed significant differences in proliferation rate, morphology and surface marker presentation. Mesenchymal stem cells are not immunogenic or immune-privileged. Individual differences highlighted through single-cell clonal cultures may be the key to finding a universal immune-privileged MSCs for allogeneic transplantation

Objectives

The molecular mechanism of rheumatoid arthritis (RA) remains elusive. We conducted a protein-protein interaction network-based integrative analysis of genome-wide association studies (GWAS) and gene expression profiles of RA.

Objectives

Osteoarthritis (OA) is the most common form of arthritis, affecting approximately 15% of the human population. Recently, increased concentration of nitric oxide in serum and synovial fluid in patients with OA has been observed. However, the exact role of nitric oxide in the initiation of OA has not been elucidated. The aim of the present study was to investigate the role of nitric oxide in innate immune regulation during OA initiation in rats.