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Advanced therapies are expected to play a crucial role in supporting repair after injury, halting the degeneration of musculoskeletal tissue to enable and promote physical activity. Despite advancements, the progress in developing advanced therapies in orthopaedics lags behind specialties like oncology, since innovative regenerative treatment strategies fall short of their expectations in musculoskeletal clinical trials. Researchers should focus on understanding the mechanism of action behind the investigated target before conducting clinical trials. Strategic research networks are needed that not only enhance scientific exchange among like-minded researchers but need to include early on commercial views, companies and venture perspectives, regulatory insights and reimbursement perspectives. Only in such collaborations essential roadblocks towards clinical trials and go-to-patients be overcome.

IntroductionOpen reduction and fixation are the standard of care for treating mandibular fractures and usually lead to successful healing. However, complications such as delayed healing, non-union, and infection can compromise patient outcomes and increase healthcare costs. The initial inflammatory response, particularly the response involving specific CD8+ T cell subpopulations, is thought to play a critical role in healing long bone fractures. In this study, we investigated the role of these immune cell profiles in patients with impaired healing of mandibular fractures.Materials and methodsIn this prospective study, we included patients with mandibular fractures surgically treated at Charité – Universitätsmedizin Berlin, Germany, between September 2020 and December 2022. We used follow-up imaging and clinical assessment to evaluate bone healing. In addition, we analyzed immune cell profiles using flow cytometry and quantified cytokine levels using electrochemiluminescence-based multiplex immunoassays in preoperative blood samples.ResultsOut of the 55 patients enrolled, 38 met the inclusion criteria (30 men and 8 women; mean age 32.18 years). Radiographic evaluation revealed 31 cases of normal healing and 7 cases of incomplete consolidation, including 1 case of non-union. Patients with impaired healing exhibited increased levels of terminally differentiated effector memory CD8+ T cells (TEMRA) and a higher TEMRA to regulatory T cell (Treg) ratio, compared with those with normal healing.ConclusionsOur analysis of mandibular fracture cases confirms our initial hypothesis derived from long bone fracture healing: monitoring the TEMRA to Treg ratio in preoperative blood can be an early indicator of patients at risk of impaired bone healing. Radiologic follow-up enabled us to detect healing complications that might not be detected by clinical assessment only. This study highlights the potential of individual immune profiles to predict successful healing and may form the basis for future strategies to manage healing complications.

Abstract Beyond SARS‐CoV2 vaccines, mRNA drugs are being explored to overcome today's greatest healthcare burdens, including cancer and cardiovascular disease. Synthetic mRNA triggers immune responses in transfected cells, which can be reduced by chemically modified nucleotides. However, the side effects of mRNA‐triggered immune activation on cell function and how different nucleotides, such as the N1‐methylpseudouridine (m1Ψ) used in SARS‐CoV2 vaccines, can modulate cellular responses is not fully understood. Here, cellular responses toward a library of uridine‐modified mRNAs are investigated in primary human cells. Targeted proteomics analyses reveal that unmodified mRNA induces a pro‐inflammatory paracrine pattern marked by the secretion of chemokines, which recruit T and B lymphocytes toward transfected cells. Importantly, the magnitude of mRNA‐induced changes in cell function varies quantitatively between unmodified, Ψ‐, m1Ψ‐, and 5moU‐modified mRNA and can be gradually tailored, with implications for deliberately exploiting this effect in mRNA drug design. Indeed, both the immunosuppressive effect of stromal cells on T‐cell proliferation, and the anti‐inflammatory effect of IL‐10 mRNA are enhanced by appropriate uridine modification. The results provide new insights into the effects of mRNA drugs on cell function and cell‐cell communication and open new possibilities to tailor mRNA‐triggered immune activation to the desired pro‐ or anti‐inflammatory application.

Abstract Skeletal muscle regeneration requires the coordinated interplay of diverse tissue-resident- and infiltrating cells. Fibro-adipogenic progenitors (FAPs) are an interstitial cell population that provides a beneficial microenvironment for muscle stem cells (MuSCs) during muscle regeneration. Here we show that the transcription factor Osr1 is essential for FAPs to communicate with MuSCs and infiltrating macrophages, thus coordinating muscle regeneration. Conditional inactivation of Osr1 impaired muscle regeneration with reduced myofiber growth and formation of excessive fibrotic tissue with reduced stiffness. Osr1-deficient FAPs acquired a fibrogenic identity with altered matrix secretion and cytokine expression resulting in impaired MuSC viability, expansion and differentiation. Immune cell profiling suggested a novel role for Osr1-FAPs in macrophage polarization. In vitro analysis suggested that increased TGFβ signaling and altered matrix deposition by Osr1-deficient FAPs actively suppressed regenerative myogenesis. In conclusion, we show that Osr1 is central to FAP function orchestrating key regenerative events such as inflammation, matrix secretion and myogenesis.

Abstract Heterotopic ossification is a disorder caused by abnormal mineralization of soft tissues in which signaling pathways such as BMP, TGFβ and WNT are known key players in driving ectopic bone formation. Identifying novel genes and pathways related to the mineralization process are important steps for future gene therapy in bone disorders. In this study, we detect an inter-chromosomal insertional duplication in a female proband disrupting a topologically associating domain and causing an ultra-rare progressive form of heterotopic ossification. This structural variant lead to enhancer hijacking and misexpression of ARHGAP36 in fibroblasts, validated here by orthogonal in vitro studies. In addition, ARHGAP36 overexpression inhibits TGFβ, and activates hedgehog signaling and genes/proteins related to extracellular matrix production. Our work on the genetic cause of this heterotopic ossification case has revealed that ARHGAP36 plays a role in bone formation and metabolism, outlining first details of this gene contributing to bone-formation and -disease.

Abstract Objective Platelet-rich plasma (PRP) therapy is increasingly popular to treat musculoskeletal diseases, including tendinopathies and osteoarthritis (OA). To date, it remains unclear to which extent PRP compositions are determined by the immune cell and cytokine profile of individuals or by the preparation method. To investigate this, we compared leukocyte and cytokine distributions of different PRP products to donor blood samples and assessed the effect of pro-inflammatory cytokines on chondrocytes. Design For each of three PRP preparations (ACP®, Angel™, and nSTRIDE® APS), products were derived using whole blood samples from twelve healthy donors. The cellular composition of PRP products was analyzed by flow cytometry using DURAClone antibody panels (DURAClone IM Phenotyping Basic and DURAClone IM T Cell Subsets). The MESO QuickPlex SQ 120 system was used to assess cytokine profiles (V-PLEX Proinflammatory Panel 1 Human Kit, Meso Scale Discovery). Primary human chondrocyte 2D and 3D in vitro cultures were exposed to recombinant IFN-γ and TNF-α. Proliferation and chondrogenic differentiation were quantitatively assessed. Results All three PRP products showed elevated portions of leukocytes compared to baseline levels in donor blood. Furthermore, the pro-inflammatory cytokines IFN-γ and TNF-α were significantly increased in nSTRIDE® APS samples compared to donor blood and other PRP products. The characteristics of all other cytokines and immune cells from the donor blood, including pro-inflammatory T cell subsets, were maintained in all PRP products. Chondrocyte proliferation was impaired by IFN-γ and enhanced by TNF-α treatment. Differentiation and cartilage formation were compromised upon treatment with both cytokines, resulting in altered messenger ribonucleic acid (mRNA) expression of collagen type 1A1 (COL1A1), COL2A1, and aggrecan (ACAN) as well as reduced proteoglycan content. Conclusions Individuals with elevated levels of cells with pro-inflammatory properties maintain these in the final PRP products. The concentration of pro-inflammatory cytokines strongly varies between PRP products. These observations may help to unravel the previously described heterogeneous response to PRP in OA therapy, especially as IFN-γ and TNF-α impacted primary chondrocyte proliferation and their characteristic gene expression profile. Both the individual’s immune profile and the concentration method appear to impact the final PRP product. Trial registration This study was prospectively registered in the Deutsches Register Klinischer Studien (DRKS) on 4 November 2021 (registration number DRKS00026175).

Abstract Particles released from cobalt-chromium-molybdenum (CoCrMo) alloys are considered common elicitors of chronic inflammatory adverse effects. There is a lack of data demonstrating particle numbers, size distribution and elemental composition of bone marrow resident particles which would allow for implementation of clinically relevant test strategies in bone marrow models at different degrees of exposure. The aim of this study was to investigate metal particle exposure in human periprosthetic bone marrow of three types of arthroplasty implants. Periprosthetic bone marrow sections from eight patients exposed to CoCrMo particles were analyzed via spatially resolved and synchrotron-based nanoscopic X-ray fluorescence imaging. These analyses revealed lognormal particle size distribution patterns predominantly towards the nanoscale. Analyses of particle numbers and normalization to bone marrow volume and bone marrow cell number indicated particle concentrations of up to 1 × 1011 particles/ml bone marrow or 2 × 104 particles/bone marrow cell, respectively. Analyses of elemental ratios of CoCrMo particles showed that particularly the particles’ Co content depends on particle size. The obtained data point towards Co release from arthroprosthetic particles in the course of dealloying and degradation processes of larger particles within periprosthetic bone marrow. This is the first study providing data based on metal particle analyses to be used for future in vitro and in vivo studies of possible toxic effects in human bone marrow following exposure to arthroprosthetic CoCrMo particles of different concentration, size, and elemental composition. Graphical abstract

Aims The aim of the HIPGEN consortium is to develop the first cell therapy product for hip fracture patients using PLacental-eXpanded (PLX-PAD) stromal cells. Methods HIPGEN is a multicentre, multinational, randomized, double-blind, placebo-controlled trial. A total of 240 patients aged 60 to 90 years with low-energy femoral neck fractures (FNF) will be allocated to two arms and receive an intramuscular injection of either 150 × 106 PLX-PAD cells or placebo into the medial gluteal muscle after direct lateral implantation of total or hemi hip arthroplasty. Patients will be followed for two years. The primary endpoint is the Short Physical Performance Battery (SPPB) at week 26. Secondary and exploratory endpoints include morphological parameters (lean body mass), functional parameters (abduction and handgrip strength, symmetry in gait, weightbearing), all-cause mortality rate and patient-reported outcome measures (Lower Limb Measure, EuroQol five-dimension questionnaire). Immunological biomarker and in vitro studies will be performed to analyze the PLX-PAD mechanism of action. A sample size of 240 subjects was calculated providing 88% power for the detection of a 1 SPPB point treatment effect for a two-sided test with an α level of 5%. Conclusion The HIPGEN study assesses the efficacy, safety, and tolerability of intramuscular PLX-PAD administration for the treatment of muscle injury following arthroplasty for hip fracture. It is the first phase III study to investigate the effect of an allogeneic cell therapy on improved mobilization after hip fracture, an aspect which is in sore need of addressing for the improvement in standard of care treatment for patients with FNF. Cite this article: Bone Jt Open 2022;3(4):340–347.

Patients suffering from musculoskeletal diseases must cope with a diminished quality of life and an increased burden on medical expenses. The interaction of immune cells and mesenchymal stromal cells during bone regeneration is one of the key requirements for the restoration of skeletal integrity. While stromal cells of the osteo-chondral lineage support bone regeneration, an excessive accumulation of cells of the adipogenic lineage is thought to promote low-grade inflammation and impair bone regeneration. Increasing evidence indicates that pro-inflammatory signaling from adipocytes is responsible for various chronic musculoskeletal diseases. This review aims to summarize the features of bone marrow adipocytes by phenotype, function, secretory features, metabolic properties and their impact on bone formation. In detail, the master regulator of adipogenesis and prominent diabetes drug target, peroxisome proliferator-activated receptor γ (PPARG), will be debated as a potential therapeutic approach to enhance bone regeneration. We will explore the possibilities of using clinically established PPARG agonists, the thiazolidinediones (TZDs), as a treatment strategy to guide the induction of a pro-regenerative, metabolically active bone marrow adipose tissue. The impact of this PPARG induced bone marrow adipose tissue type on providing the necessary metabolites to sustain osteogenic-as well as beneficial immune cells during bone fracture healing will be highlighted.

Abstract Diseases that jeopardize the musculoskeletal system and cause chronic impairment are prevalent throughout the Western world. In Germany alone, ~1.8 million patients suffer from these diseases annually, and medical expenses have been reported to reach 34.2bn Euros. Although musculoskeletal disorders are seldom fatal, they compromise quality of life and diminish functional capacity. For example, musculoskeletal disorders incur an annual loss of over 0.8 million workforce years to the German economy. Among these diseases, traumatic skeletal muscle injuries are especially problematic because they can occur owing to a variety of causes and are very challenging to treat. In contrast to chronic muscle diseases such as dystrophy, sarcopenia, or cachexia, traumatic muscle injuries inflict damage to localized muscle groups. Although minor muscle trauma heals without severe consequences, no reliable clinical strategy exists to prevent excessive fibrosis or fatty degeneration, both of which occur after severe traumatic injury and contribute to muscle degeneration and dysfunction. Of the many proposed strategies, cell‐based approaches have shown the most promising results in numerous pre‐clinical studies and have demonstrated success in the handful of clinical trials performed so far. A number of myogenic and non‐myogenic cell types benefit muscle healing, either by directly participating in new tissue formation or by stimulating the endogenous processes of muscle repair. These cell types operate via distinct modes of action, and they demonstrate varying levels of feasibility for muscle regeneration depending, to an extent, on the muscle injury model used. While in some models the injury naturally resolves over time, other models have been developed to recapitulate the peculiarities of real‐life injuries and therefore mimic the structural and functional impairment observed in humans. Existing limitations of cell therapy approaches include issues related to autologous harvesting, expansion and sorting protocols, optimal dosage, and viability after transplantation. Several clinical trials have been performed to treat skeletal muscle injuries using myogenic progenitor cells or multipotent stromal cells, with promising outcomes. Recent improvements in our understanding of cell behaviour and the mechanistic basis for their modes of action have led to a new paradigm in cell therapies where physical, chemical, and signalling cues presented through biomaterials can instruct cells and enhance their regenerative capacity. Altogether, these studies and experiences provide a positive outlook on future opportunities towards innovative cell‐based solutions for treating traumatic muscle injuries—a so far unmet clinical need.

Abstract Metallic implants are frequently used in medicine to support and replace degenerated tissues. Implant loosening due to particle exposure remains a major cause for revision arthroplasty. The exact role of metal debris in sterile peri‐implant inflammation is controversial, as it remains unclear whether and how metals chemically alter and potentially accumulate behind an insulating peri‐implant membrane, in the adjacent bone and bone marrow (BM). An intensively focused and bright synchrotron X‐ray beam allows for spatially resolving the multi‐elemental composition of peri‐implant tissues from patients undergoing revision surgery. In peri‐implant BM, particulate cobalt (Co) is exclusively co‐localized with chromium (Cr), non‐particulate Cr accumulates in the BM matrix. Particles consisting of Co and Cr contain less Co than bulk alloy, which indicates a pronounced dissolution capacity. Particulate titanium (Ti) is abundant in the BM and analyzed Ti nanoparticles predominantly consist of titanium dioxide in the anatase crystal phase. Co and Cr but not Ti integrate into peri‐implant bone trabeculae. The characteristic of Cr to accumulate in the intertrabecular matrix and trabecular bone is reproducible in a human 3D in vitro model. This study illustrates the importance of updating the view on long‐term consequences of biomaterial usage and reveals toxicokinetics within highly sensitive organs.

Numerous clinical trials of mesenchymal stromal/stem cells (MSCs) as a new treatment for coronavirus-induced disease (COVID-19) have been registered recently, most of them based on intravenous (IV) infusion. There is no approved effective therapy for COVID-19, but MSC therapies have shown first promise in the treatment of acute respiratory distress syndrome (ARDS) pneumonia, inflammation, and sepsis, which are among the leading causes of mortality in COVID-19 patients. Many of the critically ill COVID-19 patients are in a hypercoagulable procoagulant state and at high risk for disseminated intravascular coagulation, thromboembolism, and thrombotic multi-organ failure, another cause of high fatality. It is not yet clear whether IV infusion is a safe and effective route of MSC delivery in COVID-19, since MSC-based products express variable levels of highly procoagulant tissue factor (TF/CD142), compromising the cells' hemocompatibility and safety profile. Of concern, IV infusions of poorly characterized MSC products with unchecked (high) TF/CD142 expression could trigger blood clotting in COVID-19 and other vulnerable patient populations and further promote the risk for thromboembolism. In contrast, well-characterized products with robust manufacturing procedures and optimized modes of clinical delivery hold great promise for ameliorating COVID-19 by exerting their beneficial immunomodulatory effects, inducing tissue repair and organ protection. While the need for MSC therapy in COVID-19 is apparent, integrating both innate and adaptive immune compatibility testing into the current guidelines for cell, tissue, and organ transplantation is critical for safe and effective therapies. It is paramount to only use well-characterized, safe MSCs even in the most urgent and experimental treatments. We here propose three steps to mitigate the risk for these vulnerable patients: (1) updated clinical guidelines for cell and tissue transplantation, (2) updated minimal criteria for characterization of cellular therapeutics, and (3) updated cell therapy routines reflecting specific patient needs.

Donor variation is a prominent critical issue limiting the applicability of cell-based therapies. We hypothesized that batch effects during propagation of bone marrow stromal cells (BMSCs) in human platelet lysate (hPL), replacing fetal bovine serum (FBS), can affect phenotypic and functional variability. We therefore investigated the impact of donor variation, hPL- vs. FBS-driven propagation and exhaustive proliferation, on BMSC epigenome, transcriptome, phenotype, coagulation risk and osteochondral regenerative function. Notably, propagation in hPL significantly increased BMSC proliferation, created significantly different gene expression trajectories and distinct surface marker signatures, already after just one passage. We confirmed significantly declining proliferative potential in FBS-expanded BMSC after proliferative challenge. Flow cytometry verified the canonical fibroblastic phenotype in culture-expanded BMSCs. We observed limited effects on DNA methylation, preferentially in FBS-driven cultures, irrespective of culture duration. The clotting risk increased over culture time. Moreover, expansion in xenogenic serum resulted in significant loss of function during 3D cartilage disk formation and significantly increased clotting risk. Superior chondrogenic function under hPL-conditions was maintained over culture. The platelet blood group and isoagglutinins had minor impact on BMSC function. These data demonstrate pronounced batch effects on BMSC transcriptome, phenotype and function due to serum factors, partly outcompeting donor variation after just one culture passage.

Abstract Background No regenerative approach has thus far been shown to be effective in skeletal muscle injuries, despite their high frequency and associated functional deficits. We sought to address surgical trauma‐related muscle injuries using local intraoperative application of allogeneic placenta‐derived, mesenchymal‐like adherent cells (PLX‐PAD), using hip arthroplasty as a standardized injury model, because of the high regenerative and immunomodulatory potency of this cell type. Methods Our pilot phase I/IIa study was prospective, randomized, double blind, and placebo‐controlled. Twenty patients undergoing hip arthroplasty via a direct lateral approach received an injection of 3.0 × 108 (300 M, n = 6) or 1.5 × 108 (150 M, n = 7) PLX‐PAD or a placebo (n = 7) into the injured gluteus medius muscles. Results We did not observe any relevant PLX‐PAD‐related adverse events at the 2‐year follow‐up. Improved gluteus medius strength was noted as early as Week 6 in the treatment‐groups. Surprisingly, until Week 26, the low‐dose group outperformed the high‐dose group and reached significantly improved strength compared with placebo [150 M vs. placebo: P = 0.007 (baseline adjusted; 95% confidence interval 7.6, 43.9); preoperative baseline values mean ± SE: placebo: 24.4 ± 6.7 Nm, 150 M: 27.3 ± 5.6 Nm], mirrored by an increase in muscle volume [150 M vs. placebo: P = 0.004 (baseline adjusted; 95% confidence interval 6.0, 30.0); preoperative baseline values GM volume: placebo: 211.9 ± 15.3 cm3, 150 M: 237.4 ± 27.2 cm3]. Histology indicated accelerated healing after cell therapy. Biomarker studies revealed that low‐dose treatment reduced the surgery‐related immunological stress reaction more than high‐dose treatment (exemplarily: CD16+ NK cells: Day 1 P = 0.06 vs. placebo, P = 0.07 vs. 150 M; CD4+ T‐cells: Day 1 P = 0.04 vs. placebo, P = 0.08 vs. 150 M). Signs of late‐onset immune reactivity after high‐dose treatment corresponded to reduced functional improvement. Conclusions Allogeneic PLX‐PAD therapy improved strength and volume of injured skeletal muscle with a reasonable safety profile. Outcomes could be positively correlated with the modulation of early postoperative stress‐related immunological reactions.

Heterogeneous populations of human bone marrow-derived stromal cells (BMSC) are among the most frequently tested cellular therapeutics for treating degenerative and immune disorders, which occur predominantly in the aging population. Currently, it is unclear whether advanced donor age and commonly associated comorbidities affect the properties of ex vivo-expanded BMSCs. Thus, we stratified cells from adult and elderly donors from our biobank (n = 10 and n = 13, mean age 38 and 72 years, respectively) and compared their phenotypic and functional performance, using multiple assays typically employed as minimal criteria for defining multipotent mesenchymal stromal cells (MSCs). We found that BMSCs from both cohorts meet the standard criteria for MSC, exhibiting similar morphology, growth kinetics, gene expression profiles, and pro-angiogenic and immunosuppressive potential and the capacity to differentiate toward adipogenic, chondrogenic, and osteogenic lineages. We found no substantial differences between cells from the adult and elderly cohorts. As positive controls, we studied the impact of in vitro aging and inflammatory cytokine stimulation. Both conditions clearly affected the cellular properties, independent of donor age. We conclude that in vitro aging rather than in vivo donor aging influences BMSC characteristics.

Delayed type hypersensitivity (DTH) reactions are considered infrequent complications in arthroplasty, but have been recognized to be associated with devastating morbidity and substantial decrease in quality of life of affected patients. Chronic inflammation of artificial joints and associated loss of peri-implant bone often require revision surgery. Methods for the diagnosis of implant-related DTH are available but infrequently considered to the full extent. Sequential diagnostics based on exclusion of septic complications, local and systemic metal level determination, lymphocyte transformation testing (LTT), and local T cell subset analysis are required for an unequivocal DTH diagnosis. Here, we report on a patient with a history of chronic rheumatoid arthritis and an unfavorable outcome of unilateral knee arthroplasty. This case illustrates pitfalls and difficulties in the course of recurrent inflammation following joint replacement. In the early course, suspicion of low-grade bacterial infection led to three two-stage revisions. Afterwards, the joint was proven to be sterile. However, metal level quantification revealed release of especially cobalt and chromium from the joint, LTT indicated persisting cobalt and nickel sensitization and subset analysis of T cells from the synovium suggested DTH as a root cause for the inflammatory symptoms. This report aims to recommend the depicted diagnostic algorithm as an adequate tool for future DTH detection. Yet, systemic to local subset ratios for effector memory and regulatory T cells should be derived from sufficient patient numbers to establish it as a diagnostic marker. Moreover, future prospects regarding implant-related DTH diagnostics are discussed. Therapeutic options for the portrayed patient are proposed, considering pharmaceutical, cell-therapeutic and surgical aspects. Patients who experience peri-implant inflammation but do not have obvious mechanical or infectious problems remain a diagnostic challenge and are at high risk of being treated inadequately. Since potentially sensitizing materials are regularly used in arthroplasty, it is essential to detect cases of acute DTH-derived inflammation of an artificial joint at early postoperative stages. This would reduce the severity of inflammation-related long-term consequences for affected patients and may avoid unnecessary revision surgery.

Placenta-derived decidua stromal cells (DSCs) are being investigated as an alternative to other sources of mesenchymal stromal cells (MSCs) for cellular therapy. DSCs are more effective in treating acute inflammatory diseases in human and this is our preclinical safety study of human DSCs in Sprague-Dawley rats and Balb/c mice. Human DSCs were cultured and expanded from fetal membranes obtained from placentas following cesarean section. In rats, 0.5 × 106 cells/kg were injected intravenously (n = 4) or intra-aortal (n = 4). In mice, DSCs were given intravenously at doses ranging from 4–40 × 106 cells/kg (total of n = 120 mice). In vivo tracking of human cells in mice was performed by using transduced DSC with luciferin gene, and in rats by using 18F-FDG PET. Clotting parameters were determined in vitro and in vivo. All intra-arterially DSC-treated rats had normal motility and behavior and histological examination was normal for liver, spleen kidneys and thigh muscles. Mice treated with DSCs showed no immediate or long-term side effects. None of the mice died or showed acute toxicity or adverse reactions 3 and 30 days after DSC infusion. Murine blood biochemistry profiles related to liver, kidney, heart, and inflammatory indices was not influenced by DSC infusion and complete blood counts were normal. In vivo tracking of infused DSCs detected a signal in the lungs for up to 4 days post infusion. Compared to bone marrow derived MSCs, the DSCs had better viability, smaller size, but stronger clotting in human blood and plasma. Both MSC- and DSC-induced coagulation and complement activation markers, thrombin-anti-thrombin complex (TAT) and C3a, and in vitro clotting parameters were decreased by heparin supplementation. In conclusion, DSCs are safe with almost no side effects even with doses 40 times higher than are used clinically, particularly when supplemented with low-dose heparin.

Arthroplasty ranks among the greatest achievements of surgical medicine, with total hip replacement termed “the operation of the century.” Despite its wide success, arthroplasty bears risks, such as local reactions to implant derived wear and corrosion products. Prevalence of allergies across Western society increases and along the number of reported hypersensitivity reactions to orthopedic implant materials. In this context the main focus is on delayed hypersensitivity (DTH). This mechanism is mainly attributed to T cells and an overreaction of the adaptive immune system. Arthroplasty implant materials are in direct contact with bone marrow (BM), which is discussed as a secondary lymphoid organ. However, the mechanisms of sensitization toward implant wear remain elusive. Nickel and cobalt ions can form haptens with native peptides to activate immune cell receptors and are therefore common T helper allergens in cutaneous DTH. The rising prevalence of metal-related allergy in the general population and evidence for the immune-modulating function of BM allow for the assumption hypersensitivity reactions could occur in peri-implant BM. There is evidence that pro-inflammatory factors released during DTH reactions enhance osteoclast activity and inhibit osteoblast function, an imbalance characteristic for osteolysis. Even though some mechanisms are understood, hypersensitivity has remained a diagnosis of exclusion. This review aims to summarize current views on the pathomechanism of DTH in arthroplasty with emphasis on BM and discusses recent advances and future directions for basic research and clinical diagnostics.

There is increasing evidence that T lymphocytes play a key role in controlling endogenous regeneration. Regeneration appears to be impaired in case of local accumulation of CD8+ effector T cells (TEFF), impairing endogenous regeneration by increasing a primary “useful” inflammation toward a damaging level. Thus, rescuing regeneration by regulating the heightened pro-inflammatory reaction employing regulatory CD4+ T (TReg) cells could represent an immunomodulatory option to enhance healing. Hypothesis was that CD4+ TReg might counteract undesired effects of CD8+ TEFF. Using adoptive TReg transfer, bone healing was consistently improved in mice possessing an inexperienced immune system with low amounts of CD8+ TEFF. In contrast, mice with an experienced immune system (high amounts of CD8+ TEFF) showed heterogeneous bone repair with regeneration being dependent upon the individual TEFF/TReg ratio. Thus, the healing outcome can only be improved by an adoptive TReg therapy, if an unfavorable TEFF/TReg ratio can be reshaped; if the individual CD8+ TEFF percentage, which is dependent on the individual immune experience can be changed toward a favorable ratio by the TReg transfer. Remarkably, also in patients with impaired fracture healing the TEFF/TReg ratio was higher compared to uneventful healers, validating our finding in the mouse osteotomy model. Our data demonstrate for the first time the key-role of a balanced TEFF/TReg response following injury needed to reach successful regeneration using bone as a model system. Considering this strategy, novel opportunities for immunotherapy in patients, which are at risk for impaired healing by targeting TEFF cells and supporting TReg cells to enhance healing are possible.

An induced pluripotent stem cell line was generated from primary human bone marrow derived mesenchymal stromal cells of a 74 year old donor using retroviruses harboring OCT4, SOX2, KLF4 and c-MYC in combination with the following inhibitors TGFβ receptor-SB 431542, MEK-PD325901, and p53-Pifithrin α. Pluripotency was confirmed both in vitro and in vivo.

Gfi1 is a key molecule in hematopoietic lineage development and mutations in GFI1 cause severe congenital neutropenia (SCN). Neutropenia is associated with low bone mass, but the underlying mechanisms are poorly characterized. Using Gfi1 knock-out mice (Gfi1-ko/ko) as SCN model, we studied the relationship between neutropenia and bone mass upon different pathogen load conditions. Our analysis reveals that Gfi1-ko/ko mice kept under strict specific pathogen free (SPF) conditions demonstrate normal bone mass and survival. However, Gfi1-ko/ko mice with early (nonSPF) or late (SPF+nonSPF) pathogen exposure develop low bone mass. Gfi1-ko/ko mice demonstrate a striking rise of systemic inflammatory markers according to elevated pathogen exposure and reduced bone mass. Elevated inflammatory cytokines include for instance Il-1b, Il-6, and Tnf-alpha that regulate osteoclast development. We conclude that low bone mass, due to low neutrophil counts, is caused by the degree of systemic inflammation promoting osteoclastogenesis.

The amniotic epithelium consists of cells exhibiting mature epitelial cell characteristics, but also varying degrees of stemness. We tested the hypothesis that induction of epithelial-to-mesenchymal transition (EMT) in amniotic epitelial cells (AECs) derived from human placenta enhances their capacity to support the ischemic myocardium. In response to incubation with transforming growth factor-β1 (TGF-β1) protein, AECs lost their cobblestone morphology and acquired a fibroblastoid shape, associated with downregulation of E-cadherin, upregulation of N-cadherin, Akt phosphorylation, and intracellular periostin translocation. EMT—AECs displayed greatly enhanced mobility and secreted gelatinase activity compared with naive AECs. The surface presentation of CD105 and CD73 decreased, and RNA microarray analysis mirrored the loss of epithelial characteristics and transcriptional profile. Unmodified AECs and EMT—AECs were then injected intramyocardially in fully immunocompetent mice after permanent LAD ligation, and heart function was followed by MRI as well as 2D speckle tracking echocardiography after 4 weeks. EMT—AEC-treated infarct hearts displayed better global systolic function and improved longitudinal strain rate in the area of interest. Although no signals of human cells were detectable by histology, infarct size was smaller in EMT—AEC-treated hearts, associated with fewer TUNEL-positive cells and upregulation of periostin, while blood vessel density was increased in both ACE- and EMT—AEC-treated hearts. We conclude that EMT enhances the cardioprotective effects of human AECs.

Among other stressors, age and mechanical constraints significantly influence regeneration cascades in bone healing. Here, our aim was to identify genes and, through their functional annotation, related biological processes that are influenced by an interaction between the effects of mechanical fixation stability and age. Therefore, at day three post-osteotomy, chip-based whole-genome gene expression analyses of fracture hematoma tissue were performed for four groups of Sprague-Dawley rats with a 1.5-mm osteotomy gap in the femora with varying age (12 vs. 52 weeks - biologically challenging) and external fixator stiffness (mechanically challenging). From 31099 analysed genes, 1103 genes were differentially expressed between the six possible combinations of the four groups and from those 144 genes were identified as statistically significantly influenced by the interaction between age and fixation stability. Functional annotation of these differentially expressed genes revealed an association with extracellular space, cell migration or vasculature development. The chip-based whole-genome gene expression data was validated by q-RT-PCR at days three and seven post-osteotomy for MMP-9 and MMP-13, members of the mechanosensitive matrix metalloproteinase family and key players in cell migration and angiogenesis. Furthermore, we observed an interaction of age and mechanical stimuli in vitro on cell migration of mesenchymal stromal cells. These cells are a subpopulation of the fracture hematoma and are known to be key players in bone regeneration. In summary, these data correspond to and might explain our previously described biomechanical healing outcome after six weeks in response to fixation stiffness variation. In conclusion, our data highlight the importance of analysing the influence of risk factors of fracture healing (e.g. advanced age, suboptimal fixator stability) in combination rather than alone.

Mesenchymal stromal cells (MSCs) are of high relevance for the regeneration of mesenchymal tissues such as bone and cartilage. The promising role of MSCs in cell-based therapies and tissue engineering appears to be limited due to a decline of their regenerative potential with increasing donor age, their limited availability in human tissues and the need of in vitro expansion prior to treatment. We therefore aimed to determine to which degree in vitro aging and chronological aging may be similar processes or if in vitro culture-related changes at the cellular and molecular level are at least altered as a function of donor age. For that purpose we established MSCs cultures from young (yMSCs) and aged (aMSCs) rats that were cultured for more than 100 passages. These long-term MSCs cultures were non-tumorigenic and exhibited similar surface marker patterns as primary MSCs of passage 2. During in vitro expansion, but not during chronological aging, MSCs progressively lose their progenitor characteristics, e.g., complete loss of osteogenic differentiation potential, diminished adipogenic differentiation, altered cell morphology and increased susceptibility towards senescence. Transcriptome analysis revealed that long-term in vitro MSCs cultivation leads to down-regulation of genes involved in cell differentiation, focal adhesion organization, cytoskeleton turnover and mitochondria function. Accordingly, functional analysis demonstrated altered mitochondrial morphology, decreased antioxidant capacities and elevated ROS levels in long-term cultivated yMSCs as well as aMSCs. Notably, only the MSC migration potential and their antioxidative capacity were altered by in vitro as well as chronological aging. Based on specific differences observed between the impact of chronological and in vitro MSC aging we conclude that both are distinct processes.