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A misdirected or imbalanced local immune composition is often one of the reasons for unsuccessful regeneration resulting in scarring or fibrosis. Successful healing requires a balanced initiation and a timely down-regulation of the inflammation for the re-establishment of a biologically and mechanically homeostasis. While biomaterial-based approaches to control local immune responses are emerging as potential new treatment options, the extent to which biophysical material properties themselves play a role in modulating a local immune niche response has so far been considered only occasionally. The communication loop between extracellular matrix, non-hematopoietic cells, and immune cells seems to be specifically sensitive to mechanical cues and appears to play a role in the initiation and promotion of a local inflammatory setting. In this review, we focus on the crosstalk between ECM and its mechanical triggers and how they impact immune cells and non-hematopoietic cells and their crosstalk during tissue regeneration. We realized that especially mechanosensitive receptors such as TRPV4 and PIEZO1 and the mechanosensitive transcription factor YAP/TAZ are essential to regeneration in various organ settings. This indicates novel opportunities for therapeutic approaches to improve tissue regeneration, based on the immune-mechanical principles found in bone but also lung, heart, and skin.

The mechano-response of highly loaded tissues such as bones or tendons is well investigated, but knowledge regarding the mechano-responsiveness of adjacent tissues such as the subacromial bursa is missing. For a better understanding of the physiological role of the bursa as a friction-reducing structure in the joint, the study aimed to analyze whether and how bursa-derived cells respond to physiological and pathological mechanical loading. This might help to overcome some of the controversies in the field regarding the role of the bursa in the development and healing of shoulder pathologies. Cells of six donors seeded on collagen-coated silicon dishes were stimulated over 3 days for 1 or 4 h with 1, 5, or 10% strain. Orientation of the actin cytoskeleton, YAP nuclear translocation, and activation of non-muscle myosin II (NMM-II) were evaluated for 4 h stimulations to get a deeper insight into mechano-transduction processes. To investigate the potential of bursa-derived cells to adapt their matrix formation and remodeling according to mechanical loading, outcome measures included cell viability, gene expression of extracellular matrix and remodeling markers, and protein secretions. The orientation angle of the actin cytoskeleton increased toward a more perpendicular direction with increased loading and lowest variations for the 5% loading group. With 10% tension load, cells were visibly stressed, indicated by loss in actin density and slightly reduced cell viability. A significantly increased YAP nuclear translocation occurred for the 1% loading group with a similar trend for the 5% group. NMM-II activation was weak for all stimulation conditions. On the gene expression level, only the expression of TIMP2 was down-regulated in the 1 h group compared to control. On the protein level, collagen type I and MMP2 increased with higher/longer straining, respectively, whereas TIMP1 secretion was reduced, resulting in an MMP/TIMP imbalance. In conclusion, this study documents for the first time a clear mechano-responsiveness in bursa-derived cells with activation of mechano-transduction pathways and thus hint to a physiological function of mechanical loading in bursa-derived cells. This study represents the basis for further investigations, which might lead to improved treatment options of subacromial bursa-related pathologies in the future.

Musculoskeletal conditions are the leading cause of pain, suffering and disability in workplaces and have a huge financial burden. Within this study, age-dependent changes in the immune and skeletal system were investigated in parallel to evaluate the impact of aged immunity on bone structures and quality. In patients with delayed fracture healing a subtype of T cells are at higher abundance compared to normal healing patients. This raises the question of whether the experience in the adaptive immunity directly influences bone structure and formation. The skeletal and immune systems were analyzed in mice aged from 3 months to 24 months. The mice were aged in specific pathogen-free environment thus keeping a relative naïve immunity or exposed to environmental microorganisms allowing the immune system to establish memory. The immune status of the mice was examined via flow cytometry, which was then correlated to their bone structure via microCT and bone competence via biomechanical testing. Bone regeneration was analyzed in vivo in a mouse osteotomy model, and healing outcome was evaluated after 3 and 21 days. Protein analysis was done to unravel the diverging cytokine patterns after fracture. In addition, MSCs from these mice were analyzed for their differentiation potential and ECM production in the presence and absence of immune cell signaling ex vivo. Furthermore, several immunomodulatory interventions were tested in order to improve bone fracture healing under the influence of an aged/experienced adaptive immunity. Age-associated alterations in the immune profile and bone tissue could be itemized between chronological and biological aging. The memory/effector compartment of adaptive immunity was significantly increased in mice that were exposed to environmental microorganisms. The immune experience led to a significantly different bone phenotype both in structure and in competence to withstand loads. The in vivo bone formation was highly affected by age but also by the immune status. Bone tissue formation during healing was delayed in the experienced adaptive immunity group and significant changes in cytokine levels were observed 3 days post-surgery. Furthermore, the tested interventions of regulatory T cells transfer or inhibition of immune cell activation as immunomodulatory approaches laid the foundation of future treatment options. Adaptive immunity directly affects bone tissue formation and tissue remodeling leading to structural differences in bone material organization as well as mechanical competence. Such knowledge is essential for the characterization of healing settings and lays the foundation for novel diagnostics and therapeutics that aim to understand and rescue delayed bone regeneration in immunologically challenging patients. This data is the first to show that a patient’s immune experience needs to be taken into account, in the context of diagnostics as well as in therapy., Erkrankungen des Bewegungsapparates gehören zu den häufigsten Ursachen von Schmerzen, Immobilität und Einschränkungen am Arbeitsplatz. Im Rahmen dieser Studie wurden altersabhängige Veränderungen des Immun- und Skelettsystems untersucht, um den Einfluss der gealterten Immunität auf den Knochen zu bewerten. Bei Patienten mit verzögerter Frakturheilung ist die Häufigkeit eines Subtyps von T Zellen im Vergleich zu normaler Heilung erhöht. Es stellt sich die Frage, ob die Erfahrung in der adaptiven Immunität einen direkten Einfluss auf den Knochen hat. Das Skelett- und das Immunsystem wurden bei Mäusen im Alter von 3 bis 24 Monaten analysiert. Die Mäuse wurden in einer SPF Umgebung gealtert, wodurch eine relativ naive Immunität aufrechterhalten wurde, oder sie wurden Umweltmikroorganismen exponiert, wodurch das Immunsystem eine Erfahrung aufbauen konnte. Die Mäuse wurden auf ihren Immunstatus untersucht, welcher dann mit ihrer Knochenstruktur und -kompetenz korreliert wurde. Die Regeneration wurde in einem Osteotomiemodell analysiert und der Heilungsverlauf nach 3 und 21 Tagen bewertet. Eine Proteinanalyse wurde durchgeführt, um die Zytokinmuster zu entschlüsseln. Zusätzlich wurden MSCs hinsichtlich ihres Differenzierungspotentials und ihrer ECM-Produktion unter Immunzellsignalen analysiert. Darüber hinaus wurden mehrere immunmodulatorische Interventionen getestet, um die beeinträchtigte Heilung unter dem Einfluss einer gealterten/erfahrenen adaptiven Immunität zu verbessern. Eine chronologische und biologische Alterung konnte über eine Trennung der immunologischen und knochengewebsspezifischen Alterung unterschieden werden. Das Gedächtnis-Kompartiment der adaptiven Immunität war bei Mäusen unter exponierter Haltung signifikant erhöht. Diese Immunerfahrung führte zu einem signifikant unterschiedlichen Knochenphänotyp in Bezug auf Struktur und Belastbarkeit. Der Verlauf der Knochenregeneration war vom Alter und vom Immunstatus abhängig. Die Heilung war in der Gruppe mit erfahrener adaptiver Immunität beeinträchtigt, und 3 Tage nach der Osteotomie wurden signifikant veränderte Zytokinspiegel beobachtet. Darüber hinaus bildeten die getesteten Interventionen des adaptiven Transfers von regulatorischen T Zellen oder der Aktivierungshemmung von Immunzellen als Immunmodulation den Grundstein für zukünftige Behandlungsoptionen. Die adaptive Immunität wirkt sich direkt auf die Knochenbildung und den Gewebeumbau aus und führt zu strukturellen Unterschieden in der Knochenmaterialorganisation sowie in der mechanischen Kompetenz. Dieses Wissen ist für die Charakterisierung des Heilungsverlaufs von wesentlicher Bedeutung und bildet die Grundlage für neuartige Diagnostika und Therapeutika, die darauf abzielen, die beeinträchtigte Knochenregeneration bei immunologisch anspruchsvollen Patienten zu verstehen und zu behandeln. Diese Daten zeigen erstmals, dass die Immunerfahrung sowohl in der Diagnostik als auch in der Therapie berücksichtigt werden sollte.

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.

Bone formation as well as bone healing capacity is known to be impaired in the elderly. Although bone formation is outpaced by bone resorption in aged individuals, we hereby present a novel path that considerably impacts bone formation and architecture: Bone formation is substantially reduced in aged individual owing to the experience of the adaptive immunity. Thus, immune-aging in addition to chronological aging is a potential risk factor, with an experienced immune system being recognized as more pro-inflammatory. The role of the aging immune system on bone homeostasis and on the bone healing cascade has so far not been considered. Within this study mice at different age and immunological experience were analyzed toward bone properties. Healing was assessed by introducing an osteotomy, immune cells were adoptively transferred to disclose the difference in biological vs. chronological aging. In vitro studies were employed to test the interaction of immune cell products (cytokines) on cells of the musculoskeletal system. In metaphyseal bone, immune-aging affects bone homeostasis by impacting bone formation capacity and thereby influencing mass and microstructure of bone trabeculae leading to an overall reduced mechanical competence as found in bone torsional testing. Furthermore, bone formation is also impacted during bone regeneration in terms of a diminished healing capacity observed in young animals who have an experienced human immune system. We show the impact of an experienced immune system compared to a naive immune system, demonstrating the substantial differences in the healing capacity and bone homeostasis due to the immune composition. We further showed that in vivo mechanical stimulation changed the immune system phenotype in young mice toward a more naive composition. While this rescue was found to be significant in young individuals, aged mice only showed a trend toward the reconstitution of a more naive immune phenotype. Considering the immune system's experience level in an individual, will likely allow one to differentiate (stratify) and treat (immune-modulate) patients more effectively. This work illustrates the relevance of including immune diagnostics when discussing immunomodulatory therapeutic strategies for the progressively aging population of the industrial countries.