Osteogenesis imperfecta (OI), also called brittle bone disease, is a genetic osteodysplasia characterized by a defect in type 1 collagen. Often diagnosed in infancy or early childhood, young patients are affected by frequent fractures. Osteogenesis imperfecta was first named almost 200 yr ago, yet there are still no FDA-approved treatments for OI, and existing treatments target only the skeletal defects of the disease. In this review, we briefly examine current treatments and ongoing clinical trials. Then, by analyzing OI with an osteoimmunological perspective, we have compiled evidence that OI has an autoimmune component. This autoimmune component of OI remains unconsidered, even though an immunology-based therapy has shown promise in treating OI. Acknowledging an autoimmune component of OI is critical to understanding its mechanisms and allowing for the development of more efficacious treatments and novel immunotherapies. Considering the existing literature and the growing impact of immunotherapeutic therapies in cancer and other autoimmune diseases, we believe it may be time to rethink the immune aspects of this genetic disorder and develop novel immunomodulating strategies to improve the quality of life for OI patients.

Chronic inflammation at bone defect sites can impede regenerative processes, but local immune responses can be adjusted to promote healing. Regulating the osteoimmune microenvironment, particularly through macrophage polarization, has become a key focus in bone regeneration research. While bone implants are crucial for addressing significant bone defects, they are often recognized by the immune system as foreign, triggering inflammation that leads to bone resorption and implant issues like fibrous encapsulation and aseptic loosening. Developing osteoimmunomodulatory implants offers a promising approach to transforming destructive inflammation into healing processes, enhancing implant integration and bone regeneration. This review explores strategies based on tuning the physicochemical attributes and chemical composition of materials in engineering osteoimmunomodulatory and pro-regenerative bone implants.

Proximal humerus fractures (PHFs) are the third most common fractures in elderly patients. Over 70% of PHFs in patients aged over 60 are displaced fractures, often necessitating surgical treatment. However, osteosynthesis is associated with a high rate of complications, highlighting the urgent need for additional therapeutic approaches to enhance bone healing and prevent osteonecrosis. This study evaluates the safety, feasibility and potential efficacy of local prostacyclin (iloprost) to improve bone healing in patients with PHFs.

Thirty eligible patients will be randomized into one of three groups at a 1:1:1 ratio. All patients will receive angular stable locking plate fixation. Two treatment groups will receive an additional single dose of local iloprost through a 24-hour infusion postoperatively (group 1: low dose; group 2: high dose), while the control group will only receive the osteosynthesis. Patients will be monitored for 52 weeks. The primary endpoint is safety, with secondary endpoints including the preservation of the screw tip apex distance as an indicator of fracture healing, head shaft angle, necrosis rate, and patient-related outcome measures.

The Ilobone study aims to provide data on the potential for biological augmentation of osteosynthesis procedures in PHFs, prone to healing challenges and complications.

The trial is registered with ClinicalTrial.gov (NCT04543682), registered 02 Sep. 2020, https://clinicaltrials.gov/show/NCT04543682 and the German Clinical Trials Registry (DRKS00027081), registered 10 Nov. 2021 https://drks.de/search/de/trial/DRKS00027081 .

Large bone defects resulting from trauma, disease, or resection often exceed the intrinsic capacity of bones to heal. The current gold standard addressing these defects is autologous bone grafting (ABG). Procedures such as reamer-irrigator-aspirator (RIA) and conventional bone grafting from the iliac crest are widely recognized as highly effective interventions for critical-size bone defects. The early phase of fracture healing is particularly crucial, as it can determine whether a complete bony union occurs, or if delayed healing or non-unions develop. The initial composition of the bone marrow (BM)-rich ABG transplant, with its unique cellular (e.g., leukocytes, monocytes, and granulocytes) and acellular (e.g., growth factors and extracellular proteins) components, plays a key role in this process. However, despite many successful case reports, the role of ABG cells, growth factors, and their precise contributions to bone healing remain largely elusive.

We characterized the native cellularity of both solid and liquid RIA-derived ABG by analyzing primary, minimally manipulated populations of monocytes, macrophages, and T cells, as well as hematopoietic, endothelial, and mesenchymal progenitor cells by flow cytometry. Growth factor and cytokine contents were assessed through antibody arrays. Possible functional and immunomodulatory properties of RIA liquid were evaluated in functional in vitro assays.

Growth factor and protein arrays revealed a plethora of soluble factors that can be linked to specific immunomodulatory and angiogenic properties, which were evaluated for their potency using functional in vitro assays. We could demonstrate a strong M2-macrophage phenotype inducing the effect of RIA liquid on macrophages. Additionally, we observed an increase in anti-inflammatory T cell subsets generated from peripheral blood mononuclear cells and BM mononuclear cells upon stimulation with RIA liquid . Finally, in vitro endothelial tube formation assays revealed highly significant angiogenic properties of RIA liquid, even at further dilutions.

The cytokine and protein content of RIA liquid exhibits potent immunomodulatory and angiogenic properties. These findings suggest significant therapeutic potential for RIA liquid in modulating immune responses and promoting angiogenesis. Anti-inflammatory and angiogenic properties demonstrated in this study might also help to further define and understand its particular mode of action while also providing explanations to the excellent bone-healing properties of ABG in general.

Case-series (Level 4).

Last decade has witnessed increasing evidence which highlights the roles of immune cells in bone regeneration. Numerous immune cell types, including macrophages, T cells, and neutrophils are involved in fracture healing by orchestrating a series of events that modulate bone formation and remodeling. In this review, the role of T cell immunity in fracture healing has been summarized, and the modulatory effects of T cell immunity in inflammation, bone formation and remodeling have been highlighted. The review also summarizes the specific roles of different T cell subsets, including CD4+ T cells, CD8+ T cells, regulatory T cells, T helper 17 cells, and γδ T cells in modulating fracture healing. The current therapeutics targeting T cell immunity to enhance fracture healing have also been reviewed, aiming to provide insights from a translational standpoint. Overall, this work discusses recent advances and challenges in the interdisciplinary research field of T cell related osteoimmunology and its implications in fracture healing.

Delayed unions or non-unions of bone fractures remain a challenge in clinical practice. Developing a deep understanding of the roles of immune cells, including T cells, in fracture healing will facilitate the advancement of novel therapeutics of fracture nonunion. This review summarizes the current understanding of different T cell subsets involved in various phases of fracture healing, providing insights for targeting T cells as an alternative strategy to enhance bone regeneration.

Inadequate repair of the intervertebral disc (IVD) contributes to low back pain. Infiltrating immune cells into damaged tissues are critical mediators of repair, yet little is known about the identities, roles, and temporal regulation following IVD injury. By analyzing transcripts of immune cell markers, histopathologic analysis, immunofluorescence, and flow cytometry, we aimed to define the temporal cascade of infiltrating immune cells and their associations with IVD degeneration.

Caudal IVDs from 12-week-old C57BL6/J mice were injured and monitored for 42 days post-injury. Transcriptional markers identifying myeloid, B, and T immune cells, and angiogenic factors were measured from the IVDs every 2-3 days. Histopathologic degeneration of the IVD was measured throughout. Flow cytometry and immunofluorescence were used to identify and localize cells including B, T, natural killer T (NKT) cells, monocytes, neutrophils, macrophages, eosinophils, and dendritic cells.

The injured IVD revealed distinct phases of inflammation and proliferation. Robust temporal oscillation in the myeloid and T cell transcripts was observed in females. Cd3+ T cells were more abundant in females than in males. The Cd3+Cd4-Cd8- T cells that dominate the female cascade contain rare γδ T cells. Injury-mediated degeneration was prevalent in both sexes but more severe in males.

This study defines the coordinated infiltration of immune cells in the IVD following injury. We report the discovery of γδ T cells in the female IVD, and this was associated with less severe degeneration. γδ T cells have potent anti-inflammatory roles and may suppress degeneration following IVD injury.

Regulatory T cells (Tregs) are essential for maintaining immune homeostasis and facilitating tissue regeneration by fostering an environment conducive to tissue repair. However, in damaged tissues, excessive inflammatory responses can overwhelm the immunomodulatory capacity of Tregs, compromising their functionality and potentially hindering effective regeneration. Mesenchymal stem cells (MSCs) play a key role in enhancing Treg function. MSCs enhance Treg activity through indirect interactions, such as cytokine secretion, and direct interactions via membrane proteins.

This review examines the regenerative functions of Tregs across various tissues, including bone, cartilage, muscle, and skin, and explores strategies to enhance Treg functionality using MSCs. Advanced techniques, such as the overexpression of relevant genes in MSCs, are highlighted for their potential to further enhance Treg function. Additionally, emerging technologies utilizing extracellular vesicles (EVs) and cell membrane-derived vesicles derived from MSCs offer promising alternatives to circumvent the potential side effects associated with live cell therapies. This review proposes approaches to enhance Treg function and promote tissue regeneration and also outlines future research directions.

This review elucidates recent technological advancements aimed at enhancing Treg function using MSCs and examines their potential to improve tissue regeneration efficiency.

The field of osteoimmunology has primarily focused on fracture healing in isolated musculoskeletal injuries. The innate immune system is the initial response to fracture, with inflammatory macrophages, cytokines, and neutrophils arriving first at the fracture hematoma, followed by an anti-inflammatory phase to begin the process of new bone formation. This review aims to first discuss the current literature and knowledge gaps on the immune responses governing single fracture healing by encompassing the individual role of macrophages, neutrophils, cytokines, mesenchymal stem cells, bone cells, and other immune cells. This paper discusses the interactive effects of these cellular responses underscoring the field of osteoimmunology. The critical role of the metabolic environment in guiding the immune system properties will be highlighted along with some effective therapeutics for fracture healing in the context of osteoimmunology. However, compared to isolated fractures, which frequently heal well, long bone fractures in over 30 % of polytrauma patients exhibit impaired healing. Clinical evidence suggests there may be distinct physiologic and inflammatory pathways altered in polytrauma resulting in nonunion. Nonunion is associated with worse patient outcomes and increased societal healthcare costs. The dysregulated immunomodulatory/inflammatory response seen in polytrauma may lead to this increased nonunion rate. This paper will investigate the differences in immune response between isolated and polytrauma fractures. Finally, future directions for fracture studies are explored with consideration of the emerging roles of newly discovered immune cell functions in fracture healing, the existing challenges and conflicting results in the field, the translational potential of these studies in clinic, and the more complex nature of polytrauma fractures that can alter cell functions in different tissues.

Leptin is a pleiotropic hormone that regulates food intake and energy homeostasis with enigmatic effects on bone development. It is unclear if leptin promotes or inhibits bone growth. The aim of this study was to characterize the micro-architecture and mechanical competence of femur bones of leptin-deficient mice.

Right femur bones of 15-week old C57BL/6 (n = 9) and leptin-deficient (ob/ob, n = 9) mice were analyzed. Whole bones were scanned using micro-CT and morphometric parameters of the cortex and trabeculae were assessed. Elastic moduli were determined from microindentations in midshaft cross-sections. Mineral densities were determined using quantitative backscatter scanning electron microscopy. 3D models of the distal femur metaphysis, cleared from trabecular bone, were meshed and used for finite element simulations of axial loading to identify straining differences between ob/ob and C57BL/6 controls.

Compared with C57BL/6 controls, ob/ob mice had significantly shorter bones. ob/ob mice showed significantly increased cancellous bone volume and trabecular thickness. qBEI quantified a ∼7% lower mineral density in ob/ob mice in the distal femur metaphysis. Indentation demonstrated a significantly reduced Young's modulus of 12.14 [9.67, 16.56 IQR] GPa for ob/ob mice compared to 23.12 [20.70, 26.57 IQR] GPa in C57BL/6 mice. FEA revealed greater deformation of cortical bone in ob/ob as compared to C57BL/6 mice.

Leptin deficient ob/ob mice have a softer cortical bone in the distal femur metaphysis but an excessive amount of cancellous bone, possibly as a response to increased deformation of the bones during axial loading. Both FEA and direct X-ray and electron microscopy imaging suggest that the morphology and micro-architecture of ob/ob mice have inferior biomechanical properties suggestive of a reduced mechanical competence.

Open 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.

In 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.

Out 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.

Our 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.

BP has shown good potential for promoting bone regeneration. However, the understanding of the mechanisms of BP-enhanced bone regeneration is still limited. This review first summarizes the recent advances in applications of BP in bone regeneration. We further highlight the possibility that BP enhances bone regeneration by regulating the behavior of mesenchymal stem cells (MSCs), osteoblasts, vascular endothelial cells (VECs), and macrophages, mainly through the regulation of cytoskeletal remodeling, energy metabolism, oxidation resistance and surface adsorption properties, etc. In addition, moderating the physicochemical properties of BP (i.e., shape, size, and surface charge) can alter the effects of BP on bone regeneration. This review reveals the underlying mechanisms of BP-enhanced bone regeneration and provides strategies for further material design of BP-based materials for bone regeneration.

With the introduction and continuous improvement in operative fracture fixation, even the most severe bone fractures can be treated with a high rate of successful healing. However, healing complications can occur and when healing fails over prolonged time, the outcome is termed a fracture non-union. Non-union is generally believed to develop due to inadequate fixation, underlying host-related factors, or infection. Despite the advancements in fracture fixation and infection management, there is still a clear need for earlier diagnosis, improved prediction of healing outcomes and innovation in the treatment of non-union.

This review provides a detailed description of non-union from a clinical perspective, including the state of the art in diagnosis, treatment, and currently available biomaterials and orthobiologics.Subsequently, recent translational development from the biological, mechanical, and infection research fields are presented, including the latest in smart implants, osteoinductive materials, and in silico modeling.

The first challenge for future innovations is to refine and to identify new clinical factors for the proper definition, diagnosis, and treatment of non-union. However, integration of in vitro, in vivo, and in silico research will enable a comprehensive understanding of non-union causes and correlations, leading to the development of more effective treatments.

The innate immune response is crucial to inflammation, but how neutrophils and macrophages act in bone repair and tissue engineering treatment strategies await clarification. In this study, it is found that N2 neutrophils release stronger "eat me" signals to induce macrophage phagocytosis and polarize into the M2 anti-inflammatory phenotype. Guided by this biological mechanism, a mesoporous bioactive glass scaffold (MBG) is filled with hyaluronic acid methacryloyl (HAMA) hydrogel loaded with Transforming growth factor-β1 (TGFβ1) adenovirus (Ad@H), constructing a genetically engineered composite scaffold (Ad@H/M). The scaffold not only has good hydrophilicity and biocompatibility, but also provides mechanical stress support for bone repair. Adenovirus infection quickly induces N2 neutrophils, upregulating NF-κB and MAPK signaling pathways through Toll-like receptor 4 (TLR4) to promote the inflammatory response and macrophage phagocytosis. Macrophages perform phagocytosis and polarize towards the M2 phenotype, mediating the inflammatory response by inhibiting the PI3K-AKT-NF-κB pathway, maintaining homeostasis of the osteogenic microenvironment. The role of the Ad@H/M scaffold in regulating early inflammation and promoting long-term bone regeneration is further validated in vivo. In brief, this study focuses on the cascade of reactions between neutrophils and macrophage subtypes, and reports a composite scaffold that coordinates the innate immune response to promote bone repair.

Musculoskeletal diseases and injuries are among the leading causes of pain and morbidity worldwide. Broad efforts have focused on developing pro-regenerative biomaterials to treat musculoskeletal conditions; however, these approaches have yet to make a significant clinical impact. Recent studies have demonstrated that the immune system is central in orchestrating tissue repair and that targeting pro-regenerative immune responses can improve biomaterial therapeutic outcomes. However, aging is a critical factor negatively affecting musculoskeletal tissue repair and immune function. Hence, understanding how age affects the response to biomaterials is essential for improving musculoskeletal biomaterial therapies. This review focuses on the intersection of the immune system and aging in response to biomaterials for musculoskeletal tissue repair. The article introduces the general impacts of aging on tissue physiology, the immune system, and the response to biomaterials. Then, it explains how the adaptive immune system guides the response to injury and biomaterial implants in cartilage, muscle, and bone and discusses how aging impacts these processes in each tissue type. The review concludes by highlighting future directions for the development and translation of personalized immunomodulatory biomaterials for musculoskeletal tissue repair.

Inflammation is thought to be dysregulated with age leading to impaired bone fracture healing. However, broad analyses of inflammatory processes during homeostatic bone aging and during repair are lacking. Here, we assessed changes in inflammatory cell and cytokine profiles in circulation and in bone tissue to identify age- and sex-dependent differences during homeostasis and repair. During homeostatic aging, male mice demonstrated accumulation of CD4+ helper T cells and CD8+ cytotoxic T cells within bone while both pro-inflammatory "M1" and anti-inflammatory "M2" macrophage numbers decreased. Female mice saw no age-associated changes in immune-cell population in homeostatic bone. Concentrations of IL-1β, IL-9, IFNγ, and CCL3/MIP-1α increased with age in both male and female mice, whereas concentrations of IL-2, TNFα, TNFR1, IL-4, and IL-10 increased only in female mice - thus we termed these "age-accumulated" cytokines. There were no notable changes in immune cell populations nor cytokines within circulation during aging. Sex-dependent analysis demonstrated slight changes in immune cell and cytokine levels within bone and circulation, which were lost upon fracture injury. Fracture in young male mice caused a sharp decrease in number of M1 macrophages; however, this was not seen in aged male mice nor in female mice of any age. Injury itself induced a decrease in the number of CD8+ T cells within the local tissue of aged male and of female mice but not of young mice. Cytokine analysis of fractured mice revealed that age-accumulated cytokines quickly dissipated after fracture injury, and did not re-accumulate in newly regenerated tissue. Conversely, CXCL1/KC-GRO, CXCL2/MIP-2, IL-6, and CCL2/MCP-1 acted as "fracture response" cytokines: increasing sharply after fracture, eventually returning to baseline. Collectively, we classify measured cytokines into three groups: (1) age-accumulated cytokines, (2) female-specific age-accumulated cytokines, and (3) fracture response cytokines. These inflammatory molecules represent potential points of intervention to improve fracture healing outcome.

T cells are adaptive immune cells essential in pathogenic response, cancer, and autoimmune disorders. During the integration of biomaterials with host tissue, T cells modify the local inflammatory environment by releasing cytokines that promote inflammatory resolution following implantation. T cells are vital for the modulation of innate immune cells, recruitment and proliferation of mesenchymal stem cells (MSCs), and formation of functional tissue around the biomaterial implant. We have demonstrated that deficiency of αβ T cells promotes macrophage polarization towards a pro-inflammatory phenotype and attenuates MSC recruitment and proliferation in vitro and in vivo. The goal of this study was to understand how CD4+ and CD8+ T cells, subsets of the αβ T cell family, impact the inflammatory response to titanium (Ti) biomaterials. Deficiency of either CD4+ or CD8+ T cells increased the proportion of pro-inflammatory macrophages, lowered anti-inflammatory macrophages, and diminished MSC recruitment in vitro and in vivo. In addition, new bone formation at the implantation site was significantly reduced in T cell-deficient mice compared to T cell-competent mice. Deficiency of CD4+ T cells exacerbated these effects compared to CD8+ T cell deficiency. Our results show the importance of CD4+ and CD8+ T cells in modulating the inflammatory response and promoting new bone formation in response to modified Ti implants. STATEMENT OF SIGNIFICANCE: CD4+ and CD8+ T cells are essential in modulating the peri-implant microenvironment during the inflammatory response to biomaterial implantation. This study shows that deficiency of either CD4+ or CD8+ T cell subsets altered macrophage polarization and reduced MSC recruitment and proliferation at the implantation site.

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.

Inadequate repair of injured intervertebral discs (IVD) leads to degeneration and contributes to low back pain. Infiltrating immune cells into damaged musculoskeletal tissues are critical mediators of repair, yet little is known about their identities, roles, and temporal regulation following IVD injury. By analyzing longitudinal changes in gene expression, tissue morphology, and the dynamics of infiltrating immune cells following injury, we characterize sex-specific differences in immune cell populations and identify the involvement of previously unreported immune cell types, γδ and NKT cells. Cd3+Cd4-Cd8- T cells are the largest infiltrating lymphocyte population with injury, and we identified the presence of γδ T cells in this population in female mice specifically, and NKT cells in males. Injury-mediated IVD degeneration was prevalent in both sexes, but more severe in males. Sex-specific degeneration may be associated with the differential immune response since γδ T cells have potent anti-inflammatory roles and may mediate IVD repair.

The use of bone substitute materials is crucial for the healing of large bone defects. Immune response induced by bone substitute materials is essential in bone regeneration. Prior research has mainly concentrated on innate immune cells, such as macrophages. Existing research suggests that T lymphocytes, as adaptive immune cells, play an indispensable role in bone regeneration. However, the mechanisms governing T cell recruitment and specific subsets that are essential for bone regeneration remain unclear. This study demonstrates that CD4+ T cells are indispensable for ectopic osteogenesis by biphasic calcium phosphate (BCP). Subsequently, the recruitment of CD4+ T cells is closely associated with the activation of calcium channels in macrophages by BCP to release chemokines Ccl3 and Ccl17. Finally, these recruited CD4+ T cells are predominantly Tregs, which play a significant role in ectopic osteogenesis by BCP. These findings not only shed light on the immune-regenerative process after bone substitute material implantation but also establish a theoretical basis for developing bone substitute materials for promoting bone tissue regeneration. STATEMENT OF SIGNIFICANCE: Bone substitute material implantation is essential in the healing of large bone defects. Existing research suggests that T lymphocytes are instrumental in bone regeneration. However, the specific mechanisms governing T cell recruitment and specific subsets that are essential for bone regeneration remain unclear. In this study, we demonstrate that activation of calcium channels in macrophages by biphasic calcium phosphate (BCP) causes them to release the chemokines Ccl3 and Ccl17 to recruit CD4+ T cells, predominantly Tregs, which play a crucial role in ectopic osteogenesis by BCP. Our findings provide a theoretical foundation for developing bone substitute material for bone tissue regeneration.

A fundamental understanding of inflammation and tissue healing suggests that the precise regulation of the inflammatory phase, both in terms of location and timing, is crucial for bone regeneration. However, achieving the activation of early inflammation without causing chronic inflammation while facilitating quick inflammation regression to promote bone regeneration continues to pose challenges. This study reveals that black phosphorus (BP) accelerates bone regeneration by building an osteogenic immunological microenvironment. BP amplifies the acute pro-inflammatory response and promotes the secretion of anti-inflammatory factors to accelerate inflammation regression and tissue regeneration. Mechanistically, BP creates an osteoimmune-friendly microenvironment by stimulating macrophages to express interleukin 33 (IL-33), amplifying the inflammatory response at an early stage, and promoting the regression of inflammation. In addition, BP-mediated IL-33 expression directly promotes osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), which further facilitates bone repair. To the knowledge, this is the first study to reveal the immunomodulatory potential of BP in bone regeneration through the regulation of both early-stage inflammatory responses and later-stage inflammation resolution, along with the associated molecular mechanisms. This discovery serves as a foundation for the clinical use of BP and is an efficient approach for managing the immune microenvironment during bone regeneration.

The healing of a bone injury is a highly complex process involving a multitude of different tissue and cell types, including immune cells, which play a major role in the initiation and progression of bone regeneration.

We histologically analyzed the spatio-temporal occurrence of cells of the innate immune system (macrophages), the adaptive immune system (B and T lymphocytes), and bone cells (osteoblasts and osteoclasts) in the fracture area of a femoral osteotomy over the healing time. This study was performed in a bone osteotomy gap mouse model. We also investigated two key challenges of successful bone regeneration: hypoxia and revascularization.

Macrophages were present in and around the fracture gap throughout the entire healing period. The switch from initially pro-inflammatory M1 macrophages to the anti-inflammatory M2 phenotype coincided with the revascularization as well as the appearance of osteoblasts in the fracture area. This indicates that M2 macrophages are necessary for the restoration of vessels and that they also play an orchestrating role in osteoblastogenesis during bone healing. The presence of adaptive immune cells throughout the healing process emphasizes their essential role for regenerative processes that exceeds a mere pathogen defense. B and T cells co-localize consistently with bone cells throughout the healing process, consolidating their crucial role in guiding bone formation. These histological data provide, for the first time, comprehensive information about the complex interrelationships of the cellular network during the entire bone healing process in one standardized set up. With this, an overall picture of the spatio-temporal interplay of cellular key players in a bone healing scenario has been created.

A spatio-temporal distribution of immune cells, bone cells, and factors driving bone healing at time points that are decisive for this process-especially during the initial steps of inflammation and revascularization, as well as the soft and hard callus phases-has been visualized. The results show that the bone healing cascade does not consist of five distinct, consecutive phases but is a rather complex interrelated and continuous process of events, especially at the onset of healing.

Critical bone defects resulting in delayed and non-union are a major concern in the field of orthopedics. Over the past decade, mesenchymal stem cells (MSCs) have become a promising frontier for bone repair and regeneration owing to their high expansion rate and osteogenic differentiation potential ex vivo. MSCs have also long been associated with their ability to modulate immune response in the recipients. These can even skew the immune response towards pro-inflammatory or anti-inflammatory type by sensing their local microenvironment. MSCs adopt anti-inflammatory phenotype at bone injury site and secrete various immunomodulatory factors such as IDO, NO, TGFβ1 and PGE-2 which have redundant role in osteoblast differentiation and bone formation. As such, several studies have also sought to decipher the immunomodulatory effects of osteogenically differentiated MSCs. The present review discusses the immunomodulatory status of MSCs during their osteogenic differentiation and summarizes few mechanisms that cause immunosuppression by osteogenically differentiated MSCs and its implication during bone healing.

Bone fracture healing is a well-orchestrated but complex process that involves numerous regulations at different scales. This complexity becomes particularly evident during the inflammatory stage, as immune cells invade the healing region and trigger a cascade of signals to promote a favorable regenerative environment. Thus, the emergence of criticalities during this stage might hinder the rest of the process. Therefore, the investigation of the many interactions that regulate the inflammation has a primary importance on the exploration of the overall healing progression. In this context, an in silico model named COMMBINI (COmputational Model of Macrophage dynamics in the Bone INjury Immunoresponse) has been developed to investigate the mechano-biological interactions during the early inflammatory stage at the tissue, cellular and molecular levels. An agent-based model is employed to simulate the behavior of immune cells, inflammatory cytokines and fracture debris as well as their reciprocal multiscale biological interactions during the development of the early inflammation (up to 5 days post-injury). The strength of the computational approach is the capacity of the in silico model to simulate the overall healing process by taking into account the numerous hidden events that contribute to its success. To calibrate the model, we present an in silico immunofluorescence method that enables a direct comparison at the cellular level between the model output and experimental immunofluorescent images. The combination of sensitivity analysis and a Genetic Algorithm allows dynamic cooperation between these techniques, enabling faster identification of the most accurate parameter values, reducing the disparity between computer simulation and histological data. The sensitivity analysis showed a higher sensibility of the computer model to the macrophage recruitment ratio during the early inflammation and to proliferation in the late stage. Furthermore, the Genetic Algorithm highlighted an underestimation of macrophage proliferation by in vitro experiments. Further experiments were conducted using another externally fixated murine model, providing an independent validation dataset. The validated COMMBINI platform serves as a novel tool to deepen the understanding of the intricacies of the early bone regeneration phases. COMMBINI aims to contribute to designing novel treatment strategies in both the biological and mechanical domains.

Introduction: Mechanical loading is known to determine the course of bone fracture healing. We hypothesise that lower limb long bone loading differs with knee flexion angle during walking and frontal knee alignment, which affects fracture healing success. Materials and methods: Using our musculoskeletal in silico modelling constrained against in vivo data from patients with instrumented knee implants allowed us to assess internal loads in femur and tibia. These internal forces were associated with the clinical outcome of fracture healing in a relevant cohort of 178 extra-articular femur and tibia fractures in patients using a retrospective approach. Results: Mean peak forces differed with femoral compression (1,330-1,936 N at mid-shaft) amounting to about half of tibial compression (2,299-5,224 N). Mean peak bending moments in the frontal plane were greater in the femur (71-130 Nm) than in the tibia (from 26 to 43 Nm), each increasing proximally. Bending in the sagittal plane showed smaller mean peak bending moments in the femur (-38 to 43 Nm) reaching substantially higher values in the tibia (-63 to -175 Nm) with a peak proximally. Peak torsional moments had opposite directions for the femur (-13 to -40 Nm) versus tibia (15-48 Nm) with an increase towards the proximal end in both. Femoral fractures showed significantly lower scores in the modified Radiological Union Scale for Tibia (mRUST) at last follow-up (p < 0.001) compared to tibial fractures. Specifically, compression (r = 0.304), sagittal bending (r = 0.259), and frontal bending (r = -0.318) showed strong associations (p < 0.001) to mRUST at last follow-up. This was not the case for age, body weight, or localisation alone. Discussion: This study showed that moments in femur and tibia tend to decrease towards their distal ends. Tibial load components were influenced by knee flexion angle, especially at push-off, while static frontal alignment played a smaller role. Our results indicate that femur and tibia are loaded differently and thus require adapted fracture fixation considering load components rather than just overall load level.

Immune homeostasis is delicately mediated by the dynamic balance between effector immune cells and regulatory immune cells. Local deviations from immune homeostasis in the microenvironment of bone fractures, caused by an increased ratio of effector to regulatory cues, can lead to excessive inflammatory conditions and hinder bone regeneration. Therefore, achieving effective and localized immunomodulation of bone fractures is crucial for successful bone regeneration. Recent research has focused on developing localized and specific immunomodulatory strategies using local hydrogel-based delivery systems. In this review, we aim to emphasize the significant role of immune homeostasis in bone regeneration, explore local hydrogel-based delivery systems, discuss emerging trends in immunomodulation for enhancing bone regeneration, and address the limitations of current delivery strategies along with the challenges of clinical translation.

The classic two-stage masquelet technique is an effective procedure for the treatment of large bone defects. Our group recently showed that one surgery could be saved by using a decellularized dermis membrane (DCD, Epiflex, DIZG). In addition, studies with bone substitute materials for defect filling show that it also appears possible to dispense with the removal of syngeneic cancellous bone (SCB), which is fraught with complications. The focus of this work was to clarify whether the SCB can be replaced by the granular demineralized bone matrix (g-DBM) or fibrous demineralized bone matrix (f-DBM) demineralized bone matrix and whether the colonization of the DCD and/or the DBM defect filling with bone marrow mononuclear cells (BMC) can lead to improved bone healing. In 100 Sprague Dawley rats, a critical femoral bone defect 5 mm in length was stabilized with a plate and then encased in DCD. Subsequently, the defect was filled with SCB (control), g-DBM, or f-DBM, with or without BMC. After 8 weeks, the femurs were harvested and subjected to histological, radiological, and biomechanical analysis. The analyses showed the incipient bony bridging of the defect zone in both groups for g-DBM and f-DBM. Stability and bone formation were not affected compared to the control group. The addition of BMCs showed no further improvement in bone healing. In conclusion, DBM offers a new perspective on defect filling; however, the addition of BMC did not lead to better results.

IL-17A (IL-17), a driver of the inflammatory phase of fracture repair, is produced locally by several cell lineages including γδ T cells and Th17 cells. However, the origin of these T cells and their relevance for fracture repair are unknown. Here, we show that fractures rapidly expanded callus γδ T cells, which led to increased gut permeability by promoting systemic inflammation. When the microbiota contained the Th17 cell-inducing taxon segmented filamentous bacteria (SFB), activation of γδ T cells was followed by expansion of intestinal Th17 cells, their migration to the callus, and improved fracture repair. Mechanistically, fractures increased the S1P receptor 1-mediated (S1PR1-mediated) egress of Th17 cells from the intestine and enhanced their homing to the callus through a CCL20-mediated mechanism. Fracture repair was impaired by deletion of γδ T cells, depletion of the microbiome by antibiotics (Abx), blockade of Th17 cell egress from the gut, or Ab neutralization of Th17 cell influx into the callus. These findings demonstrate the relevance of the microbiome and T cell trafficking for fracture repair. Modifications of microbiome composition via Th17 cell-inducing bacteriotherapy and avoidance of broad-spectrum Abx may represent novel therapeutic strategies to optimize fracture healing.

Naturally-derived cell membranes have shown great promise in functionalizing nanoparticles to enhance biointerfacing functions for drug delivery applications. However, its potential for functionalizing macroporous scaffolds to enhance tissue regeneration in vivo remains unexplored. Engineering scaffolds with immunomodulatory functions represents an exciting strategy for tissue regeneration but is largely limited to soft tissues. Critical-sized bone defects cannot heal on their own, and the role of adaptive immune cells in scaffold-mediated healing of cranial bone defects remains largely unknown. Here, mensenchymal stem cell membrane (MSCM)-coated microribbon (µRB) scaffolds for treating critical size cranial bone defects via targeting immunomodulation are reported. Confocal imaging and proteomic analyses are used to confirm successful coating and characterize the compositions of cell membrane coating. It is demonstrated that MSCM coating promotes macrophage (Mφ) polarization toward regenerative phenotype, induces CD8+ T cell apoptosis, and enhances regulatory T cell differentiation in vitro and in vivo. When combined with a low dosage of BMP-2, MSCM coating further accelerates bone regeneration and suppresses inflammation. These results establish cell membrane-coated microribbon scaffolds as a promising strategy for treating critical size bone defects via immunomodulation. The platform may be broadly used with different cell membranes and scaffolds to enhance regeneration of multiple tissue types.

Bone morphogenetic proteins (BMPs) are used as key therapeutic agents for the treatment of difficult fractures. While their effects on osteoprogenitors are known, little is known about their effects on the immune system.

We used permutations of BMP-6 (B), vascular endothelial growth factor (V), and Hedgehog signaling pathway activator smoothened agonist (S), to treat a rat mandibular defect and investigated healing outcomes at week 8, in correlation with the cellular landscape of the immune cells in the fracture callus at week 2.

Maximum recruitment of immune cells to the fracture callus is known to occur at week 2. While the control, S, V, and VS groups remained as nonunions at week 8; all BMP-6 containing groups - B, BV, BS and BVS, showed near-complete to complete healing. This healing pattern was strongly associated with significantly higher ratios of CD4 T (CD45⁺CD3⁺CD4⁺) to putative CD8 T cells (CD45⁺CD3⁺CD4^-), in groups treated with any permutation of BMP-6. Although, the numbers of putative M1 macrophages (CD45⁺CD3^-CD11b/c⁺CD38^high) were significantly lower in BMP-6 containing groups in comparison with S and VS groups, percentages of putative - Th1 cells or M1 macrophages (CD45⁺CD4⁺IFN-γ⁺) and putative - NK, NKT or cytotoxic CD8T cells (CD45⁺CD4^-IFN-γ⁺) were similar in control and all treatment groups. Further interrogation revealed that the BMP-6 treatment promoted type 2 immune response by significantly increasing the numbers of CD45⁺CD3^-CD11b/c⁺CD38^low putative M2 macrophages, putative - Th2 cells or M2 macrophages (CD45⁺CD4⁺IL-4⁺) cells and putative - mast cells, eosinophils or basophils (CD45⁺CD4^-IL-4⁺ cells). CD45^- non-haematopoietic fractions of cells which encompass all known osteoprogenitor stem cells populations, were similar in control and treatment groups.

This study uncovers previously unidentified regulatory functions of BMP-6 and shows that BMP-6 enhances fracture healing by not only acting on osteoprogenitor stem cells but also by promoting type 2 immune response.

The use of autografts, as primary cell and tissue source, is the current gold standard approach to treat critical size bone defects and nonunion defects. The unique mixture of the autografts, containing bony compartments and bone marrow (BM), delivers promising results. Although BM mesenchymal stromal cells (BM-MSCs) still represent a major target for various healing approaches in current preclinical research and respective clinical trials, their occurrence in the human BM is typically low. In vitro expansion of this cell type is regulatory challenging as well as time and cost intensive. Compared with marginal percentages of resident BM-MSCs in BM, BM mononuclear cells (BM-MNCs) contained in BM aspirates, concentrates, and bone autografts represent a readily available abundant cell source, applicable within hours during surgical procedures without the need for time-consuming and regulatory challenging cell expansion. This benefit is one reason why autografting has become a clinical standard procedure. However, the exact anatomy and cellularity of BM-MNCs in humans, which is strongly correlated to their unique mode of action and wide application range remains to be elucidated. The aim of this review was to present an overview of the current knowledge on these specific cell types found in human BM, emphasize the contribution of BM-MNCs in bone healing, highlight donor site dependence, and discuss limitations in the current isolation and subsequent characterization procedures. Hereby, the most recent and relevant examples of human BM-MNC cell characterization, flow cytometric analyses, and findings are summarized, with a strong focus on bone therapy.

Bone has a remarkable endogenous regenerative capacity that enables scarless healing and restoration of its prior mechanical function, even under challenging conditions such as advanced age and metabolic or immunological degenerative diseases. However - despite much progress - a high number of bone injuries still heal with unsatisfactory outcomes. The mechanisms leading to impaired healing are heterogeneous, and involve exuberant and non-resolving immune reactions or overstrained mechanical conditions that affect the delicate regulation of the early initiation of scar-free healing. Every healing process begins phylogenetically with an inflammatory reaction, but its spatial and temporal intensity must be tightly controlled. Dysregulation of this inflammatory cascade directly affects the subsequent healing phases and hinders the healing progression. This Review discusses the complex processes underlying bone regeneration, focusing on the early healing phase and its highly dynamic environment, where vibrant changes in cellular and tissue composition alter the mechanical environment and thus affect the signalling pathways that orchestrate the healing process. Essential to scar-free healing is the interplay of various dynamic cascades that control timely resolution of local inflammation and tissue self-organization, while also providing sufficient local stability to initiate endogenous restoration. Various immunotherapy and mechanobiology-based therapy options are under investigation for promoting bone regeneration.

Understanding the optimal conditions required for bone healing can have a substantial impact to target the problem of non-unions and large bone defects. The combination of bioactive factors, regenerative progenitor cells and biomaterials to form a tissue engineered (TE) complex is a promising solution but translation to the clinic has been slow. We hypothesized the typical material testing algorithm used is insufficient and leads to materials being mischaracterized as promising. In the first part of this study, human bone marrow - derived mesenchymal stromal cells (hBM-MSCs) were embedded in three commonly used biomaterials (hyaluronic acid methacrylate, gelatin methacrylate and fibrin) and combined with relevant bioactive osteogenesis factors (dexamethasone microparticles and polyphosphate nanoparticles) to form a TE construct that underwent in vitro osteogenic differentiation for 28 days. Gene expression of relevant transcription factors and osteogenic markers, and von Kossa staining were performed. In the second and third part of this study, the same combination of TE constructs were implanted subcutaneously (cell containing) in T cell-deficient athymic Crl:NIH-Foxn1^rnu rats for 8 weeks or cell free in an immunocompetent New Zealand white rabbit calvarial model for 6 weeks, respectively. Osteogenic performance was investigated via MicroCT imaging and histology staining. The in vitro study showed enhanced upregulation of relevant genes and significant mineral deposition within the three biomaterials, generally considered as a positive result. Subcutaneous implantation indicates none to minor ectopic bone formation. No enhanced calvarial bone healing was detected in implanted biomaterials compared to the empty defect. The reasons for the poor correlation of in vitro and in vivo outcomes are unclear and needs further investigation. This study highlights the discrepancy between in vitro and in vivo outcomes, demonstrating that in vitro data should be interpreted with extreme caution. In vitro models with higher complexity are necessary to increase value for translational studies. STATEMENT OF SIGNIFICANCE: Preclinical testing of newly developed biomaterials is a crucial element of the development cycle. Despite this, there is still significant discrepancy between in vitro and in vivo test results. Within this study we investigate multiple combinations of materials and osteogenic stimulants and demonstrate a poor correlation between the in vitro and in vivo data. We propose rationale for why this may be the case and suggest a modified testing algorithm.

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.

The immune system is closely linked to bone homeostasis and plays a pivotal role in several pathological and inflammatory conditions. Through various pathways it modulates various bone cells and subsequently sustains the physiological bone metabolism. Myeloid-derived suppressor cells (MDSCs) are a group of heterogeneous immature myeloid-derived cells that can exert an immunosuppressive function through a direct cell-to-cell contact, secretion of anti-inflammatory cytokines or specific exosomes. These cells mediate the innate immune response to chronic stress on the skeletal system. In chronic inflammation, MDSCs act as an inner offset to rebalance overactivation of the immune system. Moreover, they have been found to be involved in processes responsible for bone remodeling in different musculoskeletal disorders, autoimmune diseases, infection, and cancer. These cells can not only cause bone erosion by differentiating into osteoclasts, but also alleviate the immune reaction, subsequently leading to long-lastingly impacted bone remodeling. In this review, we discuss the impact of MDSCs on the bone metabolism under several pathological conditions, the involved modulatory pathways as well as potential therapeutic targets in MDSCs to improve bone health.

The purpose of this review is to summarize the clinical and basic science methods used to assess fracture healing and propose a framework to improve the translational possibilities.

Mainstays of fracture healing assessment include clinical examination, various imaging modalities, and assessment of function. Pre-clinical studies have yielded insight into biomechanical progression as well as the genetic, molecular, and cellular processes of fracture healing. Efforts are emerging to identify early markers to predict impaired healing and possibly early intervention to alter these processes. Despite of the differences in clinical and preclinical research, opportunities exist to unify and improve the translational efforts between these arenas to develop and optimize our ability to assess and predict fracture healing, thereby improving the clinical care of these patients.