Early pH Changes in Musculoskeletal Tissues upon Injury-Aerobic Catabolic Pathway Activity Linked to Inter-Individual Differences in Local pH.

Local pH is stated to acidify after bone fracture. However, the time course and degree of acidification remain unknown. Whether the acidification pattern within a fracture hematoma is applicable to adjacent muscle hematoma or is exclusive to this regenerative tissue has not been studied to date. Thus, in this study, we aimed to unravel the extent and pattern of acidification in vivo during the early phase post musculoskeletal injury. Local pH changes after fracture and muscle trauma were measured simultaneously in two pre-clinical animal models (sheep/rats) immediately after and up to 48 h post injury. The rat fracture hematoma was further analyzed histologically and metabolomically. In vivo pH measurements in bone and muscle hematoma revealed a local acidification in both animal models, yielding mean pH values in rats of 6.69 and 6.89, with pronounced intra- and inter-individual differences. The metabolomic analysis of the hematomas indicated a link between reduction in tricarboxylic acid cycle activity and pH, thus, metabolic activity within the injured tissues could be causative for the different pH values. The significant acidification within the early musculoskeletal hematoma could enable the employment of the pH for novel, sought-after treatments that allow for spatially and temporally controlled drug release.

Spatio-Temporal Bone Remodeling after Hematopoietic Stem Cell Transplantation.

The interaction of hematopoietic cells and the bone microenvironment to maintain bone homeostasis is increasingly appreciated. We hypothesized that the transfer of allogeneic T lymphocytes has extensive effects on bone biology and investigated trabecular and cortical bone structures, the osteoblast reconstitution, and the bone vasculature in experimental hematopoietic stem cell transplantations (HSCT). Allogeneic or syngeneic hematopoietic stem cells (HSC) and allogeneic T lymphocytes were isolated and transferred in a murine model. After 20, 40, and 60 days, bone structures were visualized using microCT and histology. Immune cells were monitored using flow cytometry and bone vessels, bone cells and immune cells were fluorescently stained and visualized. Remodeling of the bone substance, the bone vasculature and bone cell subsets were found to occur as early as day +20 after allogeneic HSCT (including allogeneic T lymphocytes) but not after syngeneic HSCT. We discovered that allogeneic HSCT (including allogeneic T lymphocytes) results in a transient increase of trabecular bone number and bone vessel density. This was paralleled by a cortical thinning as well as disruptive osteoblast lining and loss of B lymphocytes. In summary, our data demonstrate that the adoptive transfer of allogeneic HSCs and allogeneic T lymphocytes can induce profound structural and spatial changes of bone tissue homeostasis as well as bone marrow cell composition, underlining the importance of the adaptive immune system for maintaining a balanced bone biology.

Clinical and Research Approaches to Treat Non-union Fracture.

PURPOSE OF REVIEW: Impaired healing outcomes or even non-unions after bone injury are still a highly relevant problem in the daily clinical life. Especially within an aging population, the occurrence of bone fractures increases and thus novel treatment approaches to overcome compromised bone regeneration are needed. RECENT FINDINGS: The gold standard to treat delayed or non-healing bone injuries is still the use of autologous bone grafts to foster regeneration. Besides its successful treatment outcome, it also has disadvantages: a second surgery is needed in order to harvest the bone material and the material is highly limited. Looking into the recent literature, a multitude of different research approaches were already conducted to identify new possible strategies to treat impaired bone regeneration: application of mesenchymal stromal cells, platelet lysates, growth factors, interference in the immune system, or bone formation stimulation by ultrasound. This review gives an overview of the treatment approaches actually performed in the clinic as well as at the bench in the context of compromised bone healing. It clearly highlights the complexity of the nature of non-healing bone fractures as well as patient-dependent factors influencing the healing process.

Complex Spatio-Temporal Interplay of Distinct Immune and Bone Cell Subsets during Bone Fracture Healing.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Local immune cell contributions to fracture healing in aged individuals - A novel role for interleukin 22.

With increasing age, the risk of bone fractures increases while regenerative capacity decreases. This variation in healing potential appears to be linked to adaptive immunity, but the underlying mechanism is still unknown. This study sheds light on immunoaging/inflammaging, which impacts regenerative processes in aging individuals. In an aged preclinical model system, different levels of immunoaging were analyzed to identify key factors that connect immunoaged/inflammaged conditions with bone formation after long bone fracture. Immunological facets, progenitor cells, the microbiome, and confounders were monitored locally at the injury site and systemically in relation to healing outcomes in 12-month-old mice with distinct individual levels of immunoaging. Bone tissue formation during healing was delayed in the immunoaged group and could be associated with significant changes in cytokine levels. A prolonged and amplified pro-inflammatory reaction was caused by upregulated immune cell activation markers, increased chemokine receptor availability and a lack of inhibitory signaling. In immunoaged mice, interleukin-22 was identified as a core cell signaling protein that played a central role in delayed healing. Therapeutic neutralization of IL-22 reversed this specific immunoaging-related disturbed healing. Immunoaging was found to be an influencing factor of decreased regenerative capacity in aged individuals. Furthermore, a novel therapeutic strategy of neutralizing IL-22 may successfully rejuvenate healing in individuals with advanced immune experiences.

The multifaceted roles of macrophages in bone regeneration: A story of polarization, activation and time.

To present knowledge, macrophages are found in all tissues of the human body. They are a cell population with high plasticity which come with a multitude of functions which appear to be adapted to the respective tissue niche and micro-environment in which they reside. Bone harbors multiple macrophage subpopulations, with the osteoclasts as classical representative of a bone resorbing cells and osteomacs as a bone tissue resident macrophage first described by the expression of F4/80. Both subtypes are found throughout all phases in bone healing. In vivo data on bone regeneration have demonstrated their essential role in initiating the healing cascade (inflammatory phase) but also of the later phases of healing (e.g. endochondral and intramembranous bone formation). To participate in such diverse processes macrophages have to be highly plastic in their functionality. Thus, the widely used M1/M2 paradigm to distinguish macrophage subpopulations may not mirror the comprehensive role of the dynamics of macrophage plasticity. From a clinical perspective it is especially relevant to distinguish what drives macrophages in impaired healing scenarios, implant loosening or infections, where their specific role of a misbalanced inflammatory setting is so far only partially known. With this review we aim at illustrating current knowledge and gaps of knowledge on macrophage plasticity and function during the cascades of regeneration and reconstitution of bone tissue. We propose aspects of the known biological mechanisms of macrophages and their specific subsets that might serve as targets to control their function in impaired healing and eventually support a scar-free regeneration. STATEMENT OF SIGNIFICANCE: Macrophages are essential for successful regeneration. In scar-free healing such as in bone, a complete failure of healing was shown if macrophages were depleted; the M1/M2 switch appears to be key to the progression from pro-inflammation to regeneration. However, experimental data illustrate that the classical M1/M2 paradigm does not completely mirror the complexity of observed macrophage functions during bone healing and thus demands a broader perspective. Within this review we discuss the high degree of plasticity of macrophages and the relevant contribution of the different and more specific M2 subtypes (M2a-M2f) during (bone) regeneration. It summarizes the versatile roles of macrophages in skeletal regeneration and thereby highlights potential target points for immunomodulatory approaches to enable or even foster bone repair.

Immune Modulation to Enhance Bone Healing-A New Concept to Induce Bone Using Prostacyclin to Locally Modulate Immunity.

Within an aging population, fracture incidences will rise and with the augmented risks of impaired healing the overall risk of delayed bone regeneration will substantially increase in elderly patients. Thus, new strategies to rescue fracture healing in the elderly are highly warranted. Modulating the initial inflammatory phase toward a reduced pro-inflammation launches new treatment options for delayed or impaired healing specifically in the elderly. Here, we evaluated the capacity of the prostacyclin analog Iloprost to modulate the inflammatory phase toward a pro-regenerative milieu using in vitro as well as in vivo model systems. In vitro, Iloprost administration led to a downregulation of potential unfavorable CD8+ cytotoxic T cells as well as their pro-inflammatory cytokine secretion profile. Furthermore, Iloprost increased the mineralization capacity of osteogenic induced mesenchymal stromal cells through both direct as well as indirect cues. In an in vivo approach, Iloprost, embedded in a biphasic fibrin scaffold, decreased the pro-inflammatory and simultaneously enhanced the anti-inflammatory phase thereby improving bone healing outcome. Overall, our presented data confirms a possible strategy to modulate the early inflammatory phase in aged individuals toward a physiological healing by a downregulation of an excessive pro-inflammation that otherwise would impair healing. Further confirmation in phase I/II trials, however, is needed to validate the concept in a broader clinical evaluation.

Individual Effector/Regulator T Cell Ratios Impact Bone Regeneration.

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.

Experience in the Adaptive Immunity Impacts Bone Homeostasis, Remodeling, and Healing.

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 naïve 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 naïve composition. While this rescue was found to be significant in young individuals, aged mice only showed a trend toward the reconstitution of a more naïve 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.

Macrophages in bone fracture healing: Their essential role in endochondral ossification.

In fracture healing, skeletal and immune system are closely interacting through common cell precursors and molecular mediators. It is thought that the initial inflammatory reaction, which involves migration of macrophages into the fracture area, has a major impact on the long term outcome of bone repair. Interestingly, macrophages reside during all stages of fracture healing. Thus, we hypothesized a critical role for macrophages in the subsequent phases of bone regeneration. This study examined the impact of in vivo induced macrophage reduction, using clodronate liposomes, on the different healing phases of bone repair in a murine model of a standard closed femoral fracture. A reduction in macrophages had no obvious effect on the early fracture healing phase, but resulted in a delayed hard callus formation, thus severely altering endochondral ossification. Clodronate treated animals clearly showed delayed bony consolidation of cartilage and enhanced periosteal bone formation. Therefore, we decided to backtrack macrophage distribution during fracture healing in non-treated mice, focusing on the identification of the M1 and M2 subsets. We observed that M2 macrophages were clearly prevalent during the ossification phase. Therefore enhancement of M2 phenotype in macrophages was investigated as a way to further bone healing. Induction of M2 macrophages through interleukin 4 and 13 significantly enhanced bone formation during the 3week investigation period. These cumulative data illustrate their so far unreported highly important role in endochondral ossification and the necessity of a fine balance in M1/M2 macrophage function, which appears mandatory to fracture healing and successful regeneration.

T Lymphocytes Influence the Mineralization Process of Bone.

Bone is a unique organ able to regenerate itself after injuries. This regeneration requires the local interplay between different biological systems such as inflammation and matrix formation. Structural reconstitution is initiated by an inflammatory response orchestrated by the host immune system. However, the individual role of T cells and B cells in regeneration and their relationship to bone tissue reconstitution remain unknown. Comparing bone and fracture healing in animals with and without mature T and B cells revealed the essential role of these immune cells in determining the tissue mineralization and thus the bone quality. Bone without mature T and B cells is stiffer when compared to wild-type bone thus lacking the elasticity that helps to absorb forces, thus preventing fractures. In-depth analysis showed dysregulations in collagen deposition and osteoblast distribution upon lack of mature T and B cells. These changes in matrix deposition have been correlated with T cells rather than B cells within this study. This work presents, for the first time, a direct link between immune cells and matrix formation during bone healing after fracture. It illustrates specifically the role of T cells in the collagen organization process and the lack thereof in the absence of T cells.

Immune modulation as a therapeutic strategy in bone regeneration.

We summarize research approaches and findings on bone healing and regeneration that were presented at a workshop at the 60th annual meeting of the Orthopedic Research Society (ORS) in New Orleans in 2014. The workshop was designed to discuss the role of inflammation in bone regeneration in the context of fundamental biology, and to develop therapeutic strategies that involve immune modulation. Delayed or non-healing of bone is a major clinical problem, with around 10% of fracture patients suffering from unsatisfying healing outcomes. Inflammation is traditionally seen as a defense mechanism, but was recently found essential in supporting and modulating regenerative cascades. In bone healing, macrophages and T- and B-cells interact with progenitor cells, bone forming osteoblasts and remodeling osteoclasts. Among the cells of the innate immunity, macrophages are promising candidates for targets in immune-modulatory interventions that would overcome complications in bone healing and bone-related diseases. Among the cells of the adaptive immune system, CD8+ T cells have been shown to have a negative impact on bone fracture healing outcome, whereas regulatory T cells could be promising candidates that have a positive, modulating effect on bone fracture healing. This workshop addressed recent advances and key challenges in this exciting interdisciplinary research field.

T and B cells participate in bone repair by infiltrating the fracture callus in a two-wave fashion.

Fracture healing is a regenerative process in which bone is restored without scar tissue formation. The healing cascade initiates with a cycle of inflammation, cell migration, proliferation and differentiation. Immune cells invade the fracture site immediately upon bone damage and contribute to the initial phase of the healing process by recruiting accessory cells to the injury site. However, little is known about the role of the immune system in the later stages of fracture repair, in particular, whether lymphocytes participate in soft and hard callus formation. In order to answer this question, we analyzed femoral fracture healing in mice by confocal microscopy. Surprisingly, after the initial inflammatory phase, when soft callus developed, T and B cells withdrew from the fracture site and were detectable predominantly at the femoral neck and knee. Thereafter lymphocytes massively infiltrated the callus region (around day 14 after injury), during callus mineralization. Interestingly, lymphocytes were not found within cartilaginous areas of the callus but only nearby the newly forming bone. During healing B cell numbers seemed to exceed those of T cells and B cells progressively underwent effector maturation. Both, osteoblasts and osteoclasts were found to have direct cell-cell contact with lymphocytes, strongly suggesting a regulatory role of the immune cells specifically in the later stages of fracture healing.