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.

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.

Age-dependent decline in fin regenerative capacity in the short-lived fish Nothobranchius furzeri.

The potential to regenerate declines with age in a wide range of organisms. A popular model system to study the mechanisms of regeneration is the fin of teleost fish, which has the ability to fully regrow upon amputation. Here, we used the short-lived killifish Nothobranchius furzeri to analyse the impact of aging on fin regeneration in more detail. We observed that young fish were able to nearly completely (98%) regenerate their amputated caudal fins within 4 weeks, whereas middle-aged fish reached 78%, old fish 57% and very old fish 46% of their original fin size. The difference in growth rate between young and old fish was already significant at 3 days post amputation (dpa) and increased with time. We therefore hypothesized that early events are crucial for the age-related differences in regenerative capacity. Indeed, we could observe a higher percentage of proliferating cells in early regenerating fin tissue of young fish compared with aged fish and larger fractions of apoptotic cells in aged fish. Furthermore, young fish showed peak upregulation of several genes involved in fgf and wnt/ß-catenin signalling at an earlier time point than old fish. Our findings suggest that regenerative processes are initiated earlier and that regeneration overall is more efficient in younger fish.