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.

Bioactive coatings have the potential to improve the bony integration of mechanically loaded orthopedic ceramic implants. Using the concept of mimicking the natural bone surface, four different coatings of varying thickness on a zirconia toughened alumina (ZTA) ceramic implant were investigated regarding their osseointegration in a drill-hole model in sheep. The hypothesis that a bioactive coating of ZTA ceramics would facilitate cancellous bone integration was investigated. The bioactive coatings consisted of either a layer of covalently bound multi phosphonate molecules (chemical modification = CM), a nano hydoxyapatite coating (HA), or two different bioactive glass (BG) coatings in micrometer thickness, forming a hydroxyl-carbonate apatite layer on the implant surface in vivo (dip-coated 45S5 = DipBG; sol-gel 70S30C = SGBG). Coated surfaces were characterized by scanning electron microscopy and X-ray photoelectron spectroscopy. After 12 weeks, osseointegration was evaluated via mechanical push-out testing and histology. HA enhanced the maximum push-out force (HA: mean 3573.85 ± 1119.91 N; SGBG: mean 1691.57 ± 986.76 N; p = 0.046), adhesive shear strength (HA: mean 9.82 ± 2.89 MPA; SGBG: mean 4.57 ± 2.65 MPA; p = 0.025), and energy release rate (HA: mean 3821.95 ± 1474.13 J/mm2; SGBG: mean 1558.47 ± 923.47 J/mm2; p = 0.032) compared to SGBG. The implant-bone interfacial stiffness increased by CM compared to SGBG coating (CM: mean 6258.06 ± 603.80 N/mm; SGBG: mean 3565.57 ± 1705.31 n/mm; p = 0.038). Reduced mechanical osseointegration of SGBG coated implants could be explained histologically by a foreign body reaction surrounding the implants.

The treatment of osteochondral defects in joint cartilage remains challenging due to its limited repair capacity. This study presents a metallic osteochondral plug with hydroxyapatite (HA)-coated cap edges for improved implant-tissue contact. The hypothesis was that improved attachment prevents from synovial fluid-influx and thereby avoids osteolysis and resulting implant instability.

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.

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. Summary 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.

Anne-Marie Pobloth, Hanna Schell, Ansgar Petersen, Katleen Beierlein, Christian Kleber, Katharina Schmidt-Bleek, Georg N. Duda Julius Wolff Institut, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany Berlin-Brandenburg Center for Regenerative Therapies, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Center for Musculoskeletal Surgery, Charité Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany Department of Orthopaedic and Trauma Surgery, University Hospital Carl Gustav Carus, TU Dresden, Germany

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.

Large segmental bone defect reconstruction with sufficient functional restoration is one of the most demanding challenges in orthopaedic surgery. Available regenerative treatment options, as the vascularized bone graft transfer, the Masquelet technique or the Ilizarov distraction osteogenesis, are associated with specific indications and distinct limitations. As an alternative, a hollow cylindrical ceramic-polymer composite scaffold (β-tricalcium phosphate and poly-lactid co-ε- caprolactone), facilitating a strong surface guiding effect for tissue ingrowth (group 1; n = 6) was investigated here. In combination with an additional autologous, cancellous bone graft filling, the scaffold's ability to work as an open-porous membrane to improve the defect healing process was analysed (group 2; n = 6). A novel model of a critical size (40 mm) tibia osteotomy defect stabilized with an external hybrid-ring fixator, was established in sheep. Segmental defect regeneration and tissue organization in relation to the scaffold were analysed radiologically, (immune-) histologically, and with second-harmonic generation imaging 12 weeks after surgery. The scaffold's tubular shape and open-porous structure controlled the collagen fibre orientation within the bone defect and guided the following mineralization process along the scaffold surface. In combination with the osteoinductive stimulus, a unilateral bony bridging of the critically sized defect was achieved in one third of the animals. The external hybrid-ring fixator was appropriate for large segmental defect stabilization in sheep.

To allow bone defect regeneration, autologous bone grafting still represents the gold standard. However, autograft harvesting has limitations, including an additional surgery, donor site morbidity, and limited availability. Demineralized bone matrix (DBM) would represent an alternative, yet lacks sufficient osteoinductive properties. Combining DBM with a potent agent, such as bone morphogenetic protein-2 (BMP-2) might be a feasible alternative approach, optimizing an established grafting material with strong osteoinductive properties. A unique mixing device has been developed that enables perioperative handling to reach a homogeneous and standardized paste for bone defect filling. DBM proved in vitro to be a suitable carrier for BMP-2, with a documented release over 56 days at concentrations sufficient to stimulate osteogenic differentiation. At the end of the elution experiment, 56 days, bioactive BMP was still captured within the DBM. Using a sheep drill hole defect model, DBM perioperatively mixed with BMP-2 showed strong osteoinductive properties comparable to those of autologous bone and outnumbering the one of DBM alone or empty defects. Bone defect healing was enabled at diaphyseal and metaphyseal defects and thus BMP-2-doped DBM represented an easy perioperative enriching method and an efficient carrier for BMP-2. With the comparability to the clinical gold standard autologous bone, DBM mixed with BMP-2 might serve as possible alternative grafting material enabling a controlled osteogenic stimulation.

Fracture treatment is an old endeavour intended to promote bone healing and to also enable early loading and regain of function in the injured limb. However, in today’s clinical routine the healing potential of the initial fracture haematoma is still not fully recognized. The Arbeitsgemeinschaft für Osteosynthesefragen (AO) formed in Switzerland in 1956 formulated four AO principles of fracture treatment which are still valid today. Fracture treatment strategies have continued to evolve further, as for example the relatively new concept of minimally invasive plate osteosynthesis (MIPO). This MIPO treatment strategy harbours the benefit of an undisturbed original fracture haematoma that supports the healing process. The extent of the supportive effect of this haematoma for the bone healing process has not been considered in clinical practice so far. The rising importance of osteoimmunological aspects in bone healing supports the essential role of the initial haematoma as a source for inflammatory cells that release the cytokine pattern that directs cell recruitment towards the injured tissue. In reviewing the potential benefits of the fracture haematoma, the early development of angiogenic and osteogenic potentials within the haematoma are striking. Removing the haematoma during surgery could negatively influence the fracture healing process. In an ovine open tibial fracture model the haematoma was removed 4 or 7 days after injury and the bone that formed during the first two weeks of healing was significantly reduced in comparison with an undisturbed control. These findings indicate that whenever possible the original haematoma formed upon injury should be conserved during clinical fracture treatment to benefit from the inherent healing potential.

Background and objectives The critical interdependency between the skeletal and immune system has been demonstrated for the fracture healing process. However, the current state of the art is contradictory. On one hand, lymphopenic mice display alleged better healing after injury. On the other hand, several studies attribute either a positive or negative role to T and B cells in bone biology, autoimmunity and fracture healing. The objective of this study is to elucidate the specific role of T and B cells in fracture repair toward early callus mineralization, bone quality, migration of osteoblast precursors and Collagen I deposition. Materials and methods Unilateral closed femoral fractures were produced in RAG1-/-, TCR βδ chain -/-, JHT -/- and C57BL/6 wild-type mice. Bone healing was assessed by histology, biomechanical testing and fluorochrome deposition. Deposition and structural properties of CollagenI were studied by second harmonic and confocal microscopy. Callus gene expression was determined by microarray analysis. Osteoblast precursors, T and B cell migration in the callus were visualised by confocal microscopy. Results Fractured bones of RAG -/- mice mineralized faster, but were much less capable to withstand deformation. Similarly, RAG -/- mice deposited bone quicker during the earliest phases of healing, whereas at later stages underperformed relative to controls. RAG -/- mice also failed to deposit a cross gap layer of Collagen I which correlated with diminished immigration of osteoblast precursors in the peripheral callus. The remaining network of Collagen I fibres in these mice was found to be highly disordered. Interestingly, no alteration in osteoblast precursor migration was observed when only either T or B cells were absent. However, Collagen I deposition was strongly impaired in T cell but not in B cell deficient mice. There, Collagen I fibres failed to organise across the gap in the peripheral callus displaying a highly disordered pattern of deposition. Conclusions Overall our data show that lack of an adaptive immune system does not lead to better, but rather deregulated and suboptimal bone healing. These features were recapitulated in mice that specifically lacked T and not B cells.

BACKGROUND:New tissue engineering strategies for bone regeneration need to be investigated in a relevant preclinical large animal model before making the translation into human patients. Therefore, our interdisciplinary group established a simplified large animal screening model for intramembranous bone defect regeneration in cancellous and cortical bone.METHODS:Related to a well-established model of cancellous drill hole defect regeneration in sheep, both the proximal and distal epimetaphyseal regions of the femur and the humerus were used bilaterally for eight drill hole cancellous defects (Ø 6 mm, 15 mm depth). Several improvements of the surgical procedure and equipment for an easier harvest of samples were invented. For the inclusion of cortical defect regeneration, a total of eight unicortical diaphyseal drill holes (6 mm Ø) were placed in the proximal-lateral and distal-medial parts of the metacarpal (MC) and metatarsal (MT) diaphyseal bone bilaterally. Acting moments within a normal gait cycle in the musculoskeletal lower limb model were compared with the results of the biomechanical in vitro torsion test until failure to ensure a low accidental fracture risk of utilized bones (ANOVA, p < 0.05). The model was tested in vivo, using thirteen adult, female, black-face sheep (Ø 66 kg; ± 5 kg; age ≥ 2.5 years). In a two-step surgical procedure 16 drill holes were performed for the investigation of two different time points within one animal. Defects were left empty, augmented with autologous cancellous bone or soft bone graft substitutes.RESULTS:The in vitro tests confirmed this model a high comparability between drilled MC and MT bones and a high safety margin until fracture. The exclusion of one animal from the in vivo study, due to a spiral fracture of the left MC bone led to a tolerable failure rate of 8 %.CONCLUSIONS:As a screening tool, promising biomaterials can be tested in this cancellous and cortical bone defect model prior to the application in a more complex treatment site.

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.

Scaffold architecture guides bone formation. However, in critical-sized long bone defects additional BMP-mediated osteogenic stimulation is needed to form clinically relevant volumes of new bone. The hierarchical structure of bone determines its mechanical properties. Yet, the micro- and nanostructure of BMP-mediated fast-forming bone has not been compared with slower regenerating bone without BMP. We investigated the combined effects of scaffold architecture (physical cue) and BMP stimulation (biological cue) on bone regeneration. It was hypothesized that a structured scaffold directs tissue organization through structural guidance and load transfer, while BMP stimulation accelerates bone formation without altering the microstructure at different length scales. BMP-loaded medical grade polycaprolactone–tricalcium phosphate scaffolds were implanted in 30 mm tibial defects in sheep. BMP-mediated bone formation after 3 and 12 months was compared with slower bone formation with a scaffold alone after 12 months. A multiscale analysis based on microcomputed tomography, histology, polarized light microscopy, backscattered electron microscopy, small angle X-ray scattering and nanoindentation was used to characterize bone volume, collagen fiber orientation, mineral particle thickness and orientation, and local mechanical properties. Despite different observed kinetics in bone formation, similar structural properties on a microscopic and sub-micron level seem to emerge in both BMP-treated and scaffold only groups. The guiding effect of the scaffold architecture is illustrated through structural differences in bone across different regions. In the vicinity of the scaffold increased tissue organization is observed at 3 months. Loading along the long bone axis transferred through the scaffold defines bone micro- and nanostructure after 12 months.

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.

The mechanical properties and good biocompatibility of zirconium and some of its alloys make these materials good candidates for biomedical applications. The attractive in vivo performance of zirconium is mainly due to the presence of a protective oxide layer. In this preliminary study, the surface of pure zirconium modified by anodisation in acidic media at low potentials to enhance its barrier protection given by the oxides and osseointegration. Bare, commercially pure zirconium cylinders were compared to samples anodised at 30 V through electrochemical tests and scanning electron microscopy (SEM). For both conditions, in vivo tests were performed in a rat tibial osteotomy model. The histological features and fluoro-chrome- labelling changes of newly bone formed around the implants were evaluated on the non-decalcified sections 63 days after surgery. Electrochemical tests and SEM images show that the anodisation treatment increases the barrier effect over the material and the in vivo tests show continuous newly formed bone around the implant with a different amount of osteocytes in their lacunae depending on the region. There was no significant change in bone thickness around either kind of implant but the anodised samples had a significantly higher mineral apposition, suggesting that the anodisation treatment stimulates and assists the osseointegration process. We conclude that anodisation treatment at 30 V can stimulate the implant fixation in a rat model, making zirconium a strong candidate material for permanent implants.

Experimental data indicate that hypercapnic adidosis has anti-inflammatory effects. These anti-inflammatory effects may even be a beneficial property in case of low tidal volume ventilation with consecutive hypercapnic acidosis. It is unclear whether these anti-inflammatory effects predominate in critically ill patients who suffer from multiple pro- and anti-inflammatory insults like extracorporeal organ support (pro-inflammatory), metabolic acidosis (pro- and anti-inflammatory), as well as hypoxia (pro-inflammatory). Eighteen pigs were randomized into three groups, mechanically ventilated and connected to a continuous veno-venous hemofiltration (CVVH) as pro-inflammatory insult. A reference group with normal acid-base state obtained normoventilation; a normoxemic acidemia group obtained normoxemic, mixed acidemia due to infusion of lactic and hyperchloremic acid and low tidal volume ventilation, and in a hypoxemic acidemia group the mixed acidemia was paralleled by hypoxemia. Lung histology including pulmonary leukocyte invasion, blood gases, blood cell counts, and hemodynamics were examined. The histological examination of the lungs of acidemic pigs showed a suppressed invasion of leukocytes and thinner alveolar walls compared with normoventilated and with hypoxemic pigs. Enhanced congestion and alveolar red blood cells (RBCs) combined with an increase of the pulmonary artery pressure were observed in acidemic pigs in comparison with the reference group. Normoxemic acidemia reduced the pro-inflammatory reaction to the CVVH and mechanical ventilation in the ventilated lung areas in the form of pulmonary leukocyte invasion. However, this did not result in reduced scores for lung injury. Instead, an increased score for criteria which represent lung injury (congestion and alveolar RBCs) was observed in acidemic pigs.

PURPOSE:The influence of basal graft support combined to early loading following an osteochondral autograft procedure is unclear. It was hypothesized that bottomed grafts may allow for early mobilization by preventing graft subsidence and leading to better healing.METHODS:Osteochondral autografts were press fitted in the femoral condyles of 24 sheep (one graft per animal). In the unbottomed group (n = 12), a gap of 2 mm was created between graft and recipient bone base. In the bottomed group (n = 12), the graft firmly rested on recipient bone. Animals were allowed immediate postoperative weightbearing. Healing times were 3 and 6 months per group (n = 6 per subgroup). After killing, histological and histomorphometric analyses were performed.RESULTS:Unbottomed grafts at 3 months showed significantly more graft subsidence (P = 0.024), significantly less mineralized bone (P = 0.028) and significantly worse cartilage and subchondral bone plate healing (P = 0.034) when compared to bottomed grafts. At 6 months, no differences were seen. Compared to the native situation, unbottomed grafts showed significantly more graft subsidence (P = 0.024), whereas bottomed grafts did not. Cystic lesions were seen in both groups. Osteoclasts were closely related to the degree of bone remodelling.CONCLUSION:In the animal model, in the case of early loading, bottomed osteochondral autografts have less chance of graft subsidence. Evident subsidence negatively influences the histological healing process. In the osteochondral autograft procedure, full graft support should be aimed for. This may allow for early mobilization, diminish graft subsidence and improve long-term integration.

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)