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Callus distraction is sometimes associated with a delay in the maturation process and serious complications. It is believed that these complications are often caused by instability of the bone segment fixation. Typical fixation devices, such as ring-fixators, show significant deformations in all directions under external loading and muscle forces. This leads to axial compression and tension as well as shear movements in the healing area. Herein we investigated the hypothesis that the direction of interfragmentary movement after callus distraction affects the bone formation and revascularization during the maturation process. Two custom fixator systems were designed to apply a protocol of lateral callus distraction and subsequent cyclic stimulation of the regenerate tissue. One fixator system was used to apply either compressive or tensile stimulation while the other was used to apply shearing stimulation. The fixators were applied to the tibial surface of the right hind leg of sheep specimens. During lateral callus distraction, a titanium plate was elevated by 0.275 mm perpendicular to the long axis of the bone twice daily, resulting in a 5.5 mm gap at the end of the ten-day distraction phase. Following a seven-day consolidation phase, the regenerate in the gap between tibial cortex and titanium plate was stimulated once daily by cyclic movement for 120 cycles. The stimulation was applied for 18 days with amplitudes of 0.6 mm in compression (Group C) or tension (Group T), or a 1.0 mm shear amplitude (Group S). Seven weeks postoperatively the specimens were analyzed for quantity of bone formation, the presence of cartilage and fibrous tissue, and blood vessel density. There was a significantly higher blood vessel density (4.6 ± 1.6%) in Group C than in Group T (1.2 ± 0.4%) or Group S (1.0 ± 0.5%) (p < 0.01). The amount of bone was significantly higher in Group C (25.6% ± 13.0%) than in Group T (13.5 ± 4.9%) (p < 0.05). Group S showed a similar amount of bone (14.0 ± 10.7%) to Group T. The results show that bone formation and revascularization are dependent on the direction of interfragmentary movement and that the cyclic compression best stimulates the healing process.

Improving revascularization is one of the major measures in fracture treatment. Moderate local inflammation triggers angiogenesis, whereas systemic inflammation hampers angiogenesis. Previous studies showed that Akkermansia muciniphila, a gut probiotic, ameliorates systemic inflammation by tightening the intestinal barrier. In this study, fractured mice intragastrically administrated with A. muciniphila were found to display better fracture healing than mice treated with vehicle. Notably, more preosteclasts positive for platelet-derived growth factor-BB (PDGF-BB) were induced by A. muciniphila at 2 weeks post fracture, coinciding with increased formation of type H vessels, a specific vessel subtype that couples angiogenesis and osteogenesis, and can be stimulated by PDGF-BB. Moreover, A. muciniphila treatment significantly reduced gut permeability and inflammation at the early stage. Dextran sulfate sodium (DSS) was used to disrupt the gut barrier to determine its role in fracture healing and whether A. muciniphila still can stimulate bone fracture healing. As expected, A. muciniphila evidently improved gut barrier, reduced inflammation and restored the impaired bone healing and angiogenesis in DSS-treated mice. Our results suggest that A. muciniphila reduces intestinal permeability and alleviates inflammation, which probably induces more PDGF-BB+ preosteoclasts and type H vessel formation in callus, thereby promoting fracture healing. This study provides the evidence for the involvement of type H vessels in fracture healing and suggests the potential of A. muciniphila as a promising strategy for bone healing. This article has an associated First Person interview with the first author of the paper.

Bone injury induces an inflammatory response that involves neutrophils, macrophages and other inflammatory cells. The recruitment of inflammatory cells to sites of injury occurs in response to specific signaling pathways. The CC chemokine receptor type 2 (CCR2) is crucial for recruiting macrophages, as well as regulating osteoclast function. In this study, we examined fracture healing in Ccr2−/− mice. We first demonstrated that the expression of Ccr2 transcripts and the filtration of macrophages into fracture calluses were most robust during the early phases of fracture healing. We then determined that the number of macrophages at the fracture site was significantly lower in Ccr2−/− mice compared with wild-type controls at 3 days after injury. As a result, impaired vascularization, decreased formation of callus, and delayed maturation of cartilage were observed at 7 days after injury in mutant mice. At day 14, Ccr2−/− mice had less bone in their calluses. At day 21, Ccr2−/− mice had larger calluses and more bone compared with wild-type mice, suggesting a delayed remodeling. In addition, we examined the effect of Ccr2 mutation on osteoclasts. We found that a lack of Ccr2 did not affect the number of osteoclasts within fracture calluses at 21 days after injury. However, Ccr2−/− osteoclasts exhibited a decreased ability to resorb bone compared with wild-type cells, which could contribute to the delayed remodeling of fracture calluses observed in Ccr2−/− mice. Collectively, these results indicate that a deficiency of Ccr2 reduces the infiltration of macrophages and impairs the function of osteoclasts, leading to delayed fracture healing.

To investigate the influence of the stability of an osteotomy fixation on the local vascularization and tissue differentiation in callus healing, a transverse osteotomy of the right metatarsal with a gap size of 2 mm was performed in 10 sheep and stabilized with an external fixator. This fixator permitted a defined axial movement. Two groups of 5 sheep were each operated upon to allow 0.2 mm (group A) or 1 mm (group B) of axial movement. Nine weeks after surgery, the callus was dissected and histological sections prepared. The type of tissue and the vessel distribution were determined. Larger interfragmentary movements led to significantly more fibrocartilage (small axial movement A: 6.2%, large axial movement B: 21.6%) and significantly less bone formation (A: 38.2%, B: 26.3%). On average, and particularly close to the periosteum the number of vessels in the callus healing area was greater in the group with smaller movements than in the group with larger movements. There was a significant difference between the distribution of small (<20 μm) and large (>40 μm) vessels across the whole healing area for both groups. Whereas the large vessels showed maximum density in the medullary cavity, the small vessels showed the highest frequency in the peripheral part of the periosteal callus.

G protein coupled receptor kinase 2 (GRK2) interacting protein-1 (GIT1), is a scaffold protein that plays an important role in angiogenesis and osteoclast activity. We have previously demonstrated that GIT1 knockout (GIT1 KO) mice have impaired angiogenesis and dysregulated osteoclast podosome formation leading to a reduction in the bone resorbing ability of these cells. Since both angiogenesis and osteoclast-mediated bone remodeling are involved in the fracture healing process, we hypothesized that GIT1 participates in the normal progression of repair following bone injury. In the present study, comparison of fracture healing in wild type (WT) and GIT1 KO mice revealed altered healing in mice with loss of GIT1 function. Alcian blue staining of fracture callus indicated a persistence of cartilagenous matrix in day 21 callus samples from GIT1 KO mice which was temporally correlated with increased type 2 collagen immunostaining. GIT1 KO mice also showed a decrease in chondrocyte proliferation and apoptosis at days 7 and 14, as determined by PCNA and TUNEL staining. Vascular microcomputed tomography analysis of callus samples at days 7, 14 and 21 revealed decreased blood vessel volume, number, and connection density in GIT1 KO mice compared to WT controls. Correlating with this, VEGF-A, phospho-VEGFR2 and PECAM1 (CD31) were decreased in GIT1 KO mice, indicating reduced angiogenesis with loss of GIT1. Finally, calluses from GIT1 KO mice displayed a reduced number of tartrate resistant acid phosphatase-positive osteoclasts at days 14 and 21. Collectively, these results indicate that GIT1 is an important signaling participant in fracture healing, with gene ablation leading to reduced callus vascularity and reduced osteoclast number in the healing callus.

Summary This study examined the role of estrogen receptor (ER) beta during mouse femoral fracture healing by employing ER knockout (KO) mice. The fracture healing in KO mice was enhanced in the early stage of neovascularization and the middle stage of endochondral ossification. Introduction This study was conducted to examine the role of ER beta during fracture healing. Methods Female ERbeta knockout (KO) mice (18 weeks old) and age-matched female wild-type (WT) mice underwent open osteotomy on the right femur. They were sacrificed at 1, 2, 4 and 6 weeks post-fracture. The sera and callus samples were subjected to the following analyses: micro-computed tomography (CT)-based angiography, micro-CT evaluation, histological examination, histomorphometry examination, real-time polymerase chain reaction (PCR) analysis, biochemical marker, and mechanical testing. Results Micro-CT-based angiography showed that the total vessel volume at the fracture site was larger in the KO group than the WT group at 1 and 2 weeks post-fracture. Micro-CT analysis revealed that the callus volume was significantly higher in the KO group from week 2 to week 4 post-fracture when compared with the WT group consistent with the histological data. Analysis of biochemical markers indicated that circulating P1NP levels in the KO mice were significantly higher than in the WT mice from week 2 to week 4 and that temporal expression of circulating C-terminal telopeptide of type I collagen (CTX) levels was also higher in the KO mice than in the WT mice. These results were consistent with quantitative real-time PCR analysis. The ultimate load, stiffness, and energy to failure were significantly higher in the KO mice than in the WT mice at week 4. Conclusions The fracture healing in KO mice was enhanced in the early stage of neovascularization and the middle stage of endochondral ossification, but not by the end of healing. Blockade of ERbeta can be considered as another therapeutic strategy for osteoporotic fracture and non-union fracture.

Tissue-engineered bone (TEB) has shown to be an effective alternative to conventional gold-standard autogenous bone for the repair of critically sized bone defects (CSBD). Moderate axial interfragmentary movement (IFM) has been shown to promote bone healing in conventional models. This study explored the use of IFM to enhance the capacity of TEB in the repair of CSBD using a goat model. CSBD were created in a goat model. Dynamic intramedullary rods designed to supply axial IFMs within 10% of the interfragmentary strain were used to stabilize CSBD goat femur models, whose bone defects were filled with TEB. Bone regeneration was evaluated using radionuclide bone imaging, roentgenographic analysis, periosteal callus area, computed tomography value score, biomechanical analysis, and histological observation. Compared with the static intramedullary rods, the dynamic intramedullary rod group showed an increase in early-stage callus formation and blood supply to the callus tissue, better differentiation of fibrous and cartilaginous tissue into bone tissue, improved strength and stiffness of callus tissue in late-stage healing, and overall better functional recovery of the goat femur. This showed that moderate axial IFM could promote the osteogenesis and reconstruction of TEB in vivo.

The benefits of vitamin D3 on fracture healing have been commonly demonstrated in vitamin-D3-depleted animal models. The aim of this study was to examine the effects of a single high dose of vitamin D3 on fracture healing in a healthy animal model, which has not been previously reported. Twenty healthy young adult guinea pigs were randomly divided into groups as 'control' and 'vitamin D', and their right tibias were fractured with digital manipulation. Guinea pigs in vitamin D group were injected intramuscularly with 50,000 i.u./kg of vitamin D3. The animals were killed at 7, 14, 21 and 28 days following fracture. Ultrastructural analysis of the harvested tibias revealed that a single high dose of vitamin D3 stimulated fracture healing. The observed effects at the fracture zone in a healthy animal model included advancement of blood supply, acceleration of synthesis and organization of collagen fibres, acceleration of the proliferation and differentiation of osteoprogenitor cells, and activation of the mineralization of the matrix.

Erythropoietin (EPO)/erythropoietin receptor (EPOR) signaling is involved in the development and regeneration of several non-hematopoietic tissues including the skeleton. EPO is identified as a downstream target of the hypoxia inducible factor-α (HIF-α) pathway. It is shown that EPO exerts a positive role in bone repair, however, the underlying cellular and molecular mechanisms remain unclear. In the present study we show that EPO and EPOR are expressed in the proliferating, pre-hypertrophic and hypertrophic zone of the developing mouse growth plates as well as in the cartilaginous callus of the healing bone. The proliferation rate of chondrocytes is increased under EPO treatment, while this effect is decreased following siRNA mediated knockdown of EPOR in chondrocytes. EPO treatment increases biosynthesis of proteoglycan, accompanied by up-regulation of chondrogenic marker genes including SOX9, SOX5, SOX6, collagen type 2, and aggrecan. The effects are inhibited by knockdown of EPOR. Blockage of the endogenous EPO in chondrocytes also impaired the chondrogenic differentiation. In addition, EPO promotes metatarsal endothelial sprouting in vitro. This coincides with the in vivo data that local delivery of EPO increases vascularity at the mid-stage of bone healing (day 14). In a mouse femoral fracture model, EPO promotes cartilaginous callus formation at days 7 and 14, and enhances bone healing at day 28 indexed by improved X-ray score and micro-CT analysis of microstructure of new bone regenerates, which results in improved biomechanical properties. Our results indicate that EPO enhances chondrogenic and angiogenic responses during bone repair. EPO's function on chondrocyte proliferation and differentiation is at least partially mediated by its receptor EPOR. EPO may serve as a therapeutic agent to facilitate skeletal regeneration.

Bone fracture healing is achieved through the proliferation and differentiation of stem cells, while bone marrow stem cells (BMSCs) contribute to endochondral ossification. During fracture healing, mesenchymal progenitor cells first form a cartilaginous blastema that becomes vascularized to recruit precursor cells of osteoblasts through the bone morphogenetic protein 2 (Bmp2)/Smad-dependent Runx2 pathway. Statins deplete geranylgeranyl diphosphate (GGPP), which participates in the regulation of BMSCs differentiation, through the inhibition of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, leading to impaired protein geranylgeranylation, which strongly impacts the bone synthesis induced by Bmp2. Accordingly, we would like to investigate the role of geranylgeranyl diphosphate synthase 1 (Ggps1) in bone fracture via endochondral ossification in mice. We used a Cre-loxP system, namely the tamoxifen-inducible Collagen 2-CreERT2 Ggps1 fl/fl, to eliminate specifically the Ggps1 activity in chondrocytes of 8–10-week-old mice. We found that the endochondral bone formation, calcification and vasculogenesis of the bony callus were accelerated in fractures in Ggps1−/−mice. Together, the results of this study confirm that the specific deletion of Ggps1, using the Collagen 2-CreERT2 mice, will accelerate the fracture healing process by activating the Bmp2/Smad-dependent Runx2 pathway. In addition, we managed to improve the fracture healing process by inhibiting the Ggps1 activity and its related products with statin drugs.