Longitudinal Changes in Serum Markers of Bone Metabolism and Bone Material Strength in Premenopausal Women with Distal Radial Fracture.

BACKGROUND: Markers of bone metabolism (MBM) play an important role in fracture evaluation, and changes have been associated with increased fracture risk. The purpose of the present study was to describe changes in MBM in premenopausal women with distal radial fractures. METHODS: Premenopausal women with distal radial fractures (n = 34) and without fractures (controls) (n = 39) were recruited. Serum MBM in patients with distal radial fractures were obtained at the time of the initial presentation, 6 weeks, and 3, 6, and 12 months. MBM included 25(OH) vitamin D, PTH, osteocalcin, P1NP, BSAP, CTX, sclerostin, DKK1, periostin, and TRAP5b. Areal bone mineral density (aBMD) was assessed with dual x-ray absorptiometry, and the bone material strength index (BMSi) was assessed with microindentation. RESULTS: Most MBM reached peak levels at 6 weeks after the injury, including osteocalcin (+17.7%), sclerostin (+23.5%), and DKK1 (12.6%). Sclerostin was lower (-27.4%) and DKK1 was higher (+22.2%) at 1 year after the fracture. CTX declined below baseline levels at 6 and 12 months, whereas TRAP5b, BSAP, and periostin did not significantly change. At 12 months, sclerostin was lower (p = 0.003) and DKK1 was higher (p = 0.03) in the distal radial fracture group than in the control group. Greater fracture severity was associated with greater increases in P1NP and BSAP. aBMD and BMSi were not associated with fracture. CONCLUSIONS: Distal radial fractures caused increases in several MBM, which typically peaked at 6 weeks after injury and gradually decreased over 6 months. Sclerostin and DKK1 remained below and above baseline at 1 year, respectively. Increasing fracture severity resulted in larger changes in MBM. aBMD and BMSi did not discriminate between patients with distal radial fractures and controls. Continued efforts to identify markers of skeletal fragility in young women are warranted to mitigate future fracture risk. LEVEL OF EVIDENCE: Prognostic Level II. See Instructions for Authors for a complete description of levels of evidence.

Prostate-Specific Membrane Antigen Expression in Distal Radius Fracture.

A 79-year old man with prostate cancer under active surveillance for 5 years was referred for a PSMA-PET/MRI for re-evaluation because of a rising prostate-specific antigen value. PET/MRI revealed a ribbonlike tracer accumulation in a healing fracture of the distal radius. This case illustrates that PSMA expression may occur in healing bone fractures in the distal radius. It can be assumed that benign causes of tracer accumulations in the upper extremities are missed in PET/CT due to elevated position of the arms during image acquisition.

The vitamin D receptor expression in skeletal muscle of women with distal radius fracture.

We evaluated the vitamin D receptor (VDR) expression in the forearm flexor muscle of women with distal radius fracture. High VDR expression was associated with low appendicular lean mass index. INTRODUCTION: We aimed to evaluate the relationship between the VDR expression in the muscle cell and the muscle mass in women with a distal radius fracture (DRF). METHODS: We prospectively recruited 45 women over 50 years of age (mean age, 66 years) with DRF and acquired biopsy of the forearm flexor muscle. The muscle cross-sectional area (CSA) and VDR expression were measured using immunohistochemistry staining. The clinical parameters including grip strength, gait speed, body mass index (BMI), bone mineral density (BMD), and serum vitamin D levels were compared between patients grouped by appendicular lean mass index and were correlated with the VDR expression. RESULTS: Twelve patients (27%) showed a decreased appendicular lean mass index, less than the cut-off value of 5.4 kg/m2 which was suggested by the Asian Working Group for Sarcopenia. Patients with a low appendicular lean mass index had significantly lower muscle CSA (p = 0.037), but a higher VDR expression (p = 0.045) than those with higher indices. VDR expression was negatively correlated with BMI (r = - 0.417, p = 0.004) and appendicular lean mass index (r = - 0.316, p = 0.044). CONCLUSIONS: DRF patients with low appendicular lean mass index presented high VDR expression and low CSA in forearm muscle cells. This suggests that the VDR expression might be upregulated in the attempt to compensate for the decreasing muscle mass. Further studies are necessary to explore the role of VDR in the progression of sarcopenia.

Cell-free scaffolds with different stiffness but same microstructure promote bone regeneration in rabbit large bone defect model.

To promote bone healing, bone repair biomaterials are increasingly designed to incorporate growth factors. However, the impact of matrix mechanics of cell-free scaffold independent of microstructure on the osteogenic differentiation of endogenous osteoprogenitor cells orchestrating bone repair and regeneration remains not to be fully understood. In our recent study, three-dimensional (3D) scaffolds with different stiffness but same microstructure have been successfully fabricated by coating decellularized bone with collagen/hydroxyapatite (HA) mixture with different collagen rations. It has been demonstrated that the scaffold with optimal stiffness can induce the osteogenic differentiation of MSCs in vitro and in the subcutaneous tissue. The present in vivo study further investigated the repair efficiency of these scaffolds in a rabbit radius with a critical-sized segmental defect model and its potential mechanism. Micro-computed tomography (µ-CT), X-ray and histological analysis were carried out to evaluate the repair capacity of these scaffolds. The results demonstrated that the cell-free scaffold with optimal stiffness incorporation of endogenous osteoprogenitor cells significantly promoted the repair and reconstruction quality of mass bone defect. One of the crucial mechanisms was that hypoxia and stromal cell-derived factor-1α (SDF-1α) mediated mesenchymal stem cells (MSCs) migration by which matrix mechanics exerted influence on bone fracture healing. These findings suggested that only modulating the matrix stiffness of cell-free scaffold can be one of the most attractive strategies for promoting the progression of bone healing.

Superior angiogenic potential of GDF-5 and GDF-5(V453/V456) compared with BMP-2 in a rabbit long-bone defect model.

BACKGROUND: The clinical application of bone morphogenetic proteins such as BMP-2 and GDF-5 (growth and differentiation factor-5) may improve the outcome of bone defect repair. In addition to the osteoinductivity of BMPs, their angiogenic potential is important as an adequate blood supply is a prerequisite for bone-healing. We used a rabbit long-bone defect model to investigate whether angiogenicity and osteogenicity were correlated features of a BMP molecule by comparing the induction of blood vessel and bone formation by BMP-2, GDF-5, and a previously created swap mutant GDF-5V453/V456 (BB-1) with elevated BMP receptor-IA binding. METHODS: Microcomputed tomography and immunohistochemistry were used to assess early bone formation and neovascularization in 15-mm (critical-sized) rabbit radius defects treated with a growth factor-loaded collagen carrier. RESULTS: Blood vessel volume and surface area on days 7 and 14 after surgery were significantly greater in defects treated with GDF-5 and with BB-1 compared with controls (p < 0.05); BMP-2 enhanced vascularization on day 14 (p < 0.05). Cumulative data including both time points reflected increased vessel volume, intersection surface area, and number of vessels after treatment with GDF-5 and BB-1 compared with BMP-2 (p < 0.05), corresponding to the histology results. Each of the growth factors resulted in enhanced bone formation compared with controls on day 14 (p < 0.01), with BB-1 resulting in significantly more bone compared with GDF-5 as indicated by bone volume and surface area (p = 0.006). CONCLUSIONS: Both GDF-5 and BB-1 had high angiogenicity, and BB-1 outperformed GDF-5 with respect to osteogenicity. Strong induction of bone formation by BMP-2 and BB-1 was thus associated with BMP receptor-IA-dependent signaling, whereas the vascularization outcome was not. CLINICAL RELEVANCE: Although both BMP-2 and the GDF-5 variant BB-1 are good inducers of bone formation, BB-1 is especially promising for long-bone healing if high angiogenicity is desired along with high osteogenicity to promote recreation of optimal bone architecture.

[Promotion effect of nuclear factor kappa B p65 on early fracture healing of rat radius by elevating prostaglandins E2 production and regulating inhibitor of DNA binding 2 protein expression].

OBJECTIVE: Series of complicated molecule signal pathway are involved in the bone regeneration. To explore the possibility of nuclear factor kappa B (NF-kappaB) which is taken as the "key activation" during the fracture healing and provide the laboratory evidence for the gene therapy of nonunion or delayed union of fractures. METHODS: Thirty-three adult male Wistar rats (weighing 180-220 g) were selected and divided randomly into 4 groups: group A (the control group, n=3), the rigth lower segments of radius were injected with normal saline 0.3 mL for 7 days, once per day; group B (Bay 11-7082 injection group, n=6), the middle and distal radius were injected with normal saline containing 50 pmol/L NF-kappaB inhibitor Bay 11-7082 0.3 mL for 7 days, once per day; group C (fracture group, n=12), the right middle and distal radius were cut by a sharp scissors to form per fracture model; and group D (Bay 11-7082 treatment group, n=12), based on group C, 0.3 mL of 50 micromol/L Bay 11-7082 were injected into the fracture site for 7 days, once per day. The callus tissues were harvested at 3, 7, 14, and 28 days after fracture for Western blot analysis, alkaline phosphatase (ALP) activity assessment, prostaglandins E2 (PGE2) production assay, and histological observation. RESULTS: The rats of all groups were survival till the experiment completion. At 3 and 7 days after injection, there was no significant difference in the ALP activity and PGE2 production between group B and group A (P > 0.05); but group C was significantly higher than group A (P < 0.01) and group D was significantly lower than group A (P < 0.01). The expressions of NF-kappaB p65, bone morphogenetic protein 7 (BMP-7), and inhibitor of DNA binding 2 (Id2) were observed at fracture sites of 4 groups. There was no significant difference in the expressions of NF-kappaB p65, BMP-7, and Id2 between group B and group A (P > 0.05); the expressions of NF-kappaB p65 and BMP-7 were significantly higher and the expression of Id2 was significantly lower in group C than group A (P < 0.01); and the expressions of NF-kappaB p65 and BMP-7 were significantly lower and the expression of Id2 was significantly higher in group D than group A (P < 0.01). The histological observation showed that a lot of osseous callus formed in group C at 14 and 28 days. CONCLUSION: but osseous callus just began to form in group D at 28 days. NF-kappaB p65 can facilitate early fracture healing of rat radius by elevating the PGE2 production and regulating BMP-7 and Id2 expression.

[Expression of vascular endothelial growth factor and microvessel density in rabbit radius defects repaired with allogeneic and autogenic bone].

OBJECTIVE: To investigate the expression levels and significance of vascular endothelial growth factor (VEGF) and microvessel density (MVD) in rabbit radius defects repaired with allogeneic and autogenic bone. METHODS: Forty adult New Zealand rabbits were chosen, and 10 mm bone defect model was created in the bilateral radii of 28 experimental rabbits. The other 12 rabbits provided allogeneic bone under the standard of American Association of Tissue Bank. In the left side, allogeneic bone were used to repair bone defect (experimental group), equal capacity autogenous iliac bone was used in the right side (control group). Animals were sacrificed at 2, 4, 8, and 12 weeks postoperatively. Immunohistochemical method was used to determine the expression of VEGF, CD34 protein and MVD counting. Bone histomorphometric parameters, including percent trabecular area (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were measured by von Kossa staining undecalcified slices. The relation was analyzed between VEGF and MVD, histomorphometric parameters. RESULTS: The positive signals of VEGF protein were detected in cytoplasm of vascular endothelial cells, chondrocytes, osteoblasts, fibroblasts and osteoclasts. At 2 weeks, there was no significant difference in VEGF protein expression between experimental group and control group (P > 0.05); at 4 and 8 weeks, the expression of VEGF in control group was significantly higher than that in experimental group (P < 0.05); and at 12 weeks, there was no significant difference between two groups (P > 0.05). There was a positive correlation (P < 0.01) between VEGF expression and MVD value in two groups at 2, 4, 8, and 12 weeks postoperatively. There was no significant difference in bone histomorphometric parameters (BV/TV, Tb.Th, Tb.N, Tb.Sp) between two groups at 12 weeks postoperatively (P > 0.05), but there was a positive correlation between VEGF expression and parameters of BV/TV, Tb.Th, and Tb.N (P < 0.01); and a negative correlation between VEGF and Tb.Sp (P < 0.01). CONCLUSION: VEGF can express diversity at different time and positions, and the different expressions indicated various biology significances in the process of the bone healing. It can coordinate growth of cartilage and bone and profit vascular reconstruction of allogeneic bone. VEGF may participate in promoting osteogenesis in the course of allogeneic bone transplantation.

The effect of zymosan and the protective effect of various antioxidants on fracture healing in rats.

AIM: To investigate the effects of free oxygen radicals and various antioxidants on bone healing after experimental formation of fracture. MATERIALS AND METHODS: Fifty male rats were used and divided into five groups (ten rats in each). The right forelimbs of the rats were broken by bimanual compression method. One hour before this procedure, 5 ml/kg of intraperitoneal (i.p.) physiologic saline were given to the control Group 1. All 40 rats in the experimental Groups 2, 3, 4 and 5 were treated with i.p. zymosan at a dosage of 100 mg/kg to induce the production of free radicals by stimulating NADPH oxidase in polymorphonuclear leukocytes. Zymosan induction was stopped on the fifth post-fracture day. In addition to the zymosan, i.p. 1 g/kg/day of dimethyl sulfoxide were given to the animals in Group 3, 50 mg/kg/d of Ginko biloba Extract (EGb 761) in Group 4 and 500 mg/kg/day of vitamin C in Group 5. Radiographs of the fractures of all animals were obtained to assess callus formation, remodeling and bridging bone formation under ether anesthetics on postfracture day 7, 14 and 21. All rats were euthanized on day 22, and sections of the radius and ulna were examined both histologically with light and electron microscopy and ultrastructurally. Statistical analysis was made with Kruskal-Wallis variance analyze test and comparison between groups was performed by Dunn's multiple comparison test. RESULTS: An impairment of bone healing was observed in Group 2 inducted with purely zymosan. Variable results were obtained for bone healing in the groups treated with various antioxidants. There was very significant difference of fracture healing between Groups 1 and 2 both histologically and radiologically (P < 0.001). There was significant difference between Groups 2 and 5 radiologically (P < 0.05). CONCLUSION: Free oxygen radicals demonstrate a negative effect on fracture healing and vitamin C (an antioxidant) partially prevents the negative effect of zymosan on fracture healing.

Tracking expression of virally mediated BMP-2 in gene therapy for bone repair.

Ex vivo gene therapy using stem cells transduced with viral vectors is a useful method for delivering a therapeutic protein to augment bone repair in animal models. However, the duration of cell-mediated protein production and the fate of the transduced cells are unknown. We constructed an adenoviral vector encoding Myc epitope tagged bone morphogenetic protein (BMP)-2 gene (AdBMP-2). Rat bone marrow cells transduced with this vector produced biologically active BMP-2 protein, which was confirmed by Western blot analysis and alkaline phosphatase assay. Implantation of bone marrow cells infected ex vivo with AdBMP-2 caused orthotopic bone formation in mouse hindlimbs and bony union of critical-sized mouse radial defects. Immunohistochemical analysis revealed that rBMCs expressed Myc epitope-tagged BMP-2 protein for 14 days in vivo and became incorporated in the endochondral fracture callus. This novel adenovirus encoding for epitope-tagged BMP-2 can be used for immunohistochemical tracking of transduced cells in ex vivo gene therapy for bone repair.

Expression and activation of the BMP-signaling components in human fracture nonunions.

BACKGROUND: The potential use of bone morphogenetic proteins (BMPs) to promote bone-healing is of great interest to orthopaedic surgeons. Although the complex mechanism leading from the local presence of BMP (whether endogenous or exogenous) to bone formation is increasingly understood, limited information is available as to whether endogenous BMPs, their receptors, or other molecules involved in their signal transduction, such as Smad1, are present or disappear during the development of fracture nonunions. The purpose of the present study was to determine, by immunohistochemical analysis, whether BMPs, BMP receptors, or Smad1 disappear from tissues during the development of a fracture nonunion. METHODS: Twenty-one patients (average age, sixty-one years; range, thirty to eighty-five years) with a delayed union (four patients) or a nonunion (seventeen patients) were included. The average duration of the delayed union or nonunion was twenty-two months (range, 3.5 to 120 months). With use of immunohistochemical analysis, we studied the localization of BMP-2, BMP-4, and BMP-7 and their receptors BMPR-IA, BMPR-IB, and BMPR-II as well as pSmad1. With use of a pSmad1 antibody, we also studied whether the BMP receptors that were expressed were activated. RESULTS: The immunohistochemical localization of all seven BMP-signaling components was demonstrated in seventeen (81%) of the twenty-one patients. The remaining four patients lacked one or more of the components. Areas of newly formed bone had the highest percentage of positively staining cells, with the staining generally decreasing in areas remote from bone formation. However, even in areas of dense fibrous tissue and in specimens that lacked newly formed bone, immunostaining was still present. The staining patterns showed co-localization of the BMP-2, BMP-4, and BMP-7 proteins with the BMP receptors. The presence of pSmad1 signified the activated state of the BMP receptors, which implies that the BMP signal is transduced inside the cell.

[The effect of wumingyi chongji on BMP content in callus].

A kind of Chinese herb medicine--Wumingyi chongji could promote fracture healing had been proved. In order to disclose the mechanism, a standard fracture model was produced in 50 healthy male Newzealand rabbits. The animals were randomly divided into experimental group and control group. Another 5 rabbits without operation act as nomal. By immunohistochemical study, the bone morphogenetic protein (BMP) content in callus was calculated by computerized interacive morphometry (CIM) in the 1st, 2nd, 3rd, 4th, 5th week after fracture. The results were: BMP in callus was much more than in nomal (P < 0.01); BMP in callus increased from the 1st week to the 3rd week, decreased from 4th week to 5th week, and was more in experimental group in the 1st and 2nd week than in the control group (P < 0.01). The highest BMP content appeared in the 2nd week in the experimental group, while in the 3rd week in the control group. It was concluded that Wumingyi chongji could promote osteoblast to synthetize BMP, BMP induce preosteoblast change into osteoblast. Thus the rate of fracture healing could be increased.

[The experimental study on expression of BMP3 gene during fracture healing].

The role of BMP3 in fracture repair and its basic mechanisms at the molecular aspect has been studied. Fourty-eight New Zealand white rabbits with the fractures in the middle of bilateral radial shafts were used as animal models, and divided randomly into six groups for calluses at the 1st, 2nd, 3rd, 4th, 6th and 8th week after the onset of fracture. The levels and the cellular localizations of expression of BMP3 mRNA were investigated with the nucleotide hybridization techniques. The results revealed that BMP3 gene expression was highly increased in the early phase of fracture repair, and reached its peak at the second week (about 3.4-fold of that of the normal control). The strong expression of BMP3 gene was localized in mesenchymal cells, chondroblasts and osteoblasts. The results suggest that BMP3 plays an important role of bone-induction in the early stage of fracture repair and it works by the way of autocrine or/and paracrine pathway.

[An experiment study of the guided bone regeneration].

During the process of wound healing, different cellular components have varied speeds of migrating. By implanting a membrane, a space was created for selected cells, that is so called guided tissue regeneration. Since bone had the potential of regeneration, the concept of guided tissue regeneration was used in the process of bone regeneration in the present study, namely, guided bone regeneration. Defects of 10 mm-long were produced on bilateral radii of 10 adult New Zealand rabbits by surgery. The defects on experimental sides were bridged with silicone tube. The opposite side served as the control. Radiography of forearms of rabbits was taken weekly. Samples were treated with 3-point bending test and histology respectively. On experimental sides, by 3-4 weeks, new bone from the fractured ends grew into bone defect, seven of ten healed within 6-8 weeks. The other 2 only had a gap less than 1 mm left. No one healed on the control sides. The maximum value of 3-point bending test on experimental sides was 11.7 times greater than that of the control sides. It was shown that bone regenerated in the tube, and no callus formed out the tube by both gross samples and histologic examinations. On histology, the gap less than 1 mm on X-ray films was fibrocartilage zone, which connected regenerated bone from both distal and proximal bone ends. The defects on the control were occupied by connective tissue. In conclusion, guided bone regeneration also presents in long bone, that may provide a new method in the treatment of bone defect and promote fracture healing.

[Distribution and effectiveness of endogenic bone morphogenetic protein (BMP) in bone defect].

BMP is one of important factors in the pathophysiology of bone regeneration. Fifteen New Zealand rabbits were used in this experiment. We studied the distribution and effectiveness of endogenic BMP on a 10 mm bone defect of radius, by utilizing immunohistochemistry of BMP and quantitative computer imaging system. On the 3rd day, death of osteocytes and BMP positive blood clot were observed. The mesenchymal cells from periosteum and endoosteum, and osteoblast were also BMP positive. By quantitative study, we found there was a gradient distribution of BMP in bone defect, i.e, the value of BMP decreased gradually along the distance from the fracture ends. The maximal value of BMP was noted at the 1st week postoperation. In conclusion, two sources of endogenic BMP were found, one was from the absorption of necrotic tissue of fracture ends, the other was from the secretion of osteogenic mesenchymal cells during the process of bone regeneration. Nonunion of bone defect was caused in part by the gradient distribution of BMP. Accordingly, the concept of effective quantity of endogenic BMP was drawn rosen. It might be a new method in the treatment of bone defect by increasing the concentration of endogenic BMP and improving its distribution.

Regional bone mineral density changes after Colles' and forehand fractures.

Patients who sustain a second Colles' fracture only in one of five instances refracture the previously injured wrist. In those who have sustained fractures of the metacarpals or phalanges of the hand (forehand) subsequent fractures of the forehand are twice as likely to be ipsilateral. We investigated whether persisting regional bone mineral changes could be the mechanism underlying these observations. Bilateral bone mineral density measurements were performed on twenty patients who had sustained a Colles' fracture and twenty-nine who had sustained forehand fractures more than one year previously. Among Colles' fracture patients there was an increase in bone mineral density in the distal radius of the fractured side when compared to the uninjured side of thirty-nine percent. The protection of these patients from subsequent ipsilateral Colles' fracture seems to be due to increased bone strength induced by the healing process. Among patients with forehand fractures no significant bone mineral changes could be demonstrated.