Improved osteogenesis in rat femur segmental defects treated with human allograft and zinc adjuvants.

Bone allograft is widely used to treat large bone defects or complex fractures. However, processing methods can significantly compromise allograft osteogenic activity. Adjuvants that can restore the osteogenic activity of processed allograft should improve clinical outcomes. In this study, zinc was tested as an adjuvant to increase the osteogenic activity of human allograft in a Rag2 null rat femoral defect model. Femoral defects were treated with human demineralized bone matrix (DBM) mixed with carboxy methyl cellulose containing ZnCl2 (0, 75, 150, 300 µg) or Zn stearate (347 µg). Rat femur defects treated with DBM-ZnCl2 (75 µg) and DBM-Zn stearate (347 µg) showed increased calcified tissue in the defect site compared to DBM alone. Radiograph scoring and µCT (microcomputed tomography) analysis showed an increased amount of bone formation at the defects treated with DBM-Zn stearate. Use of zinc as an adjuvant was also tested using human cancellous bone chips. The bone chips were soaked in ZnCl2 solutions before being added to defect sites. Zn adsorbed onto the chips in a time- and concentration-dependent manner. Rat femur defects treated with Zn-bound bone chips had more new bone in the defects based on µCT and histomorphometric analyses. The results indicate that zinc supplementation of human bone allograft improves allograft osteogenic activity in the rat femur defect model.

The assessment of xenogeneic bone immunotoxicity and risk management study.

BACKGROUND: Xenogeneic bone has been widely used in a variety of clinical bone-related disease to promote bone healing and restore bone defects. However, the adverse effects of immune system limit its application in the clinic. The aim of this study was to evaluate xenogeneic bone safety of immunotoxicity and explore the methods for immune risk supervision. RESULTS: Xenogeneic bone, which is freeze-dried bovine cancellous bone, was implanted into the muscle of mice. On day 7, 14 and 28, the effects of xenogeneic bone were examined on humoral immunity and cellular immunity, including the levels of IgG, IgM, C3, inflammatory factors (TNF-α, IL-6), alkaline phosphatase (ALP) and the lymphocyte phenotype. The data showed that xenogeneic bone implantation had no potential to induce immune responses not only in humoral immunity but also in cellular immunity. To reveal the risk of immunogenicity, the residual DNA and the clearance of α-gal epitope were analyzed in 2 different bones (bone 1 is deproteinized bone, bone 2 is acellular and defatted bone). It was suggested that DNA of xenogeneic bone can be limited to < 50 ng per mg dry weight for the repair or regeneration with the acceptable immune risk. And α-gal clearance of xenogeneic bone could be an effective risk factor for improving xenograft quality management. CONCLUSIONS: Through the detection of xenogeneic bone immunotoxicity, our findings indicated that the supervisions of risk factors could contribute to reduce the immune risk. And the risk factors under the acceptable limitation could decrease or replace animal experiment. However, it still needs to be studied on the limitation of α-gal epitope to predict rejection of xenogeneic bone more accurately.

T cell immunomodulation by clinically used allogeneic human cancellous bone fragments: a potential novel immunotherapy tool.

Multipotential stromal cells (MSCs) demonstrate strong immunomodulation capabilities following culture expansion. We have previously demonstrated that human cancellous bone fragments (CBFs) clinically used as viable allografts for spinal fusion have resident MSCs that exhibit T cell immunomodulation after monolayer expansion. This study investigated the immunomodulatory ability of these CBFs without MSC culture-expansion. CD4 positive T cells were induced to proliferate using CD3/CD28 stimulation and added to CBFs at different ratios of T cells per gram of CBF. A dose-dependent suppressive effect on T cell proliferation was evident and correlated with increased culture supernatant levels of TGF-ß1, but not PGE2. CBF-driven immunosuppression was reduced in co-cultures with TGF-ß neutralising antibodies and was higher in cell contact compared to non-contact cultures. CBF gene expression profile identified vascular cell adhesion molecule-1, bone marrow stromal antigen 2/CD317 and other interferon signalling pathway members as potential immunomodulatory mediators. The CD317 molecule was detected on the surface of CBF-resident cells confirming the gene expression data. Taken together, these data demonstrate that human clinically used CBFs are inherently immunomodulatory and suggest that these viable allografts may be used to deliver therapeutic immunomodulation for immune-related diseases.

Evaluation of the osteoprotective potential of whey derived-antioxidative (YVEEL) and angiotensin-converting enzyme inhibitory (YLLF) bioactive peptides in ovariectomised rats.

Milk contains various bioactive components with osteoanabolic properties. This study investigates the comparative effect of the whey-derived antioxidative (YVEEL) and angiotensin-converting enzyme inhibitory (YLLF) bioactive peptides on bone remodelling in ovariectomised (OVX) osteoporotic rat model. OVX animals were administered with antioxidative (AO) (500 µg kg-1 day-1) and angiotensin-converting enzyme inhibitory (ACE inhibitory) (50 µg kg-1 day-1) peptides for eight weeks. Trabecular microarchitectural parameters of femoral and tibiae bone were determined using micro-CT scan. Bone formation, resorption, turnover markers (ALP, RANKL, OCN) and inflammatory cytokines (TNF-α, TGF-ß, IFN-γ) were determined by ELISA. Both AO and ACE inhibitory peptides inhibited the increase in bone turnover and inflammatory cytokines while increased the bone formation markers. The altered morphometric parameters of femoral and tibiae bones due to OVX were strikingly attenuated by the peptide administration. The results indicated that AO peptide exerts more osteoprotective potential than ACE inhibitory peptide by suppressing inflammatory status and enhancing bone formation markers.

Impairment of Bone Remodeling in LIGHT/TNFSF14-Deficient Mice.

Multiple cytokines produced by immune cells induce remodeling and aid in maintaining bone homeostasis through differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts. Here, we investigate bone remodeling controlled by the tumor necrosis factor (TNF) superfamily cytokine LIGHT. LIGHT-deficient mice (Tnfsf14-/- ) exhibit spine deformity and reduced femoral cancellous bone mass associated with an increase in the osteoclast number and a slight decrease of osteoblasts compared with WT mice. The effect of LIGHT in bone cells can be direct or indirect, mediated by both the low expression of the anti-osteoclastogenic osteoprotegerin (OPG) in B and T cells and reduced levels of the pro-osteoblastogenic Wnt10b in CD8+ T cells in Tnfsf14-/- mice. LIGHT stimulation increases OPG levels in B, CD8+ T, and osteoblastic cells, as well as Wnt10b expression in CD8+ T cells. The high bone mass in Light and T- and B-cell-deficient mice (Rag- /Tnfsf14- ) supports the cooperative role of the immune system in bone homeostasis. These results implicate LIGHT as a potential target in bone disease. © 2017 American Society for Bone and Mineral Research.

Temporal role of macrophages in cancellous bone healing.

Macrophages are important phagocytosing and cytokine producing cells with effects on fracture healing. We used clodronate-containing liposomes to reduce the number of macrophages, in order to study their role in the early phases of cancellous bone healing. Holes were drilled bilaterally into the cancellous bone of the proximal metaphysis of the tibia of 60 mice. A screw was inserted in the hole in the right tibia. The day of surgery was day 0. Clodronate-containing liposomes were injected intraperitoneally as a single injection either day -4 or -1 (before surgery) or day 1 or 3 (after surgery). A control group underwent surgery as above, but received no clodronate. The mice were killed day 7. The mechanical quality of the new formed cancellous bone holding the screw was evaluated by a pull-out test. The contents of the drill hole in the left tibia was analyzed by microCT. Another set of 20 mice received a drill hole in the metaphysis of the right tibia, and were given either clodronate or saline injections days -3 and -2. The animals were killed day 1 and 3. Flow cytometry was used to analyze the composition of macrophage subpopulations in the regenerating tissue. Flow cytometry showed that clodronate injections day -3 and -2 led to a decrease in mature monocytes day 1 together with an increase in immature monocytes. On day 3 this effect had mostly disappeared, suggesting that the effect of the injections lasted 3 to 5days. Mechanical testing revealed that the injections prior to surgery decreased the strength of the new formed bone, holding the screw, by about half. Bone density in the drill hole was similarly reduced. In contrast, the injections given day 1 and 3 had smaller and statistically insignificant effects. Since their depletion at later time points failed to produce a significant effect, it seems that the role of macrophages in cancellous bone is most crucial during the first two days after trauma.

An Antibody to Notch2 Reverses the Osteopenic Phenotype of Hajdu-Cheney Mutant Male Mice.

Notch receptors play a central role in skeletal development and bone remodeling. Hajdu-Cheney syndrome (HCS), a disease characterized by osteoporosis and fractures, is associated with gain-of-NOTCH2 function mutations. To study HCS, we created a mouse model harboring a point 6955C>T mutation in the Notch2 locus upstream of the proline, glutamic acid, serine, and threonine domain, leading to a Q2319X change at the amino acid level. Notch2Q2319X heterozygous mutants exhibited cancellous and cortical bone osteopenia. Microcomputed tomography demonstrated that the cancellous and cortical osteopenic phenotype was reversed by the administration of antibodies generated against the negative regulatory region (NRR) of Notch2, previously shown to neutralize Notch2 activity. Bone histomorphometry revealed that anti-Notch2 NRR antibodies decreased the osteoclast number and eroded surface in cancellous bone of Notch2Q2319X mice. An increase in osteoclasts on the endocortical surface of Notch2Q2319X mice was not observed in the presence of anti-Notch2 NRR antibodies. The anti-Notch2 NRR antibody decreased the induction of Notch target genes and Tnfsf11 messenger RNA levels in bone extracts and osteoblasts from Notch2Q2319X mice. In vitro experiments demonstrated increased osteoclastogenesis in Notch2Q2319X mutants in response to macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand, and these effects were suppressed by the anti-Notch2 NRR. In conclusion, Notch2Q2319X mice exhibit cancellous and cortical bone osteopenia that can be corrected by the administration of anti-Notch2 NRR antibodies.

Skeletal inflammation and attenuation of Wnt signaling, Wnt ligand expression, and bone formation in atherosclerotic ApoE-null mice.

ApoE-null (ApoE-KO) mice fed a high-fat diet (HFD) develop atherosclerosis, due in part to activation of vascular inflammation by oxidized low-density lipoprotein. Since bone loss also occurs in these mice, we used them to investigate the impact of oxidized lipids on bone homeostasis and to search for underlying pathogenic pathways. Four-month-old female ApoE-KO mice fed a HFD for three months exhibited increased levels of oxidized lipids in bone, as well as decreased femoral and vertebral trabecular and cortical bone mass, compared with ApoE-KO mice on normal diet. Despite HFD-induced increase in expression of Alox15, a lipoxygenase that oxidizes LDL and promotes atherogenesis, global deletion of this gene failed to ameliorate the skeletal impact of HFD. Osteoblast number and function were dramatically reduced in trabecular and cortical bone of HFD-fed mice, whereas osteoclast number was modestly reduced only in trabecular bone, indicating that an imbalance in favor of osteoclasts was responsible for HFD-induced bone loss. These changes were associated with decreased osteoblast progenitors and increased monocyte/macrophages in the bone marrow as well as increased expression of IL-1ß, IL-6, and TNF. HFD also attenuated Wnt signaling as evidenced by reduced expression of Wnt target genes, and it decreased expression of pro-osteoblastogenic Wnt ligands. These results suggest that oxidized lipids decrease bone mass by increasing anti-osteoblastogenic inflammatory cytokines and decreasing pro-osteoblastogenic Wnt ligands.

Antigen-free bovine cancellous bone loaded with recombinant human bone morphogenetic protein-2 for the repair of tibial bone defects in goat model.

Antigen-free bovine cancellous bone has good performances of porous network structures and mechanics with antigen extracted. To develop a bioactive scaffold for enhancing bone repair and evaluate its biological property, rhBMP-2 loaded with antigen-free bovine cancellous bone was used to treat tibial bone defect. Twenty-four healthy adult goats were chosen to establish goat defects model and randomly divided into four groups. The goats were treated with rhBMP-2/antigen-free bovine cancellous bone scaffolds (group A), autogenous cancellous bone graft (group B), porous tricalciumphosphate scaffolds (group C) and nothing (group D). Animals were evaluated with radiological and histological methods at 4, 8 and 12 weeks after surgery. The gray value of radiographs was used to evaluate the healing of the defects, which revealed that the group A had a better outcome of defect healing compared with group C at 4, 8 and 12 weeks, respectively (p < 0.05), while the difference between groups A and B was without significance at each time (p > 0.05). The newly formed bone area was calculated from histological sections, and the results indicated that the amount of new bone in group A increased significantly compared with that in group C (p < 0.05) but was similar to that in group B (p > 0.05) at 4, 8 and 12 weeks, respectively. In addition, the expression of collagen I and vascular endothelial growth factor by real-time polymerase chain reaction at 12 weeks in group A was significantly higher than that in group C (p = 0.034, p = 0.032, respectively), but no significant differences were found when compared with that in group B (p = 0.36, p = 0.54, respectively). At the same time, group C presented better results than group D on bone defects healing. Therefore, the composites of antigen-free bovine cancellous bone loaded with rhBMP-2 have a good osteoinductive activity and capacity to promote the repair of bone defects.

Contradictory Role of CD97 in Basal and Tumor Necrosis Factor-Induced Osteoclastogenesis In Vivo.

OBJECTIVE: CD97, a member of the 7-transmembrane epidermal growth factor family of adhesion G protein-coupled receptors, is expressed on various cell types. This study was undertaken to elucidate the functions of CD97 in bone and inflammation in an experimental mouse model, by examining the effect of CD97 on osteoclastogenesis in vitro, characterizing the skeletal phenotype of CD97-deficient (CD97-knockout [KO]) mice, and assessing the responses to tumor necrosis factor (TNF) treatment. METHODS: Femoral tissue and bone marrow (BM)-derived cells from CD97-KO and wild-type (WT) mice were assessed using histomorphometric analyses, in vitro cultures, and reverse transcription-polymerase chain reaction. Serum cytokine and chemokine levels in the presence or absence of TNF challenge were analyzed by multiplex assay. RESULTS: In cultures of mouse BM-derived macrophages in vitro, RANKL induced the expression of CD97. In vivo, the trabecular bone volume of the femurs of female CD97-KO mice was increased, and this was associated with a decrease in the number of osteoclasts. Compared to WT mice, CD97-KO mice had a reduced potential to form osteoclast-like cells in vitro. Furthermore, TNF treatment augmented the formation of osteoclasts in the calvaria of CD97-KO mice in vivo, by increasing the production of RANKL and other cytokines and chemokines and by reducing the production of osteoprotegerin by calvarial cells. CONCLUSION: These findings demonstrate that CD97 is a positive regulator of osteoclast-like cell differentiation, a mechanism that influences bone homeostasis. However, the presence of CD97 may be essential to suppress the initial osteoclastogenesis that occurs in response to acute and local inflammatory stimuli.