Increased Bone Volume Research Articles

Osteoinductive 3D printed scaffold healed 5\xa0cm segmental bone defects in the ovine metatarsus

Autologous bone grafts are considered the gold standard grafting material for the treatment of nonunion, but in very large bone defects, traditional autograft alone is insufficient to induce repair. Recombinant human bone morphogenetic protein 2 (rhBMP-2) can stimulate bone regeneration and enhance the healing efficacy of bone grafts. The delivery of rhBMP-2 may even enable engineered synthetic scaffolds to be used in place of autologous bone grafts for the treatment of critical size defects, eliminating risks associated with autologous tissue harvest. We here demonstrate that an osteoinductive scaffold, fabricated by combining a 3D printed rigid polymer/ceramic composite scaffold with an rhBMP-2-eluting collagen sponge can treat extremely large-scale segmental defects in a pilot feasibility study using a new sheep metatarsus fracture model stabilized with an intramedullary nail. Bone regeneration after 24 weeks was evaluated by micro-computed tomography, mechanical testing, and histological characterization. Load-bearing cortical bridging was achieved in all animals, with increased bone volume observed in sheep that received osteoinductive scaffolds compared to sheep that received an rhBMP-2-eluting collagen sponge alone.

Sclerostin antibody increases trabecular bone and bone mechanical properties by increasing osteoblast activity damaged by whole-body irradiation in mice.

Irradiation therapy causes bone deterioration and increased risk for skeletal-related events. Irradiation interferes with trabecular architecture through increased osteoclastic activity, decreased osteoblastic activity, and increased adipocyte expansion in the bone marrow (BM), which further compounds bone-related disease. Neutralizing antibodies to sclerostin (Scl-Ab) increase bone mass and strength by increasing bone formation and reducing bone resorption. We hypothesized that treatment with Scl-Ab would attenuate the adverse effects of irradiation by increasing bone volume and decreasing BM adipose tissue (BMAT), resulting in better quality bone. In this study, 12-week-old female C57BL/6J mice were exposed to 6Gy whole-body irradiation or were non-irradiated, then administered Scl-Ab (25mg/kg) or vehicle weekly for 5weeks. Femoral μCT analysis confirmed that the overall effect of IR significantly decreased trabecular bone volume/total volume (Tb.BV/TV) (2-way ANOVA, p<0.0001) with a -43.8% loss in Tb.BV/TV in the IR control group. Scl-Ab independently increased Tb.BV/TV by 3.07-fold in non-irradiated and 3.6-fold in irradiated mice (2-way ANOVA, p<0.0001). Irradiation did not affect cortical parameters, although Scl-Ab increased cortical thickness and area significantly in both irradiated and non-irradiated mice (2-way ANOVA, p<0.0001). Femoral mechanical testing confirmed Scl-Ab significantly increased bending rigidity and ultimate moment independently of irradiation (2-way ANOVA, p<0.0001). Static and dynamic histomorphometry of the femoral metaphysis revealed osteoblast vigor, not number, was significantly increased in the irradiated mice treated with Scl-Ab. Systemic alterations were assessed through serum lipidomic analysis, which showed that Scl-Ab normalized lipid profiles in the irradiated group. This data supports the theory of sclerostin as a novel contributor to the regulation of osteoblast activity after irradiation. Overall, our data support the hypothesis that Scl-Ab ameliorates the deleterious effects of whole-body irradiation on bone and adipose tissue in a mouse model. Our findings suggest that future research into localized and systemic therapies after irradiation exposure is warranted.

The Effect of Megakaryocytes and Platelets Derived from Human-Induced Pluripotent Stem Cells on Bone Formation.

Introduction Platelet-rich plasma (PRP) is drawing attention as a substance that can promote bone formation. The growth factors present in PRP are stable for a long time after freeze-drying. However, the effects of PRP are inconsistent, and its effects on bone union in spinal surgery remain controversial. The immortalized megakaryocyte cell lines (imMKCLs) derived from human-induced pluripotent stem cells (hiPSCs) have been developed to produce numerous stable and clinically functional platelets. In this study, growth factors present in freeze-dried hiPSC-derived imMKCLs and platelets (iPS-MK/Plts) were evaluated, and their ability to promote bone formation was examined using a rat lumbar artificial bone grafting model. Methods We prepared freeze-dried iPS-MK/Plts and quantified their growth factors by enzyme-linked immunosorbent assays. Surgical grafting of artificial bone to the lumbar transverse processes was performed in 8-week-old female rats, which were divided into two groups: artificial bone graft (control) and artificial bone graft plus freeze-dried iPS-MK/Plts (iPS group). Transplantation was performed only on the left side. Eight weeks after the surgery, we captured computed tomography images and compared bilateral differences in the bone volume of the graft site in each rat. We also compared the left side/right side bone volume ratio between the two groups. Results The freeze-dried iPS-MK/Plts contained numerous growth factors. While there was no significant increase in bone volume on the transplanted side than that on the non-grafted side in the control group, bone volume significantly increased on the transplanted side in the iPS group, as evidenced by augmented mean left/right bone volume ratio of the iPS group compared with that of the control group. But the new bone observed in the iPS group was histologically normal. Conclusions Freeze-dried hiPSC-derived MKCLs and platelets contain several stable growth factors and have the potential for promoting new bone formation.

Early B-cell Factor1 (Ebf1) promotes early osteoblast differentiation but suppresses osteoblast function.

Early B cell factor 1 (Ebf1) is a transcription factor that regulates B cell, neuronal cell and adipocyte differentiation. We and others have shown that Ebf1 is expressed in osteoblasts and that global deletion of Ebf1 results in increased bone formation in vivo. However, as Ebf1 is expressed in multiple tissues and cell types, it has remained unclear, which of the phenotypic changes in bone are derived from bone cells. The aim of this study was to determine the cell-autonomous and differentiation stage-specific roles of Ebf1 in osteoblasts. In vitro, haploinsufficient Ebf1+/- calvarial cells showed impaired osteoblastic differentiation indicated by lower alkaline phosphatase (ALP) activity and reduced mRNA expression of osteoblastic genes, while overexpression of Ebf1 in wild type mouse calvarial cells led to enhanced osteoblast differentiation with increased expression of Osterix (Osx). We identified a putative Ebf1 binding site in the Osterix promoter by ChIP assay in MC3T3-E1 osteoblasts and showed that Ebf1 was able to activate Osx-luc reporter construct that included this Ebf1 binding site, suggesting that Ebf1 indeed regulates osteoblast differentiation by inducing Osterix expression. To reconcile our previous data and that of others with our novel findings, we hypothesized that Ebf1 could have a dual role in osteoblast differentiation promoting early but inhibiting late stages of differentiation and osteoblast function. To test this hypothesis in vivo, we generated conditional Ebf1 knockout mice, in which Ebf1 deletion was targeted to early or late osteoblasts by crossing Ebf1fl/fl mice with Osx- or Osteocalcin (hOC)-Cre mouse lines, respectively. Deletion of Ebf1 in early Ebf1Osx-/- osteoblasts resulted in significantly increased bone volume and trabecular number at 12weeks by μCT analysis, while Ebf1hOC-/- mice did not have a bone phenotype. To conclude, our data demonstrate that Ebf1 promotes early osteoblast differentiation by regulating Osterix expression. However, Ebf1 inhibits bone accrual in the Osterix expressing osteoblasts in vivo but it is redundant in the maintenance of mature osteoblast function.

Ibandronate Reduces the Surface Bone Resorption of Mandibular Bone Grafts: A Randomized Trial With Internal Controls.

ABSTRACTAutologous bone grafts are considered the gold standard for reconstruction of the edentulous alveolar ridges. However, this procedure is associated with unpredictable bone loss caused by physiological bone resorption. Bisphosphonates are antiresorptive drugs that act specifically on osteoclasts, thereby maintaining bone density, volume, and strength. It was hypothesized that the resorption of bone grafts treated with an ibandronate solution would be less advanced than bone grafts treated with saline. Ten patients who underwent bilateral sagittal split osteotomy were included in a randomized double‐blind trial with internal controls. Each patient received a bone graft treated with a solution of ibandronate on one side and a graft treated with saline (controls) contralaterally. Radiographs for the measurement of bone volume were obtained at 2 weeks and at 6 months after surgery. The primary endpoint was the difference in the change of bone volume between the control and the ibandronate bone grafts 6 months after surgery. All of the bone grafts healed without complications. One patient was excluded because of reoperation. In eight of the nine patients, the ibandronate bone grafts showed an increase in bone volume compared with baseline, with an average gain of 126 mm3 (40% more than baseline) with a range of +27 to +218 mm3. Only one ibandronate‐treated graft had a decrease in bone volume (8%). In the controls, an average bone volume loss of −146 mm3 (58% of baseline) with a range of −29 to −301 mm3 was seen. In the maxillofacial field, the reconstructions of atrophic alveolar ridges, especially in the esthetical zones, are challenging. These results show that bone grafts locally treated with ibandronate solution increases the remaining bone volume. This might lead to new possibilities for the maxillofacial surgeons in the preservation of bone graft volumes and for dental implant installations. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Neural crest-derived mesenchymal progenitor cells enhance cranial allograft integration.

Replacement of lost cranial bone (partly mesodermal and partly neural crest‐derived) is challenging and includes the use of nonviable allografts. To revitalize allografts, bone marrow‐derived mesenchymal stromal cells (mesoderm‐derived BM‐MSCs) have been used with limited success. We hypothesize that coating of allografts with induced neural crest cell‐mesenchymal progenitor cells (iNCC‐MPCs) improves implant‐to‐bone integration in mouse cranial defects. Human induced pluripotent stem cells were reprogramed from dermal fibroblasts, differentiated to iNCCs and then to iNCC‐MPCs. BM‐MSCs were used as reference. Cells were labeled with luciferase (Luc2) and characterized for MSC consensus markers expression, differentiation, and risk of cellular transformation. A calvarial defect was created in non‐obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and allografts were implanted, with or without cell coating. Bioluminescence imaging (BLI), microcomputed tomography (μCT), histology, immunofluorescence, and biomechanical tests were performed. Characterization of iNCC‐MPC‐Luc2 vs BM‐MSC‐Luc2 showed no difference in MSC markers expression and differentiation in vitro. In vivo, BLI indicated survival of both cell types for at least 8 weeks. At week 8, μCT analysis showed enhanced structural parameters in the iNCC‐MPC‐Luc2 group and increased bone volume in the BM‐MSC‐Luc2 group compared to controls. Histology demonstrated improved integration of iNCC‐MPC‐Luc2 allografts compared to BM‐MSC‐Luc2 group and controls. Human osteocalcin and collagen type 1 were detected at the allograft‐host interphase in cell‐seeded groups. The iNCC‐MPC‐Luc2 group also demonstrated improved biomechanical properties compared to BM‐MSC‐Luc2 implants and cell‐free controls. Our results show an improved integration of iNCC‐MPC‐Luc2‐coated allografts compared to BM‐MSC‐Luc2 and controls, suggesting the use of iNCC‐MPCs as potential cell source for cranial bone repair.

Dihydroartemisinin attenuates osteoarthritis by inhibiting abnormal bone remodeling and angiogenesis in subchondral bone.

The present study aimed to investigate whether dihydroartemisinin (DHA) alleviates osteoarthritis (OA) in a mouse model of OA. Ten-week-old female C57BL/6j mice were used to establish OA models by anterior cruciate ligament transection (ACLT) and ovariectomized (OVX). DHA was then used to treat the OA in the ACLT and OVX mice. Safranin O-fast green staining and Osteoarthritis Research Society International (OARSI)-modified Mankin scores were used to grade articular cartilage degeneration. Expression of metalloproteinase-13 (MMP-13) and vascular endothelial growth factor (VEGF) in the articular cartilage and leukemia inhibitory factor (LIF), sclerostin, and β-catenin in the subchondral bone were analyzed by immunohistochemistry. Expression of RANKL and CD31 were detected by immunofluorescence. Micro-computed tomography was used to ascertain alterations in the microarchitecture of the subchondral bone. The results demonstrated that DHA decreased MMP-13 and VEGF expression in the articular cartilage. DHA decreased OARSI scores and reduced articular cartilage degeneration. In addition, DHA reduced abnormal subchondral bone remodeling, as demonstrated by a reduction in trabecular separation (Tb.Sp), increased bone volume fractions (BV/TV), as well as bone mineral densities (BMD) compared with the ACLT+vehicle group and the OVX+vehicle group. Furthermore, DHA decreased the inhibition of sclerostin through reduction of LIF secretion by osteoclasts and, hence, attenuated aberrant bone remodeling and inhibited angiogenesis in subchondral bone, further reducing the progression of OA. The present study demonstrated that DHA attenuated OA by inhibiting abnormal bone remodeling and angiogenesis in subchondral bone, which may be a potential therapeutic target for this disease.

Resorbable additively manufactured scaffold imparts dimensional stability to extraskeletally regenerated bone

Dimensionally stable vertical bone regeneration outside of the existing bony envelope is a major challenge in the field of orofacial surgery. In this study, we demonstrate that a highly porous, resorbable scaffold fabricated using additive manufacturing techniques enables reproducible extra-skeletal bone formation and prevents bone resorption. An additively manufactured medical grade polycaprolactone (mPCL) biphasic scaffold mimicking the architecture of the jaw bone, consisting of a 3D-printed outer shell overlying an inner highly porous melt electrowritten scaffold, was assessed for its ability to support dimensionally stable bone regeneration in an extraskeletal ovine calvarial model. To investigate bone formation capacity (stage 1), 7 different constructs placed under a protective dome were assessed 8 weeks post-implantation: Empty control, Biphasic scaffold with hydrogel (PCL-Gel), PCL-Gel with 75 or 150 μg of BMP-2 (PCL-BMP-75 and PCL-BMP-150), hydrogel only (Gel), Gel containing 75 or 150 μg of BMP-2 (Gel-BMP-75 and Gel-BMP-150). To assess dimensional stability (stage 2), in a separate cohort, 5 animals were similarly implanted with 2 samples of each of the Gel-BMP-150 and PCL-BMP-150 groups, and after 8 weeks of healing, the protective domes were removed and titanium implants were placed in the regenerated bone and allowed to heal for a further 8 weeks. Bone formation and osseointegration were assessed using micro-computed tomography, histology and histomorphometry. In stage 1, enhanced bone formation was found in the BMP-2 containing groups, especially the PCL-BMP constructs whereby regeneration of full bone height was achieved in a reproducible manner. There was no significant bone volume increase with the higher dose of BMP-2. In the dimensional stability assessment (stage 2), after the rtemoval of the protective dome, the biphasic scaffold prevented bone resorption whereas in the absence of the scaffold, the bone previously formed in the hydrogel underwent extensive resorption. This was attributed to the space maintenance properties and dimensional stability of the biphasic scaffold. Titanium implants osseointegrated into the newly formed bone within the biphasic scaffolds. In conclusion, additively manufactured biphasic scaffolds functionalized with BMP-2 facilitated dimensionally stable bone regeneration that supported dental implant osseointegration.

Peptide Enhanced Bone Graft Substitute Presents Improved Short-Term Increase in Bone Volume and Construct Stiffness Compared to Iliac Crest Autologous Bone in an Ovine Lumbar Interbody Fusion Model.

Study design:Preclinical ovine model.Objective:To assess the in vivo efficacy and safety of the P-15 L bone graft substitute and compare its performance to autologous iliac crest bone graft (ICBG) for lumbar interbody fusion indications.Methods:Thirty skeletally mature sheep underwent lumbar interbody fusion surgery. Half of the sheep received autologous ICBG and the other half the peptide enhanced bone graft substitute (P-15 L). Following termination at 1, 3, and 6 months after surgery, the operated segments were analyzed using micro computed tomography (µCT), histology, and destructive mechanical testing. Additional systemic health monitoring was performed for the P-15 L group.Results:One month after surgery, there was only minor evidence of bone remodeling and residual graft material could be clearly observed within the cage. There was active bone remodeling between 1 and 3 months after surgery. At 3 months after surgery significantly denser and stiffer bone was found in the P-15 L group, whereas at 6 months, P-15 L and ICBG gave similar fusion results. The P-15 L bone graft substitute did not have any adverse effects on systemic health.Conclusions:The drug device combination P-15 L was demonstrated to be effective and save for lumbar interbody fusion as evidenced by this ovine model. Compared to autologous ICBG, P-15 L seems to expedite bone formation and remodeling but in the longer-term fusion results were similar.

Effects of Limosilactobacillus fermentum CCFM1139 on experimental periodontitis in rats.

Periodontitis is a polymicrobial inflammatory disease often characterized by the excessive colonization of Porphyromonas gingivalis and Fusobacterium nucleatum, which causes alveolar bone resorption and advanced oral inflammation. This study aimed to evaluate the effect of Limosilactobacillus fermentum CCFM1139 on experimental periodontitis induced following ligature and infection with P. gingivalis and F. nucleatum in vivo. The results showed that L. fermentum CCFM1139 significantly reduced weight loss associated with periodontal inflammation (p < 0.05), while decreasing both the P. gingivalis and F. nucleatum populations within the oral cavity of rats (p < 0.05) and regulating the expression of tumor necrosis factor-alpha, interleukin (IL)-1 beta, and IL-8 in the periodontal tissue (p < 0.05). Microcomputed tomography (micro-CT) and histopathological examination revealed that L. fermentum CCFM1139 supplementation reduced the level of alveolar bone loss and bone porosity and increased bone volume (p < 0.05) in the experimental animals. Furthermore, L. fermentum CCFM1139 exhibited promising effects in preventing the deepening of the periodontal pocket and the increase in the gap between adjacent molars. Thus L. fermentum CCFM1139 was shown to have solid potential as an oral probiotic for protection against periodontitis suggesting that this may be a good candidate in the production of a new functional food for improving periodontitis.

The Dual Effects of Reactive Oxygen Species on the Mandibular Alveolar Bone Formation in SOD1 Knockout Mice: Promotion or Inhibition.

The status of reactive oxygen species (ROS) correlates closely with the normal development of the oral and maxillofacial tissues. Oxidative stress caused by ROS accumulation not only affects the development of enamel and dentin but also causes pathological changes in periodontal tissues (periodontal ligament and alveolar bone) that surround the root of the tooth. Although previous studies have shown that ROS accumulation plays a pathologic role in some oral and maxillofacial tissues, the effects of ROS on alveolar bone development remain unclear. In this study, we focused on mandibular alveolar bone development of mice deficient in superoxide dismutase1 (SOD1). Analyses were performed using microcomputerized tomography (micro-CT), TRAP staining, immunohistochemical (IHC) staining, and enzyme-linked immunosorbent assay (ELISA). We found for the first time that slightly higher ROS in mandibular alveolar bone of SOD1(-/-) mice at early ages (2-4 months) caused a distinct enlargement in bone size and increased bone volume fraction (BV/TV), trabecular thickness (Tb.Th), and expression of alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2), and osteopontin (OPN). With ROS accumulation to oxidative stress level, increased trabecular bone separation (Tb.Sp) and decreased expression of ALP, Runx2, and OPN were found in SOD1(-/-) mice at 6 months. Additionally, dosing with N-acetylcysteine (NAC) effectively mitigated bone loss and normalized expression of ALP, Runx2, and OPN. These results indicate that redox imbalance caused by SOD1 deficiency has dual effects (promotion or inhibition) on mandibular alveolar bone development, which is closely related to the concentration of ROS and the stage of growth. We present a valuable model here for investigating the effects of ROS on mandibular alveolar bone formation and highlight important roles of ROS in regulating tissue development and pathological states, illustrating the complexity of the redox signal.

The antioxidant effect of triptolide contributes to the therapy in a collagen-induced arthritis rat model

ABSTRACT Background As a chronic autoimmune disease, rheumatoid arthritis (RA) is related to oxidative stress, which may lead to the occurrence and persistence of inflammation in RA. The purpose of this study is to evaluate the potential antioxidant effect of triptolide in collagen-induced arthritis (CIA) rat model. Methods We examined the severity of arthritis, levels of local and systemic oxidative stress, periarticular bone erosion and weight of organs in CIA rats treated with triptolide. Results We found that triptolide decreased the paw thickness and clinical arthritis score, significantly. The mRNA expression and activity of myeloperoxidase and inducible nitric oxide synthase were remarkably decreased in the paws of the CIA rats after triptolide treatment. Triptolide significantly inhibited the levels of nitrite and nitrate in serum, as well as the urinary level of dityrosine. Triptolide treatment also markedly increased bone volume of tibia, but suppressed epiphyseal plate thickness of both femur and tibia. In addition, there was no significant difference in the weight of organs after the therapy, except decreased spleen weight. Conclusions These results suggested that the local and systemic oxidative stress was enhanced in the CIA rats and the therapeutic dose of triptolide had a definite antioxidant effect.

A longitudinal characterization of sex-specific somatosensory and spatial memory deficits in HIV Tg26 heterozygous mice.

The pathogenesis of human immunodeficiency virus associated neurological disorders is still not well understood, yet is known to result in neurological declines despite combination anti-retroviral therapy. HIV-1 transgenic (Tg26) mice contain integrated non-infectious HIV-1 proviral DNA. We sought to assess the integrity of neurocognitive function and sensory systems in HIV-1 Tg26 mice using a longitudinal design, in both sexes, to examine both age- and sex-related disease progression. General neurological reflexive testing showed only acclimation to repeated testing by all groups. Yet, at 2.5 months of age, female Tg26 +/- mice showed hyposensitivity to noxious hot temperatures, compared to wild types (both sexes) and male Tg26 +/- mice, that worsened by 10 months of age. Female Tg26 +/- mice had short-term spatial memory losses in novel object location memory testing at 2.5 and 7 months, compared to female wild types; changes not observed in male counterparts. Female Tg26 +/- mice showed mild learning deficits and short- and long-term spatial memory deficits in olfactory and visually cued Barnes Maze testing at 3 months of age, yet greater learning and memory deficits by 8 months. In contrast, male Tg26 +/- mice displayed no learning deficits and fewer spatial memory deficits (mainly heading errors in nontarget holes). Thus, greater sex-specific temperature hyposensitivity and spatial memory declines were observed in female HIV Tg26 +/- mice, than in male Tg26 +/- mice, or their wild type littermates, that increased with aging. Additionally, tibial bones were examined using ex vivo micro-CT after tissue collection at 11 months. Sex-dependent increases in bone volume and trabecular number were seen in males, matching their greater weights at this age. These results indicate that HIV-1 Tg26 mice is a promising model in which to study neuropathic mechanisms underlying peripheral pathology as well as cognitive deficits seen with HIV.

Cornelian cherry extract ameliorates osteoporosis associated with hypercholesterolemia in New Zealand rabbits.

Results of animal studies show that a high-cholesterol diet increases bone resorption and decreases bone formation, thus leading to osteoporosis. Previously, we reported on the beneficial influence of Cornelian cherry (Cornus mas L.) fruit on lipid profile in an animal model of diet-induced hipercholesterolemia. To investigate the influence of Cornus mas L. extract and loganic acid (LA) on cholesterol-induced bone changes. The study was conducted on 50 New Zealand rabbits. The animals were given either standard chow (group P) or the same standard chow enriched with 1% cholesterol (other groups). Additionally, the group CHOL+EX received Cornus mas L. extract, group CHOL+LA - loganic acid, and group CHOL+SIM - simvastatin. Serum concentration of bone turnover markers, bone mineral density (BMD) and bone micro-computed tomography (microCT) were assessed. In the CHOL group, a decrease in osteocalcin (OC) and an increase in C-terminated telopeptide of type I collagen (CTX) levels were detected (CHOL vs P 0.674 ±0.159 ng/mL vs 1.003 ±0.297 ng/mL and 10.049 ±1.276 ng/mL vs 7.721 ±1.187 ng/mL, respectively). The EX and LA ameliorated cholesterol-induced changes in serum OC (0.857 ±0.160 ng/mL and 1.103 ±0.356 ng/mL, respectively) and CTX (7.735 ±1.045 ng/mL and 8.128 ±1.106 ng/mL, respectively). There was a significant decrease in femoral BMD in CHOL group (0.429 ±0.11 g/cm² vs 0.449 ±0.020 g/cm²). The EX and LA ameliorated those changes (0.458 ±0.016 g/cm² and 0.449 ±0.021 g/cm², respectively). The microCT revealed increased bone volume ratio (BV/TV) and trabecular thickness (Tb.Th.) in the CHOL+EX group. Cornus mas L. inhibited bone resorption and stimulated bone formation, thereby preventing the development of cholesterol-induced osteoporosis.

The Radiographic Effect of Bone Marrow Aspirate Concentrate on Bone Maturation During Mandibular Distraction Osteogenesis.

This study aims to evaluate the effect of bone marrow aspirate concentrate (BMAC) during distraction on the consolidation period. The study had 2 groups each had 6 patients. In the control, the distraction had no enhancement; while, the study group was enhanced by BMAC. The bone quality and quantity were assessed using cone beam computed tomography (CBCT). The assessment of bone density showed non-significant (NS) (P = 0.06) increase in bone density in the study group (M = 293 ± 100 HU) compared to the control group (M = 176 ± 94 HU). The Assessment of bone volume showed a NS (P = 0.15) increase in bone volume in Study group with average bone volume/total volume (M = 49.47% ± 4.5%) compared to Control group (M = 43.9% ± 7.5%). Further examination is recommended to evaluate the effect of BMAC on the distracted bone. The addition of BMAC made a non-significant improvement in bone quantity and quality.

Local injections of \u03b2-NGF accelerates endochondral fracture repair by promoting cartilage to bone conversion

There are currently no pharmacological approaches in fracture healing designed to therapeutically stimulate endochondral ossification. In this study, we test nerve growth factor (NGF) as an understudied therapeutic for fracture repair. We first characterized endogenous expression of Ngf and its receptor tropomyosin receptor kinase A (TrkA) during tibial fracture repair, finding that they peak during the cartilaginous phase. We then tested two injection regimens and found that local β-NGF injections during the endochondral/cartilaginous phase promoted osteogenic marker expression. Gene expression data from β-NGF stimulated cartilage callus explants show a promotion in markers associated with endochondral ossification such as Ihh, Alpl, and Sdf-1. Gene ontology enrichment analysis revealed the promotion of genes associated with Wnt activation, PDGF- and integrin-binding. Subsequent histological analysis confirmed Wnt activation following local β-NGF injections. Finally, we demonstrate functional improvements to bone healing following local β-NGF injections which resulted in a decrease in cartilage and increase of bone volume. Moreover, the newly formed bone contained higher trabecular number, connective density, and bone mineral density. Collectively, we demonstrate β-NGF’s ability to promote endochondral repair in a murine model and uncover mechanisms that will serve to further understand the molecular switches that occur during cartilage to bone transformation.

Functional Scaffold-Free Bone Equivalents Induce Osteogenic and Angiogenic Processes in a Human In Vitro Fracture Hematoma Model.

After trauma, the formed fracture hematoma within the fracture gap contains all the important components (immune/stem cells, mediators) to initiate bone regeneration immediately. Thus, it is of great importance but also the most susceptible to negative influences. To study the interaction between bone and immune cells within the fracture gap, up-to-date in vitro systems should be capable of recapitulating cellular and humoral interactions and the physicochemical microenvironment (eg, hypoxia). Here, we first developed and characterized scaffold-free bone-like constructs (SFBCs), which were produced from bone marrow-derived mesenchymal stromal cells (MSCs) using a macroscale mesenchymal condensation approach. SFBCs revealed permeating mineralization characterized by increased bone volume (μCT, histology) and expression of osteogenic markers (RUNX2, SPP1, RANKL). Fracture hematoma (FH) models, consisting of human peripheral blood (immune cells) mixed with MSCs, were co-cultivated with SFBCs under hypoxic conditions. As a result, FH models revealed an increased expression of osteogenic (RUNX2, SPP1), angiogenic (MMP2, VEGF), HIF-related (LDHA, PGK1), and inflammatory (IL6, IL8) markers after 12 and 48 hours co-cultivation. Osteogenic and angiogenic gene expression of the FH indicate the osteoinductive potential and, thus, the biological functionality of the SFBCs. IL-6, IL-8, GM-CSF, and MIP-1β were detectable within the supernatant after 24 and 48 hours of co-cultivation. To confirm the responsiveness of our model to modifying substances (eg, therapeutics), we used deferoxamine (DFO), which is well known to induce a cellular hypoxic adaptation response. Indeed, DFO particularly increased hypoxia-adaptive, osteogenic, and angiogenic processes within the FH models but had little effect on the SFBCs, indicating different response dynamics within the co-cultivation system. Therefore, based on our data, we have successfully modeled processes within the initial fracture healing phase in vitro and concluded that the cross-talk between bone and immune cells in the initial fracture healing phase is of particular importance for preclinical studies. © 2021 American Society for Bone and Mineral Research (ASBMR).

Heterogeneity in microstructural deterioration following spinal cord injury.

Modelling and remodelling adapt bone morphology to accommodate strains commonly encountered during loading. If strains exceed a threshold threatening fracture, modelling-based bone formation increases bone volume reducing these strains. If unloading reduces strains below a threshold that inhibits resorption, increased remodelling-based bone resorption reduces bone volume restoring strains, but at the price of compromised bone volume and microstructure. As weight-bearing regions are adapted to greater strains, we hypothesized that microstructural deterioration will be more severe than at regions commonly adapted to low strains following spinal cord injury. We quantified distal tibial, fibula and radius volumetric bone mineral density (vBMD) using high-resolution peripheral quantitative computed tomography in 31 men, mean age 43.5years (range 23.5-75.0), 12 with tetraplegia and 19 with paraplegia of 0.7 to 18.6years duration, and 102 healthy age- and sex-matched controls. Differences in morphology relative to controls were expressed as standardized deviation (SD) scores (mean±SD). Standardized between-region differences in vBMD were expressed as SDs (95% confidence intervals, CI). Relative to controls, men with tetraplegia had deficits in total vBMD of -1.72±1.38 SD at the distal tibia (p<0.001) and-0.68±0.69 SD at distal fibula (p=0.041), but not at the distal radius, despite paralysis. Deficits in men with paraplegia were -2.14±1.50 SD (p<0.001) at the distal tibia and -0.83±0.98 SD (p=0.005) at the distal fibula while distal radial total vBMD was 0.23±1.02 (p=0.371), not significantly increased, despite upper limb mobility. Comparing regions, in men with tetraplegia, distal tibial total vBMD was 1.04 SD (95%CI 0.07, 2.01) lower than at the distal fibula (p=0.037) and 1.51 SD (95%CI 0.45, 2.57) lower than at the distal radius (p=0.007); the latter two sites did not differ from each other. Results were similar in men with paraplegia, but total vBMD at the distal fibula was 1.06 SD (95%CI 0.35, 1.77) lower than at the distal radius (p=0.004). Microarchitectural deterioration following spinal cord injury is heterogeneous, perhaps partly because strain thresholds regulating the cellular activity of mechano-transduction are region specific.

Vitamin D supplementation improves bone mineralisation independent of dietary phosphate in male X-linked hypophosphatemic (Hyp) mice.

The disorder of X-linked hypophosphatemia (XLH), results in the supressed renal production of active 1α,25-dihydroxyvitamin D (1,25(OH)2D) due to elevated fibroblast growth factor-23 (FGF23) levels. While adequate 25(OH)D levels are generally associated with improved mineralisation of the skeleton independent of circulating 1,25(OH)2D levels, it is unclear whether raising 25(OH)D to sufficiently high levels through dietary vitamin D3 administration contributes to improving bone mineralisation in the murine homolog for XLH, Hyp mice. Three-week-old male Hyp mice were fed one of four diets containing either 1000IU (C) or 20,000IU (D) vitamin D3/kg diet with either 0.35% phosphate or 1.25% phosphate (P) until 12weeks of age (n=12/group). When compared to C-fed mice, D-fed mice significantly elevated serum 25(OH)D levels to 72.8±4.9nmol/L (2-fold, p<0.001) and increased both cortical bone mineral density (15%, p<0.01), and vertebral trabecular BV/TV% (80%, p<0.001), despite persistent hypophosphatemia and normocalcemia. The increase in bone volume was associated with improved Tb.Th (12%, p<0.01) and Tb.N (63%, p<0.001). Unlike with D-diet, P-fed mice resulted in increased femoral (15%, p<0.001) and vertebral (12%, p<0.001) length, and a 34% increase in vertebral trabecular BV/TV% when compared to control fed animals (p<0.001). However, the addition of the high P diet to the high D diet did not result in additive effects on bone mineralisation when compared to the effects of D diet alone, despite serum 25(OH)D levels elevated to 118.8±8.6nmol/L. In D-fed mice, the increase in bone mineral density and volume was associated with reduced osteoid volume, reduced ObS/BS, and a trend for reduced serum PTH levels, suggesting reduced bone turnover in these animals. Thus, elevating serum 25(OH)D levels independently improves bone mineralisation in Hyp mice without causing hypercalcemia, suggesting that further studies are required in XLH patients to establish the role of increasing 25(OH)D levels in improving bone mineralisation.

Gelatin-Modified Calcium/Strontium Hydrogen Phosphates Stimulate Bone Regeneration in Osteoblast/Osteoclast Co-Culture and in Osteoporotic Rat Femur Defects-In Vitro to In Vivo Translation.

The development and characterization of biomaterials for bone replacement in case of large defects in preconditioned bone (e.g., osteoporosis) require close cooperation of various disciplines. Of particular interest are effects observed in vitro at the cellular level and their in vivo representation in animal experiments. In the present case, the material-based alteration of the ratio of osteoblasts to osteoclasts in vitro in the context of their co-cultivation was examined and showed equivalence to the material-based stimulation of bone regeneration in a bone defect of osteoporotic rats. Gelatin-modified calcium/strontium phosphates with a Ca:Sr ratio in their precipitation solutions of 5:5 and 3:7 caused a pro-osteogenic reaction on both levels in vitro and in vivo. Stimulation of osteoblasts and inhibition of osteoclast activity were proven during culture on materials with higher strontium content. The same material caused a decrease in osteoclast activity in vitro. In vivo, a positive effect of the material with increased strontium content was observed by immunohistochemistry, e.g., by significantly increased bone volume to tissue volume ratio, increased bone morphogenetic protein-2 (BMP2) expression, and significantly reduced receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio. In addition, material degradation and bone regeneration were examined after 6 weeks using stage scans with ToF-SIMS and µ-CT imaging. The remaining material in the defects and strontium signals, which originate from areas exceeding the defect area, indicate the incorporation of strontium ions into the surrounding mineralized tissue. Thus, the material inherent properties (release of biologically active ions, solubility and degradability, mechanical strength) directly influenced the cellular reaction in vitro and also bone regeneration in vivo. Based on this, in the future, materials might be synthesized and specifically adapted to patient-specific needs and their bone status.