A porcine collagen-derived matrix as a carrier for recombinant human bone morphogenetic protein-2 enhances spinal fusion in rats

Background contextRecombinant bone morphogenetic proteins (rhBMPs) have been used successfully in clinical trials. However, large doses of rhBMPs were required to induce adequate bone repair. Collagen sponges (CSs) have failed to allow a more sustained release of rhBMPs. Ongoing research aims to design carriers that allow a more controlled and sustained release of the protein. E-Matrix is a injectable scaffold matrix that may enhance rhBMP activity and stimulate bone regeneration.PurposeThe purpose of this study was to test E-Matrix as a carrier for rhBMPs in a CS and examine its feasibility in clinical applications by using a rat spinal fusion model.Patient sampleA total of 80 Lewis rats aged 8–16 weeks were divided into nine groups.Study design/settingRat spinal fusion model.Outcome measuresRadiographs were obtained at 4, 6, and 8 weeks. The rats were sacrificed and their spines were explanted and assessed by manual palpation, high-resolution microcomputed tomography (micro-CT), and histologic analysis.MethodsGroup I animals were implanted with CS alone (negative control); Group II animals with CS containing 10 μg rhBMP-2 (positive control); Group III animals with CS containing 3 μg rhBMP-2; Group IV animals with CS containing 3 μg rhBMP-2 and E-Matrix; Group V animals with CS containing 1 μg rhBMP-2; Group VI animals with CS containing 1 μg rhBMP-2 and E-Matrix; Group VII animals with CS containing 0.5 μg rhBMP-2; Group VIII animals with CS containing 0.5 μg rhBMP-2 and E-Matrix; and Group IX animals with CS and E-Matrix without rhBMP-2.ResultsRadiographic evaluation, micro-CT, and manual palpation revealed spinal fusion in all rats in the BMP-2 and E-Matrix groups (IV, VI, and VIII) and high-dose BMP-2 groups (II and III). Four spines in the 3 μg rhBMP-2 group (V) fused, and one spine in the 0.5 μg rhBMP-2 group (VII) exhibited fusion. No spines were fused in Groups I (CS alone) and IX (E-Matrix alone). The volume of new bone in the area between the tip of the L4 transverse process and the base of the L5 transverse process in Group IV was equivalent to the volumes observed in Group II.ConclusionE-matrix enhances spinal fusion as a carrier for rhBMP-2 in a rat spinal fusion model. The results of this study suggest that E-Matrix as a growth factor carrier may be applicable to spinal fusion and may improve rhBMP-2's activity at the fusion site.     

Investigating the synergistic efficacy of BMP-7 and zoledronate on bone allografts using an open rat osteotomy model

Bone grafts are well-established in the treatment of fracture non-unions but union is still not always achieved. Harvesting autograft is associated with donor site morbidity and the available amount of bone is limited. Allograft is more easily obtained and available in greater quantities but lacks the osteoinductive characteristics of autograft. We have previously shown a synergistic effect of bone morphogenetic protein (BMP-7), systemic bisphosphonates and autograft. In the present study we hypothesized that the combination of allograft + BMP-7 + systemic ZA is more effective than autograft alone, which is currently the most frequently used aid in augmenting fracture and non-union healing.Femoral osteotomies were performed on 82 male Sprague Dawley rats and fixed with intramedullary K-wires. The rats were randomized into 7 groups: i. saline, ii. autograft, iii. allograft, iv. allograft + BMP-7, v. autograft + zoledronate (ZA), vi. allograft + ZA and vii. allograft + BMP-7 + ZA. Autografts were harvested from the contralateral tibia. Allografts were obtained from donor rats and frozen. BMP-7 was administered locally in the form of a putty placed circumferentially around the osteotomy. At 2 weeks, the rats were injected with a single dose of either saline or ZA. The rats were sacrificed at 6 weeks and the femurs were evaluated using radiography, histology, μCT and three-point bending tests.Complete radiological healing was seen in all rats in the BMP-7 groups. The callus volume was larger and the calluses were denser with allograft + BMP-7 + ZA than in all other groups (μCT, p < 0.001). Mechanical testing yielded a substantially higher peak force with the allograft + BMP-7 + ZA combination than all other groups (p < 0.01, p < 0.001). This was further reinforced in the 59% increase in the peak force observed in the osteotomized femurs of the allograft + BMP-7 + ZA group compared to the control femurs (p < 0.01), whereas significant decreases of 22–27% were observed in the saline or bone-graft alone groups (p < 0.01, p < 0.05). Thus our results suggest that allograft combined with the anabolic effect of BMP-7 and the anti-catabolic effect of zoledronate is more efficient than autograft alone.     

Manipulation of anabolic and catabolic responses with bone morphogenetic protein and zoledronic acid in a rat spinal fusion model

Bone fusion involves a complex set of regulated signaling pathways that control the formation of new bone matrix and the resorption of damaged bone matrix at the surgical site. It has been reported that systemically administering a single dose of zoledronic acid (ZA) at the optimal time increases the strength of the bone morphogenetic protein (BMP)–mediated callus. In the present study, we aimed to investigate the effect of BMP-2 and ZA in a rat spinal model. Sixty-seven rats were divided into 6 groups: group I (n = 11) animals were implanted with a carrier alone, group II (n = 12) animals were implanted with a carrier and a subcutaneous injection of ZA was administered 2 weeks after surgery, group III (n = 12) animals were implanted with a carrier containing 1 μg of rhBMP-2, group IV (n = 12) animals were implanted with a carrier containing 1 μg of rhBMP-2 and a subcutaneous injection of ZA was administered 2 weeks after surgery, group V (n = 10) animals were implanted with a carrier containing 3 μg of rhBMP-2, and group VI (n = 10) animals were implanted with a carrier containing 3 μg of rhBMP-2 and a subcutaneous injection of ZA was administered 2 weeks after surgery. The rats were euthanized after 6 weeks, and their spines were explanted and assessed by manual palpation, radiography, high-resolution micro-computerized tomography (micro-CT), and histologic analysis. The fusion rates in group VI (60%) were considerably higher than those in the groups I (0%), II (0%), III (12.5%), IV (20.8%), and V (35%), (P < 0.05). Additionally, the radiographic scores of group VI were higher than those in the other groups, (P < 0.05). In micro-CT analysis, the tissue and bone volumes of the callus were significantly higher in group VI than those in the other groups, (P < 0.05). The trabecular number was significantly higher and the trabecular spacing was significantly lower in group VI than those in the other groups, (P < 0.05). The combination of rhBMP-2 and ZA administered systemically as a single dose at the optimal time was efficacious in our rat spinal fusion model. Our results suggest that this combination facilitates spinal fusion and has potential clinical application.     

Inkjet-based biopatterning of SDF-1β augments BMP-2-induced repair of critical size calvarial bone defects in mice

BackgroundA major problem in craniofacial surgery is non-healing bone defects. Autologous reconstruction remains the standard of care for these cases. Bone morphogenetic protein-2 (BMP-2) therapy has proven its clinical utility, although non-targeted adverse events occur due to the high milligram-level doses used. Ongoing efforts explore the use of different growth factors, cytokines, or chemokines, as well as co-therapy to augment healing.MethodsHere we utilize inkjet-based biopatterning to load acellular DermaMatrix delivery matrices with nanogram-level doses of BMP-2, stromal cell-derived factor-1β (SDF-1β), transforming growth factor-β1 (TGF-β1), or co-therapies thereof. We tested the hypothesis that bioprinted SDF-1β co-delivery enhances BMP-2 and TGF-β1-driven osteogenesis both in-vitro and in-vivo using a mouse calvarial critical size defect (CSD) model.ResultsOur data showed that BMP-2 bioprinted in low-doses induced significant new bone formation by four weeks post-operation. TGF-β1 was less effective compared to BMP-2, and SDF-1β therapy did not enhance osteogenesis above control levels. However, co-delivery of BMP-2 + SDF-1β was shown to augment BMP-2-induced bone formation compared to BMP-2 alone. In contrast, co-delivery of TGF-β1 + SDF-1β decreased bone healing compared to TGF-β1 alone. This was further confirmed in vitro by osteogenic differentiation studies using MC3T3-E1 pre-osteoblasts.ConclusionsOur data indicates that sustained release delivery of a low-dose growth factor therapy using biopatterning technology can aid in healing CSD injuries. SDF-1β augments the ability for BMP-2 to drive healing, a result confirmed in vivo and in vitro; however, because SDF-1β is detrimental to TGF-β1-driven osteogenesis, its effect on osteogenesis is not universal.     

Apatite-coated silk fibroin scaffolds to healing mandibular border defects in canines

Tissue engineering has become a new approach for repairing bony defects. Highly porous osteoconductive scaffolds perform the important role for the success of bone regeneration. By biomimetic strategy, apatite-coated porous biomaterial based on silk fibroin scaffolds (SS) might provide an enhanced osteogenic environment for bone-related outcomes. To assess the effects of apatite-coated silk fibroin (mSS) biomaterials for bone healing as a tissue engineered bony scaffold, we explored a tissue engineered bony graft using mSS seeded with osteogenically induced autologous bone marrow stromal cells (bMSCs) to repair inferior mandibular border defects in a canine model. The results were compared with those treated with bMSCs/SS constructs, mSS alone, SS alone, autologous mandibular grafts and untreated blank defects. According to radiographic and histological examination, new bone formation was observed from 4 weeks post-operation, and the defect site was completely repaired after 12 months for the bMSCs/mSS group. In the bMSCs/SS group, new bone formation was observed with more residual silk scaffold remaining at the center of the defect compared with the bMSCs/mSS group. The engineered bone with bMSCs/mSS achieved satisfactory bone mineral densities (BMD) at 12 months post-operation close to those of normal mandible (p > 0.05). The quantities of newly formed bone area for the bMSCs/mSS group was higher than the bMSCs/SS group (p < 0.01), but no significant differences were found when compared with the autograft group (p > 0.05). In contrast, bony defects remained in the center with undegraded silk fibroin scaffold and fibrous connective tissue, and new bone only formed at the periphery in the groups treated with mSS or SS alone. The results suggested that apatite-coated silk fibroin scaffolds combined with bMSCs could be successfully used to repair mandibular critical size border defects and the premineralization of these porous silk fibroin protein scaffolds provided an increased osteoconductive environment for bMSCs to regenerate sufficient new bone tissue.     

The effects of multiple high-resolution peripheral quantitative computed tomography scans on bone healing in a rabbit radial bone defect model

The use of in vivo high-resolution computed tomography (CT) scanners provides the unique opportunity for evaluating temporal progression in healing of bone defects. However, these in vivo scanners impose ionizing radiation that could affect the healing and morphology of the bone. The primary objective of this study was to determine the effects of in vivo scanning at 2-week intervals on bone healing of a critical sized radial defect in rabbits and to investigate the effect of this radiation protocol on bone marrow cell viability using clinically applicable radiation doses. Thirty male rabbits were randomized into three groups: two groups received a 15 mm defect in the left radius that was filled with an autologous bone graft (DEF-CT and DEF-SHAM), and one group acted as an intact control (INT-CT). The duration of the study was 6 weeks. DEF-CT and INT-CT had high-resolution CT scans performed at 2-week intervals. The total cumulative radiation dose was 81.6 mGy per animal. DEF-SHAM received sham CT scans at the same time points. In group DEF-CT, the bone volume (BV) in the defect increased significantly over time (p ≤ 0.002, for all comparisons); the bone mineral density (BMD) in the defect decreased over time and was significantly lower at weeks 4 and 6 than at weeks 0 and 2 (p < 0.001, for all comparisons). In group INT-CT, BV and BMD did not change over time (p = 1, for all comparison). The BV (p = 0.50) and the BMD (p = 0.37) in the defect as measured by microCT scan during ex vivo analysis was not significantly different between DEF-CT and DEF-SHAM. Similarly, histomorphometry showed no significant difference in the total bone area (p = 0.22) and percentage bone within the defect (p = 0.24) between these groups.Bone marrow analysis of the left (radiated) and right (non-radiated) radius of the INT-CT group via a Colony Forming Units (CFU) assay demonstrated an average of 25.3 and 28.5 colonies for radiated and non-radiated radii, respectively (p = 0.72).In conclusion, there was no significant difference in bone healing between radiated and non-radiated radius defects in rabbits. This is an important finding as it demonstrates that serial in vivo high resolution-CT imaging can not only provide accurate tissue regeneration data, but it can also be used to reduce the number of temporal cohorts within an experimental design.     

Sclerostin antibody and interval treadmill training effects in a rodent model of glucocorticoid-induced osteopenia

Glucocorticoids have a beneficial anti-inflammatory and immunosuppressive effect, but their use is associated with decreased bone formation, bone mass and bone quality, resulting in an elevated fracture risk. Exercise and sclerostin antibody (Scl-Ab) administration have both been shown to increase bone formation and bone mass, therefore the ability of these treatments to inhibit glucocorticoid-induced osteopenia alone or in combination were assessed in a rodent model. Adult (4 months-old) male Wistar rats were allocated to a control group (C) or one of 4 groups injected subcutaneously with methylprednisolone (5 mg/kg/day, 5 days/week). Methylprednisolone treated rats were injected subcutaneously 2 days/week with vehicle (M) or Scl-Ab-VI (M + S: 25 mg/kg/day) and were submitted or not to treadmill interval training exercise (1 h/day, 5 days/week) for 9 weeks (M + E, M + E + S). Methylprednisolone treatment increased % fat mass and % apoptotic osteocytes, reduced whole body and femoral bone mineral content (BMC), reduced femoral bone mineral density (BMD) and osteocyte lacunae occupancy. This effect was associated with lower trabecular bone volume (BV/TV) at the distal femur. Exercise increased BV/TV, osteocyte lacunae occupancy, while reducing fat mass, the bone resorption marker NTx, and osteocyte apoptosis. Exercise did not affect BMC or cortical microarchitectural parameters. Scl-Ab increased the bone formation marker osteocalcin and prevented the deleterious effects of M on bone mass, further increasing BMC, BMD and BV/TV to levels above the C group. Scl-Ab increased femoral cortical bone parameters at distal part and midshaft. Scl-Ab prevented the decrease in osteocyte lacunae occupancy and the increase in osteocyte apoptosis induced by M. The addition of exercise to Scl-Ab treatment did not result in additional improvements in bone mass or bone strength parameters. These data suggest that although our exercise regimen did prevent some of the bone deleterious effects of glucocorticoid treatment, particularly in trabecular bone volume and osteocyte apoptosis, Scl-Ab treatment resulted in marked improvements in bone mass across the skeleton and in osteocyte viability, resulting in decreased bone fragility.     

Effects of combined treatment with eldecalcitol and alendronate on bone mass,mechanical properties,and bone histomorphometry in ovariectomized rats: A comparison with alfacalcidol and alendronate

Eldecalcitol (ELD), a 2β-hydroxypropyloxy derivative of 1α,25 (OH) 2D3, inhibits bone resorption more potently than alfacalcidol (ALF) while maintaining osteoblastic function in an ovariectomized (OVX) osteoporosis rat model. Alendronate (ALN), which is the most common bisphosphonate used for the treatment of osteoporosis, increases the bone mineral density (BMD) by suppressing bone resorption. In this study, we investigated the effects of combination treatments with ELD and ALN or with ALF and ALN on bone mass and strength in OVX rats. Seventy female rats, 32 weeks old, were assigned to seven groups: (1) a sham-operated control group; (2) an OVX-control group; (3) an ELD group; (4) an ALF group; (5) an ALN group; (6) an ELD + ALN group; and (7) an ALF + ALN group. OVX rats were orally treated with ELD (0.015 μg/kg), ALF (0.0375 μg/kg), or ALN (0.2 mg/kg) daily for 12 weeks. In both the lumbar spine and the femur, ELD and ALF monotherapy significantly increased the BMD, and ELD + ALN and ALF + ALN significantly increased the BMD, compared with ALN monotherapy, as an additive effect. In particular, ELD + ALN resulted in a significantly higher BMD than ALF + ALN in the femur. On mechanical testing of the lumbar spine, ELD and ALF monotherapy significantly increased the ultimate load, and ELD + ALN and ALF + ALN significantly increased the ultimate load compared with ALN monotherapy. In the femur, ELD, ELD + ALN, and ALF + ALN treatment significantly increased the ultimate load, compared with the OVX-control group, and ELD + ALN resulted in a significantly higher ultimate load than ALN monotherapy. A histomorphometric analysis showed that ELD monotherapy and ELD + ALN combination therapy had a potent inhibitory effect on bone resorption parameters (osteoclast surface and eroded surface), while maintaining bone formation parameters (osteoblast surface and osteoid surface). By contrast, ALF and ALF + ALN significantly lowered the histological parameters of both bone resorption and formation. These results suggested that ELD or ALF used in combination with ALN has therapeutic advantages over ALN monotherapy, with ELD + ALN combination treatment producing an especially beneficial anti-osteoporotic effect by inhibiting osteoclastic bone resorption and maintaining osteoblastic function, compared with ALF + ALN combination treatment.     

Whole body vibration during fracture healing intensifies the effects of estradiol and raloxifene in estrogen-deficient rats

Current osteoporosis therapies aim to delay bone destruction and have additional anabolic effects. While they have demonstrated some positive effects on bone healing, more progress is needed in this area. This study used the well-known osteoporotic agents estrogen (E) and raloxifene (R) in conjunction with biomechanical whole body vibration (WBV) at a frequency of 70 Hz twice daily for six weeks to stimulate bone healing. Eighty-four 3-month old female Sprague–Dawley rats (12 per group) were bilaterally ovariectomized to develop osteopenia within eight weeks. Osteotomy of the metaphyseal tibiae was performed and fracture healing was then studied using mechanical tests, histomorphometry, computed tomography (μCT), and gene analysis. We found that E and R improved the structure of osteopenic bones as did WBV alone, although significant levels for WBV were seldom reached. Combination treatments significantly enhanced stiffness (R + WBV; p < 0.05), endosteal bone (R + WBV; p < 0.01), and trabecular density (E + WBV; p < 0.05, R + WBV; p < 0.05). In addition, the expression of osteoclast-specific Trap was significantly reduced after treatment with E, R, or their combination with WBV (p < 0.01). The effects were additive and not inhibitory, leading us to conclude that the combined applications of WBV with E or R may improve the healing of osteopenic bones. The therapies studied are all currently approved for human use, suggesting ready applicability to clinical practice. To better understand the effects of WBV on osteopenic bones, the ideal vibration regime will require further study.     

Alteration of Masquelet's induced membrane characteristics by different kinds of antibiotic enriched bone cement in a critical size defect model in the rat's femur

The Masquelet technique for the treatment of large bone defects consists of a 2-stage procedure. In the first stage, a polymethylmethacrylate (PMMA) cement spacer is inserted into the bony defect of a rat's femur and over a period of 2–4 weeks a membrane forms that encapsulates the defect/spacer. In a second operation the membrane is opened, the PMMA spacer is removed and the resulting cavity is filled with autologous bone. Different kinds of bone cements are available, with or without supplemental antibiotics. Both might influence the development and the characteristics of the induced membrane which might affect the bone healing response. Hence, this comparative study was performed to elucidate the effect of different bone cements with or without supplemental antibiotics on the development of an induced membrane in a critical size femur defect model in rats.A total of 72 male SD rats received a 10 mm critical size defect of the femur which was stabilised by a plate osteosynthesis and filled with either Palacos + Gentamycin, Copal Gentamycin + Vancomycin, Copal + Gentamycin + Clindamycin or Copal Spacem. The induced membranes were analysed after two, four and six weeks (wks) after insertion of the cement spacers (n = 6/group). Paraffin embedded histological sections of the membrane were microscopically analysed for membrane thickness, elastic fibres, vascularisation and proliferation by an independent observer blinded to the group setup.The thickness of the induced membrane increased significantly from 2 wks (553 μm) to 6 wks (774 μm) in group Palacos + Gentamycin whereas membrane thickness decreased significantly in groups Copal + Gentamycin + Clindamycin (682–329 μm) and Copal Spacem (916 μm to 371 μm). The comparison between the groups revealed significantly increased membrane thickness in group Palacos + Gentamycin and Copal Gentamycin + Vancomycin in comparison to group Copal + Gentamycin + Clindamycin six weeks after induction. However, the fraction of elastic fibres was significantly increased in groups Copal + Gentamycin + Clindamycin (71%, 80%) and Copal Spacem (82%, 81%) after 2 and 4 weeks in comparison to the groups Palacos + Gentamycin (56%, 57%) and Copal Gentamycin + Vancomycin (63%, 69%). Those differences however were partly diminished after 6 wks. The ratio of immature (vWF+) to more mature (CD31+) blood vessels increased significantly in groups Palacos + Gentamycin and Copal Gentamycin + Vancomycin whereas no significant alterations were noted in groups Copal + Gentamycin + Clindamycin and Copal Spacem.For the first time we demonstrated that thickness and proportion of elastic fibres in induced membranes were influenced by the type of cement and the kind of supplemental antibiotics being used. Whether these alterations of the induced membrane have an effect on bone healing remains to be proven in future studies.     

Embryonic stem cell therapy improves bone quality in a model of impaired fracture healing in the mouse; tracked temporally using in vivo micro-CT

In the current study, we used an estrogen-deficient mouse model of osteoporosis to test the efficacy of a cell-generated bone tissue construct for bone augmentation of an impaired healing fracture. A reduction in new bone formation at the defect site was observed in ovariectomized fractures compared to the control group using repeated measures in vivo micro-computed tomography (μCT) imaging over 4 weeks. A significant increase in the bone mineral density (BMD), trabecular bone volume ratio, and trabecular number, thickness and connectivity were associated with fracture repair in the control group, whereas the fractured bones of the ovariectomized mice exhibited a loss in all of these parameters (p < 0.001). In a separate group, ovariectomized fractures were treated with murine embryonic stem (ES) cell-derived osteoblasts loaded in a three-dimensional collagen I gel and recovery of the bone at the defect site was observed. A significant increase in the trabecular bone volume ratio (p < 0.001) and trabecular number (p < 0.01) was observed by 4 weeks in the fractures treated with cell-loaded collagen matrix compared to those treated with collagen I alone. The stem cell-derived osteoblasts were identified at the fracture site at 4 weeks post-implantation through in situ hybridization histochemistry. Although this cell tracking method was effective, the formation of an ectopic cellular nodule adjacent to the knee joints of two mice suggested that alternative in vivo cell tracking methods should be employed in order to definitively assess migration of the implanted cells. To our knowledge, this study is the first of its kind to examine the efficacy of stem cell therapy for fracture repair in an osteoporosis-related fracture model in vivo. The findings presented provide novel insight into the use of stem cell therapies for bone injuries.     

Dynamic acoustic radiation force retains bone structural and mechanical integrity in a functional disuse osteopenia model

Disuse osteopenia and bone loss have been extensively reported in long duration space mission and long term bed rest. The pathology of the bone loss is similar to osteoporosis but highly confined to weight bearing bones. The current anabolic and/or anti-resorptive drugs have systemic effects and are costly over extended time, with concerns of long term fracture risk. This study use Low Intensity Pulsed Ultrasound (LIPUS) as a non-invasive acoustic force and anabolic stimulus to countermeasure disuse induced bone loss. Four-month old C57BL/6 mice were randomized into five groups, 1) age-matched (AM), 2) non-suspended sham (NS), 3) non-suspended-LIPUS (NU), 4) suspended sham (SS), and 5) suspended-LIPUS (SU) groups. After four weeks of suspension, μCT analyses showed significant decreases in trabecular bone volume fraction (BV/TV) (− 36%, p < 0.005), bone tissue mineral density (TMD) (− 3%, p < 0.05), trabecular thickness (Tb.Th) (− 12.5%, p < 0.005), and increase in bone surface/bone volume (+ BS/BV) (+ 16%, p < 0.005), relative to age-matched (AM). The application of LIPUS for 20 min/day for 5 days/week, significantly increased TMD (+ 3%, p < 0.05), Tb.Th (+ 6%, p < 0.05), and decreased BS/BV (− 10%, p < 0.005), relative to suspension alone (SS) mice. Histomorphometry analyses showed a breakdown of bone microstructure under disuse conditions consist with μCT results. In comparison to SS mice, LIPUS treated bone showed increased structural integrity with increased bone formation rates at metaphysical endosteal and trabecular surfaces (+ 0.104 ± 0.07 vs 0.031 ± 0.30 μm³/μm²/day) relative to SS. Four-point bending mechanical tests of disused SS femurs showed reduced elastic modulus (− 53%, p < 0.05), yield (− 33%, p < 0.05) and ultimate strength (− 45%, p < 0.05) at the femoral diaphysis relative to AM bone. LIPUS stimulation mitigated the adverse effects of disuse on bone elastic modulus (+ 42%, p < 0.05), yield strength (+ 29%, p < 0.05), and ultimate strength (+ 39%, p < 0.05) relative to SS femurs. LIPUS provides the essential mechanical stimulus to retain bone morphological and mechanical integrity in disuse conditions. This study demonstrates LIPUS potential as regional therapeutic agent to countermeasure disuse induced bone loss while maintaining bone's integrity.     

Effects of a moderately high-protein diet and interval aerobic training combined with strength-endurance exercise on markers of bone metabolism,microarchitecture and turnover in obese Zucker rats

BackgroundWeight loss is a public health concern in obesity-related diseases such as metabolic syndrome, and the protein level of the diets seem to be crucial for the development and maintenance of bone. The nature of exercise and whether exercise in combination with moderately high-protein dietary interventions could protect against potential bone mass deficits remains unclear.ObjectivesTo investigate the effects of a moderately high-protein diet and interval aerobic training combined with strength-endurance exercise (IASE) protocol on bone status, and to assess potential interaction effects (i.e. diet*IASE).MethodsMale Zucker fatty rats were randomized distributed into 4 groups (n = 8): normoprotein + sedentary; normoprotein + exercise; moderately high-protein + sedentary, and moderately high-protein + exercise. Training groups conducted an IASE program, 5 days/week for 2 months. Markers of bone metabolism were measured in plasma. Parameters of bone mass and 3D outcomes for trabecular and cortical bone microarchitecture were assessed by micro-computed tomography.ResultsFemur length, plasma osteocalcin, sclerostin, osteoprotegerin, receptor activator of nuclear factor kappa-B ligand, insulin, leptin, PTH, uric acid and urinary phosphorus levels were lower in the moderately high-protein compared to the normoprotein groups (all, p < 0.05), whereas plasma alkaline phosphatase, aspartate aminotransferase, alanine transaminase, and urinary uric acid concentrations, and cortical total volume (TV) and bone volume (BV) were higher in the moderately high-protein (all, p < 0.01). Final body weight and alkaline phosphatase levels were lower in the exercise compared to the sedentary (both, p < 0.05), whereas femur length and weight, aminoterminal propeptides of type I procollagen and C-terminal telopeptides of type I collagen concentrations, and cortical TV and BV were higher in the exercise compared to the sedentary groups (all, p < 0.05).ConclusionThe combination of interventions may be effective to enhance trabecular bone microarchitecture and BMD, and has a partial impact on cortical bone in obese rats. Nevertheless, they do not induce any alteration on the bone turnover markers.     

Prostate-only Versus Whole-pelvis Radiation with or Without a Brachytherapy Boost for Gleason Grade Group 5 Prostate Cancer: A Retrospective Analysis

BackgroundThe role of elective whole-pelvis radiotherapy (WPRT) remains controversial. Few studies have investigated it in Gleason grade group (GG) 5 prostate cancer (PCa), known to have a high risk of nodal metastases.ObjectiveTo assess the impact of WPRT on patients with GG 5 PCa treated with external-beam radiotherapy (EBRT) or EBRT with a brachytherapy boost (EBRT + BT).Design, setting, and participantsWe identified 1170 patients with biopsy-proven GG 5 PCa from 11 centers in the United States and one in Norway treated between 2000 and 2013 (734 with EBRT and 436 with EBRT + BT).Outcome measurements and statistical analysisBiochemical recurrence-free survival (bRFS), distant metastasis-free survival (DMFS), and prostate cancer-specific survival (PCSS) were compared using Cox proportional hazards models with propensity score adjustment.Results and limitationsA total of 299 EBRT patients (41%) and 320 EBRT + BT patients (73%) received WPRT. The adjusted 5-yr bRFS rates with WPRT in the EBRT and EBRT + BT groups were 66% and 88%, respectively. Without WPRT, these rates for the EBRT and EBRT + BT groups were 58% and 78%, respectively. The median follow-up was 5.6 yr. WPRT was associated with improved bRFS among patients treated with EBRT + BT (hazard ratio [HR] 0.5, 95% confidence interval [CI] 0.2–0.9, p = 0.02), but no evidence for improvement was found in those treated with EBRT (HR 0.8, 95% CI 0.6–1.2, p = 0.4). WPRT was not significantly associated with improved DMFS or PCSS in the EBRT group (HR 1.1, 95% CI 0.7–1.7, p = 0.8 for DMFS and HR 0.7, 95% CI 0.4–1.1, p = 0.1 for PCSS), or in the EBRT + BT group (HR 0.6, 95% CI 0.3–1.4, p = 0.2 for DMFS and HR 0.5 95% CI 0.2–1.2, p = 0.1 for PCSS).ConclusionsWPRT was not associated with improved PCSS or DMFS in patients with GG 5 PCa who received either EBRT or EBRT + BT. However, WPRT was associated with a significant improvement in bRFS among patients receiving EBRT + BT. Strategies to optimize WPRT, potentially with the use of advanced imaging techniques to identify occult nodal disease, are warranted.Patient summaryWhen men with a high Gleason grade prostate cancer receive radiation with external radiation and brachytherapy, the addition of radiation to the pelvis results in a longer duration of prostate-specific antigen control. However, we did not find a difference in their survival from prostate cancer or in their survival without metastatic disease. We also did not find a benefit for radiation to the pelvis in men who received radiation without brachytherapy.     

Membrane depolarization regulates intracellular RANKL transport in non-excitable osteoblasts

Parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D₃ (VD₃) are important factors in Ca^2 + homeostasis, and promote osteoclastogenesis by modulating receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA expression. However, their contribution to RANKL intracellular transport (RANKLiT), including the trigger for RANKL lysosomal vesicle (RANKL-lv) fusion to the cell membrane, is unclear. In neurons, depolarization of membrane potential increases the intracellular Ca^2 + level ([Ca^2 +]_i) and promotes neurotransmitter release via fusion of the synaptic vesicles to the cell membrane. To determine whether membrane depolarization also regulates cellular processes such as RANKLiT in MC3T3-E1 osteoblasts (OBs), we generated a light-sensitive OB cell line and developed a system for altering their membrane potential via delivery of a blue light stimulus. In the membrane fraction of RANKL-overexpressing OBs, PTH and VD₃ increased the membrane-bound RANKL (mbRANKL) level at 10 min after application without affecting the mRNA expression level, and depolarized the cell membrane while transiently increasing [Ca^2 +]_i. In our novel OB line stably expressing the channelrhodopsin-wide receiver, blue light-induced depolarization increased the mbRANKL level, which was reversed by treatment of blockers for L-type voltage-gated Ca^2 + channels and Ca^2 + release from the endoplasmic reticulum. In co-cultures of osteoclast precursor-like RAW264.7 cells and light-sensitive OBs overexpressing RANKL, light stimulation induced an increase in tartrate-resistant acid phosphatase activity and promoted osteoclast differentiation. These results indicate that depolarization of the cell membrane is a trigger for RANKL-lv fusion to the membrane and that membrane potential contributes to the function of OBs. In addition, the non-genomic action of VD₃-induced RANKL-lv fusion included the membrane-bound VD₃ receptor (1,25D₃-MARRS receptor). Elucidating the mechanism of RANKLiT regulation by PTH and VD₃ will be useful for the development of drugs to prevent bone loss in osteoporosis and other bone diseases.     

Zoledronate prevents lactation induced bone loss and results in additional post-lactation bone mass in mice

In rodents, lactation is associated with a considerable and very rapid bone loss, which almost completely recovers after weaning. The aim of the present study was to investigate whether the bisphosphonate Zoledronate (Zln) can inhibit lactation induced bone loss, and if Zln interferes with recovery of bone mass after lactation has ceased.Seventy-six 10-weeks-old NMRI mice were divided into the following groups: Baseline, Pregnant, Lactation, Lactation + Zln, Recovery, Recovery + Zln, and Virgin Control (age-matched). The lactation period was 12 days, then the pups were removed, and thereafter recovery took place for 28 days. Zln, 100 μg/kg, was given s.c. on the day of delivery, and again 4 and 8 days later. Mechanical testing, μCT, and dynamic histomorphometry were performed. At L4, lactation resulted in a substantial loss of bone strength (− 55% vs. Pregnant, p < 0.01), BV/TV (− 40% vs. Pregnant, p < 0.01), and trabecular thickness (Tb.Th) (− 29% vs. Pregnant, p < 0.001). Treatment with Zln completely prevented lactation induced loss of bone strength, BV/TV, and Tb.Th at L4. Full recovery of micro-architectural and mechanical properties was found 28 days after weaning in vehicle-treated mice. Interestingly, the recovery group treated with Zln during the lactation period had higher BV/TV (+ 45%, p < 0.01) and Tb.Th (+ 16%, p < 0.05) compared with virgin controls. Similar results were found at the proximal tibia and femur. This indicates that Zln did not interfere with the bone formation taking place after weaning. On this background, we conclude that post-lactation bone formation is not dependent on a preceding lactation induced bone loss.     

Effects of osteogenic medium on healing of the experimental critical bone defect in a rabbit model

Today, finding an ideal biomaterial to treat the large bone defects, delayed unions and non-unions remains a challenge for orthopedic surgeons and researchers. Several studies have been carried out on the subject of bone regeneration, each having its own advantages. At the same time, a variety of disadvantages still remain. The present study has been designed in vivo to evaluate the effects of osteogenic medium on healing of experimental critical bone defect in a rabbit model. Twenty New Zealand albino rabbits, 12 months old, of both sexes, weighing 2.0 ± 0.5 kg were used in this study. An approximately 10 mm segmental defect was created in the mid portion of each radius as a critical size bone defect. In the osteogenic medium group (n = 5) 1 ml osteogenic medium, in the maintenance medium group (n = 5) 1 ml maintenance medium, and in the normal saline group (n = 5) 1 ml normal saline were injected in the defected area while the defects of the rabbits of the control group (n = 5) were left empty. Radiological evaluation was done on the 1st day and then at the 2nd, 4th, 6th and 8th weeks post injury. Biomechanical and histopathological evaluations were done 8 weeks post injury. The radiological, histological and biomechanical findings of the present study indicated a superior bone healing capability in the osteogenic and maintenance medium groups, by the end of 8 weeks post-surgery, in comparison to the normal saline and control groups. In conclusion, this study demonstrated that the osteogenic medium and maintenance medium could promote bone regeneration in long bone defects better than the control group in rabbit model.     

Ibrutinib suppresses alloantibody responses in a mouse model of allosensitization

BackgroundIbrutinib is a Bruton's tyrosine Kinase (BTK) antagonist that inhibits B cell receptor (BCR) signaling. Complete BTK deficiency is associated with absence of B-cells. Ibrutinb is currently approved by FDA for treatment of B-cell malignancies, including Waldenström macroglobulinaemia. We recently carried out studies to determine if ibrutinib could modify alloantibody responses.Materials and methodsA mouse model of allogenic sensitization using a C57BL/6 mouse as the recipient of a skin allograft from an HLA-A2 transgenic mouse was utilized to examine the effects of ibrutinib on alloantibody responses and B cell effector functions. Donor-specific antibody (DSA) levels were measured in a flow-cytometric antibody binding assay. Splenic T and B cell subsets and plasma cells were analyzed in flow cytometry.ResultsControl mice developed peak levels of DSA IgM at day 14 PTx while the ibrutinib treated mice had significantly lower levels of DSA IgM (p = 0.0047). Control mice developed HLA.A2-specific IgG antibodies at day 14 (230 ± 60 MFI) and reached peak levels at day 21 (426 ± 61 MFI). In contrast, mice in the treatment group had low levels of HLA.A2-specific IgG at day 14 (109 ± 59 MFI, p = 0.004) and day 21 (241 ± 86 MFI, p = 0.003). FACS analysis found a reduction of B220⁺ or CD19⁺ B cell population (p < 0.05). In addition, ibrutinib attenuated recall DSA IgG responses to re-sensitization (p < 0.05) and reduced CD38⁺ CD138⁺ plasma cells (p < 0.05) in the spleens.ConclusionsIbrutinib is effective in suppressing alloantibody responses through blocking BTK-mediated BCR signaling, leading to reduction of B cells and short-lived plasma cells in the spleens. Use of ibrutinib may provide benefits to HLA-sensitized transplant patients for alloantibody suppression.     

The effect on improvement of recovery and pain scores of paravertebral block immediately before breast surgery

ObjectivesParavertebral block (PVB) has the potential to reduce postoperative pain after breast surgery. The aim of the study was to investigate whether PVB performed immediately before surgery could affect the postoperative morbidities in terms of pain and emesis, and improve the quality of recovery (QoR) in patients after surgery for breast cancer.MethodsPostoperative data were collected prospectively from two groups of patients undergoing unilateral breast surgery during the study period of 1 month. Forty consecutive patients received either solely general anesthesia (GA group, n = 25) or GA plus ultrasound-guided PVB (GA + PVB group, n = 15) for the surgery. Pain scores and areal distribution of pain were compared between the two groups 1 hour and 6 hours postoperatively and on the midmorning of postoperative Day 1 (POD1). The QoR scores were compared between the two groups 6 hours postoperatively and on the midmorning of POD1. Incidence of postoperative nausea and vomiting and doses of analgesics and narcotics given were also compared.ResultsPain scores at rest were significantly lower in the GA + PVB group at all designated time points [1 hour (p < 0.0001), 6 hours (p < 0.0001), and on midmorning of POD1 (p = 0.041)]. Pain scores with movements was also significantly lower at all time points in the GA + PVB group (1 hour, p < 0.0001; 6 hours, p < 0.0001; midmorning of POD1, p = 0.0012). Areal distribution of pain at rest and with movement was wider in the GA group 1 hour and 6 hours postoperately but was identical to that of GA+ PVB group on the mid-morning of POD1 [1 hour postoperatively at rest (p < 0.0001), with movement (p < 0.0001); 6 hours postoperatively at rest (p = 0.0018), with movement (p = 0.0048)]. The QoR scores were significantly higher in the GA + PVB group at 6 hours (p < 0.0001) and on midmorning of POD1 (p = 0.0079). The incidences of postoperative nausea and vomiting were significantly lower in the GA + PVB group (p = 0.0004). Doses of postoperative analgesics and narcotics were significantly less in the GA + PVB group (p < 0.0001 and p = 0.001, respectively). Time to first request for analgesics was significantly longer in the GA + PVB group (p = 0.0002).ConclusionsPVB given before surgery in combination with GA could provide better postoperative analgesia and better QoR than did GA alone in patients undergoing surgery for unilateral breast cancer.