Effect of gamma irradiation on the osteoinductivity of morphogenetic protein extract from reindeer bone

BACKGROUND: Bone morphogenetic proteins (BMPs), which are capable of stimulating the production of new bone, must be sterilized before preclinical and clinical use to reduce the risk of infections and associated complications. In this study, we investigated the effects of gamma sterilization on the osteoinductivity of native reindeer BMP extract in the Balb/C mouse thigh muscle pouch model. METHODS: 5 mg of native reindeer BMP extract and 5 mg of bovine serum albumin were administered separately either in gelatine capsules or mixed with gelatine as injections. The dose of gamma irradiation was 4.1 Mrad. Unsterile capsules and injections served as controls. New bone formation was evaluated based on the incorporation of Ca45 and also radiographically 3 weeks after implantation. RESULTS: Albumin-containing implants and injections did not induce new bone formation, as monitored in radiographs. Gamma sterilization did not reduce the osteoinductivity of native BMP extract in capsules, but a significant decrease in osteoinductivity--measured as area (50%) and Ca45 incorporation of new bone (27%)--was seen after injection. Gamma sterilization had no effect on the optical density of new bone induced by native BMP extract administered in capsules or by injection. INTERPRETATION: We conclude that, as gamma irradiation did not reduce the osteoinductivity of reindeer BMP extract in gelatine capsules, this method appears to be suitable for sterilization of BMPs to be given in capsule form. Native reindeer BMP extract was more sensitive to irradiation in soluble collagen (gelatine) than BMP in gelatine capsules. This finding must be given serious consideration regarding treatment of patients, but the remaining activity may be sufficient for the induction of bone formation in preclinical and clinical situations.     

Reindeer BMP extract in the healing of critical-size bone defects in the radius of the rabbit

Background?Native BMP extracts from reindeer effectively induce ectopic new bone formation in vivo, but their bone healing properties have not yet been evaluated. We investigated the effect of reindeer BMP extracts on the healing of long bone defects.

Methods?The implants tested contained 5?mg or 10?mg of unsterilized BMP extract from reindeer and 10?mg of gamma-sterilized BMP extract administered with collagen carrier (Lyostypt, B. Braun, Germany). 70 μg of rhBMP-2 with collagen carrier (InductOs; Wyeth Europa) served as positive control, and collagen implants (Lyostypt) and untreated defects served as negative controls. New Zealand White rabbits with 1.5?cm of critical-size radius bone defects were used, with 8 weeks of follow-up.

Results?Radiographic analysis showed bone formation (BF) to be higher in all groups containing BMPs than in the untreated controls. BF was also higher in the rhBMP-2 group, and marginally higher in the group treated with 10?mg of unsterilized reindeer BMP extract (p = 0.06) as compared to the collagen controls. Bone union (BU) was better in the unsterilized BMP extract groups and rhBMP-2 group than in the untreated controls. BU was also better in the implants with 10?mg of unsterilized reindeer BMP extract and rhBMP-2 than in the collagen-treated implants. The mean area of new bone at the site of the defect proved to be higher in all implants containing BMP than in the untreated defects. It was also higher in the groups with 10?mg of unsterilized reindeer BMP extract and rhBMP-2 than in the collagen-treated controls. Mechanical tests showed torsional stiffness of the bones to be higher in the group with 10?mg of unsterilized BMP extract than in the collagen group. The mean cross-sectional bone area measured by pQCT densitometry was higher in the rhBMP-2 group than in the collagen group. The mean bone density at the defect area was higher in the group with 10?mg of unsterilized BMP than in the rhBMP-2 group.

Interpretation?We conclude that both reindeer BMP extract and rhBMP-2 induced improved healing of the rabbit radius bone defects at the doses used. Gamma sterilization of reindeer BMP extract reduced osteoinductivity slightly, but not significantly.     

Gamma irradiation and ethylene oxide in the sterilization of native reindeer bone morphogenetic protein extract.

BACKGROUND AND AIMS: For human use, it is necessary to sterilize bone morphogenetic proteins (BMPs), in order to reduce the risk of infections and associated complications. We compared the effects of ethylene oxide and gamma irradiation in the sterilization of native reindeer BMP extract with regard to bone induction in the Balb/C mouse thigh muscle pouch model. MATERIALS AND METHODS: BMP extract, sterilized with ethylene oxide gas (Steri-Vac 4XL, temperature 29 degrees C, exposure time 4 h, ethylene oxide concentration 860 mg/l), or gamma irradiation at doses of 3.15 MRad was administered in implants containing 5 or 10 mg of BMP extract with collagen carrier. Non-sterilized collagen implants served as controls. New bone formation was evaluated based on the incorporation of Ca45 and radiographically three weeks after implantation. RESULTS: The collagen was not able to induce new bone visible in radiographs. The mean Ca45 incorporation in the gamma sterilized group containing 5 mg of BMP extract was 30% (p = 0.04) and that containing 10 mg of BMP extract was 60% (p = 0.02) higher than seen in the corresponding ethylene oxide sterilized groups. The mean new bone areas were 45% higher in the gamma sterilized groups than in the corresponding ethylene oxide sterilized groups, but the differences were not significant. The mean optical density of new bone in the gamma sterilized group containing 5 mg of BMP extract was 75% (p = 0.00) and in that containing 10 mg of BMP extract was 70% (p = 0.00) higher than seen in the corresponding ethylene oxide sterilized groups. CONCLUSION: Native reindeer BMP extract is more sensitive to the effects of ethylene oxide gas sterilization than gamma irradiation. These results suggest that gamma irradiation is recommendable for the sterilization of BMP extracts.     

The effect of different mineral frames on ectopic bone formation in mouse hind leg muscles induced by native reindeer bone morphogenetic protein

Introduction Bone morphogenetic proteins (BMPs) require carrier material for slow release and framing material for osteoconduction.Materials and methods The effect of a frame on early bone formation induced by partially purified native reindeer BMP in composite implants containing 3 mg of BMP, type IV collagen and tricalcium phosphate (TCP/Col/BMP) or hydroxyapatite (HA/Col/BMP) or biphasic tricalcium phosphate-hydroxyapatite (TCP/HA/Col/BMP) or biocoral (NC/Col/BMP) was evaluated using a mouse hind leg muscle pouch model. Collagen with native reindeer BMP (Col/BMP) and corresponding implants without native reindeer BMP served as controls. Evaluation was done by incorporation of ⁴⁵Ca, radiographically and histologically 3 weeks after the implantation.Results None of the implants without native reindeer BMP were able to induce new bone visible on radiographs. The area of new bone formation in the Col/BMP (p=0.026) and TCP/HA/Col/BMP (p=0.012) groups was significantly greater than in the TCP/Col/BMP group. The optical density of the new bone area was significantly greater in the TCP/HA/Col/BMP group than in the TCP/Col/BMP (p=0.036) or Col/BMP (p=0.02) groups. ⁴⁵Ca incorporation was many times greater in all the groups containing native reindeer BMP than in the corresponding groups without BMP. In the Col/BMP (p=0.046) and TCP/HA/Col/BMP (p=0.046) groups, ⁴⁵Ca incorporation was significantly greater than in the TCP/Col/BMP group. No significant differences were found in any parameters between HA/Col/BMP and NC/Col/BMP groups and the other BMP-containing groups.Conclusions Hydroxyapatite, biocoral and biphasic tricalciumphosphate-hydroxyapatite are equally good as framing material for native reindeer BMP, while tricalciumphosphate is somewhat worse. Osteoinduction of native reindeer BMP works well with collagen alone.     

11 kGy gamma irradiated demineralized bone matrix enhances osteoclast activity

Background

Demineralized bone matrix (DBM) allografts are widely used in orthopaedic clinics. However, the biological impact on its osteoinductivity after its sterilization process by gamma irradiation is not well studied. Furthermore, little is known about the relationship between residual calcium levels on osteoinductivity.

Hypothesis

We hypothesize that low-dose gamma irradiation retains the osteoinducitivity properties of DBM and causes ectopic bone formation.

Materials and methods

A randomised animal trial was performed to compare tissue and molecular responses of low-dose (11 kGy) gamma irradiated and non-irradiated human DBM at 6 weeks post-intramuscular implantation using an athymic rat model. In addition, we correlated residual calcium levels and bone formation in gamma irradiated DBM.

Results

A modified haematoxylin and eosin stain identified ectopic bony capsules at all implanted sites with no significant difference on the amount of new bone formed between the groups. Statistically significantly lower ratio of alkaline phosphatase expression over tartrate-resistant acid phosphatase and/or cathepsin K expressions was found between the groups.

Discussion

This study found that low-dose gamma irradiated DBM, which provides a sterility assurance level of 10^?6 for bone allografts, retained osteoinductivity but exhibited significantly enhanced osteoclastic activity. Furthermore, this is the first study to find a positive correlation between residual calcium levels and bone formation in gamma irradiated DBM.     

Irradiation has no effect on the incorporation of impacted morselized bone: a bone chamber study in goats

Background Gamma irradiation has been widely used for sterilization of bone allografts. However, gamma irradiation alters proteins. This is favorable when it reduces immunogenicity, but is undesirable when osteoinductive proteins are damaged. Although the effect of gamma irradiation on BMPs has been studied, the effect of irradiation on the process of incorporation of morselized bone chips remains unclear. We studied the effects of sterilization by gamma irradiation on the incorporation of impacted morselized allografts.

Methods Bone chambers with impacted allografts, rinsed impacted allografts, allografts that were rinsed and subsequently irradiated, and an empty control were implanted in proximal medial tibiae of goats. Incorporation was evaluated using histology and histomorphometry.

Results Histology revealed evidence of bone graft incorporation, which proceeded in a similar way in unprocessed, rinsed, and both rinsed and irradiated bone grafts. After 12 weeks, no difference in bone and tissue ingrowth was found between the unprocessed, the rinsed, and the rinsed and subsequently irradiated allografts. The amount of unresorbed graft remnant was highest in the unprocessed bone grafts.

Interpretation We conclude that sterilization with gamma irradiation does not influence the incorporation of impacted rinsed bone allografts.     

Effect of sterilization on bone morphogenetic protein

Demineralized bone matrix and bone morphogenetic protein have been used clinically to accelerate bone regeneration. However, the best method of sterilization has been the subject of controversy. Some investigators have used ethylene oxide, but others have reported that doses adequate for sterilization destroyed the osteoinductivity of demineralized bone matrix and that gamma irradiation was less harmful in this respect. We used partially purified bone morphogenetic protein and type-I collagen to investigate the effects of sterilization by ethylene oxide and gamma irradiation on the activity of bone morphogenetic protein. Osteoinductivity was reduced considerably after sterilization by gamma irradiation at 2.5 Mrad and by ethylene oxide at 37°C for 4 hours and at 55°C for 1 hour; however, the reduction induced by ethylene oxide at 29°C for 5 hours was about half of the control values. This study showed that ethylene oxide at 29°C for 5 hours can be used clinically for sterilization of bone morphogenetic protein. We also investigated the effect of gamma irradiation on bone morphogenetic protein and the collagen carrier separately and found that collagen was far more labile than bone morphogenetic protein.     

Ethylene oxide does not extinguish the osteoinductive capacity of demineralized bone: A reappraisal in rats

We examined the influence of ethylene oxide (EO) and gamma irradiation on the osteoinductive capacity of demineralized bone. Demineralized bone powder prepared from Wistar rats was exposed to EO (55 °C or 40 °C) or gamma irradiation (25 KGy) or was preserved in ethanol. Sterilely-prepared bones served as controls. The powder was packed in a gelatin capsule and implanted for 6 weeks in muscles of 6-week-old female rats. Exposure of demineralized bone particles to EO 55 °C resulted in an almost complete loss of osteoinductivity. Irradiated bones lost about 40% of their osteoinductive capacity, while sterilization with EO at 40 °C resulted in only a slight alteration of the osteoinductivity, as assessed by the recovered weight ratio, calcium content, alkaline phosphatase activity measurements and histo-morphometry. Ethanol treatment had no influence on the new bone yield when compared to controls.

As EO exposure at 40 °C is a true sterilization procedure, it can be recommended in a clinical setting for its small effect on osteoinductive capacity as assessed experimentally in rats.     

A novel application of high-dose (50 kGy) gamma irradiation for demineralized bone matrix: effects on fusion rate in a rat spinal fusion model

BACKGROUND CONTEXT: The safety of allograft material has come under scrutiny because of recent reports of allograft-associated bacterial and viral infections in tissue recipients. Gamma irradiation, although being one of the most effective ways of terminal sterilization, has been shown to affect the biomechanical properties of allograft bone. It may also have detrimental effects on the osteoinductivity of allograft material such as demineralized bone matrix (DBM) by the denaturation of proteins because of heat generated by irradiation. Sterilization of DBM material is an important variable in processing graft materials. This is considered to be one of the factors leading to different fusion rates observed with different commercially available DBM products, as the sterilization procedure itself may affect the osteoinductivity of the material. Currently, there is no ideal sterilization technique that limits the detrimental effect on osteoinductivity and fusion rates. PURPOSE: To evaluate the effects of a range of hydrogen peroxide exposures with or without the controlled high-dose gamma irradiation after processing with radioprotectant solutions (Clearant radiation sterilization procedure) on the fusion rates of human DBM. STUDY DESIGN: A prospective in vivo animal study. METHODS: Eighty mature athymic nude female rats were used for this study, which formed 10 equal groups. Human DBM exposed to hydrogen peroxide for different time periods (0, 1, 6, and 24 hours) was divided into two major subgroups. One group was further treated with controlled high-dose radiation using radioprotectants (radiation treated), whereas the other group was frozen immediately without specific treatment (non-radiation treated). Both radiation-treated and non-radiation-treated DBM material from each group of hydrogen peroxide exposure times were implanted between L4 and L5 transverse processes of the rats forming eight test groups including eight animals in each. The remaining 16 rats were divided into two additional groups to form negative (only decortication, n=8) and positive (bone morphogenetic protein [BMP]-2, n=8) control groups. The rats were evaluated for fusion by radiographs (2, 4, and 8 weeks), manual palpation (8 weeks), and histological analysis after sacrificing. Comparison of fusion rate among all groups was made using these three evaluation methods. RESULTS: Increasing the time period of hydrogen peroxide (0, 1, 6, or 24 hours) exposure for preparation of DBM from bone allograft did not affect the fusion rates significantly (p<.05), although there was a trend toward decreasing fusion rates with longer exposure times. When the hydrogen peroxide washed DBM preparations were also radiation treated, the resulting fusion rates were again not significantly different (p<.05). Agreement among fusion detection methods was found to be high. CONCLUSIONS: Hydrogen peroxide processing was not detrimental to fusion rates. The additional terminal sterilization technique with special gamma irradiation protocols (Clearant process) also did not decrease the fusion rates but could provide an additional margin of safety.     

Are Endogenous BMPs Necessary for Bone Healing during Distraction Osteogenesis?

Previous reports suggest the application of exogenous BMPs can accelerate bone formation during distraction osteogenesis (DO). However, there are drawbacks associated with the use of exogenous BMPs. A possible alternative to the use of exogenous BMPs is to upregulate the expression of endogenous BMPs. Since DO results in spontaneously generated de novo bone formation in a uniform radiographic, histological, and biomechanical temporal sequence, a genetically engineered model lacking endogenous BMP2 should have measurable deficits in bone formation at different time points. We performed DO on BMP2  ^fl/+ and BMP2  ^{fl/+ cre} mice using a miniature Ilizarov fixator. Distracted samples were collected at various time points and analyzed using Real Time-quantitative PCR, μCT, radiology, immunohistochemistry, histology, and biomechanical testing. Immunohistochemical studies of 34-day heterozygous samples showed reduced expression of BMP2, BMP7, BMPR1a, ACTR1, and ACTR2b. μCT analysis of 51-day heterozygous samples revealed a decrease in trabecular number and increase in trabecular separation. Biomechanical testing of 51-day heterozygous samples revealed decreased stiffness and increased ultimate displacement. Radiological analysis showed the heterozygotes contained a decreased bone fill score at 17, 34, and 51 days. These data suggest endogenous BMPs are important for bone healing and manipulating endogenous BMPs may help accelerate bone consolidation during DO.     

The use of bone morphogenetic protein in spine fusion

Background contextBecause pseudarthrosis remains a clinically significant complication after spinal arthrodesis, the role of recombinant bone morphogenetic proteins (BMPs) is continually evaluated in spine surgery.PurposeThis article reviews the important literature in clinical research involving the use of BMPs in the augmentation of spinal fusion.Study design/settingReview article.MethodsA literature search was performed via MEDLINE through PubMed with the dates January 1960 to July 2007 using the keywords “bone morphogenetic protein, BMP, spinal arthrodesis, and/or bone healing.” Pertinent preclinical and clinical publications were chosen based on relevance and quality for inclusion in this study.ResultsPublications focused on the historical context and potential clinical applications using BMP were selected to delineate the risks, benefits, and current indications for the augmentation of spinal arthrodesis.ConclusionsAlthough multiple commercially available recombinant BMPs have demonstrated clinical success in interbody and posterolateral fusions, the associated costs preclude its routine use in spinal arthrodesis. The spine surgeon must assess each patient individually based on age, bone quality, diagnosis, comorbidities, and risks of nonunion to determine the cost effectiveness of the use of BMP to augment spinal fusion.     

Immune response and effect of adenovirus-mediated human BMP-2 gene transfer on the repair of segmental tibial bone defects in goats

Background?Tissue-engineered bone may be used for filling bone defects. There are, however, no reports on this technique used in large animals.

Methods?We evaluated the effectiveness of, and immune response in repairing diaphyseal bone defects by gene transfer using bone morphogenetic proteins (BMPs). We used adenovirus-mediated human BMP-2 (Adv-hBMP-2) gene-transduced bone marrow stromal cells (BMSCs) to repair 2.1-cm segmental tibial bone defects in goats (group I, n = 7). An Adv-ßgal-transduced BMSC group (group II, n = 5), a non-transduced BMSC group (group III, n = 5), and an untreated group (group IV, n = 2) were used as controls. Self-secreted extracellular matrix was used as cellular carrier.

Results?Radiographic and histomorphometric examination demonstrated more callus in the bone defects of group I compared to other groups.Week 24 after implantation, the defect healing rates of groups I, II, III, and IV were 6/7, 1/5, 2/5, and 0/2, respectively. The maximum compressive strength of new tissue in the bone defects of group I was higher than those of groups II and III. Temporary cellular and persistent humoral immune responses against adenovirus were detected after hBMP-2 gene transfer.

Interpretation?We found that Adv-hBMP-2 genetransduced BMSCs had superior osteoinductivity in the repair of tibial bone defects in goats, but it could cause temporary cellular and persistent humoral immune responses against adenovirus.     

Parathyroid hormone induces differentiation of mesenchymal stromal/stem cells by enhancing bone morphogenetic protein signaling

Parathyroid hormone (PTH) stimulates bone remodeling and induces differentiation of bone marrow mesenchymal stromal/stem cells (MSCs) by orchestrating activities of local factors such as bone morphogenetic proteins (BMPs). The activity and specificity of different BMP ligands are controlled by various extracellular antagonists that prevent binding of BMPs to their receptors. Low-density lipoprotein receptor-related protein 6 (LRP6) has been shown to interact with both the PTH and BMP extracellular signaling pathways by forming a complex with parathyroid hormone 1 receptor (PTH1R) and sharing common antagonists with BMPs. We hypothesized that PTH-enhanced differentiation of MSCs into the osteoblast lineage through enhancement of BMP signaling occurs by modifying the extracellular antagonist network via LRP6. In vitro studies using multiple cell lines, including Sca-1⁺CD45^–CD11b^–MSCs, showed that a single injection of PTH enhanced phosphorylation of Smad1 and could also antagonize the inhibitory effect of noggin. PTH treatment induced endocytosis of a PTH1R/LRP6 complex and resulted in enhancement of phosphorylation of Smad1 that was abrogated by deletion of PTH1R, β-arrestin, or chlorpromazine. Deletion of LRP6 alone led to enhancement of pSmad1 levels that could not be further increased with PTH treatment. Finally, knockdown of LRP6 increased the exposure of endogenous cell-surface BMP receptor type II (BMPRII) significantly in C2C12 cells, and PTH treatment significantly enhanced cell-surface binding of ¹²⁵I-BMP2 in a dose- and time-dependent manner, implying that LRP6 organizes an extracellular network of BMP antagonists that prevent access of BMPs to BMP receptors. In vivo studies in C57BL/6J mice and of transplanted green fluorescent protein (GFP)-labeled Sca-1⁺CD45^–CD11b^–MSCs into the bone marrow cavity of Rag2^−/− immunodeficient mice showed that PTH enhanced phosphorylation of Smad1 and increased commitment of MSCs to osteoblast lineage, respectively. These data demonstrate that PTH enhancement of MSC differentiation to the osteoblast lineage occurs through a PTH- and LRP6-dependent pathway by endocytosis of the PTH1R/LRp6 complex, allowing enhancement of BMP signaling. © 2012 American Society for Bone and Mineral Research.     

Influence of ethylene oxide sterilization on the activity of native reindeer bone morphogenetic protein

We studied the effects of ethylene oxide sterilization (Steri-Vac 4XL, temperature 29°C, exposure time 4 h 10 min, ethylene oxide concentration 860 mg/l) on the osteoinductivity of partially purified native reindeer bone morphogenetic protein (BMP) in a hind leg muscle pouch model of male NMRI mice. BMP was administered in implants containing 3 mg in a collagen carrier. Implants without sterilization and without BMP served as controls. New bone formation was evaluated based on the calcium yield, radiographic and histological examination 3 weeks after implantation. The implants without BMP were not able to induce new bone visible in radiographs. In the sterilized BMP group, the mean area of new bone was 35% (p=0.004) and density 32% (p=0.000) smaller than in the nonsterilized group. Calcium yield was 20% lower in the sterilized group than in the nonsterilized group, but this difference was not significant (p=0.22). It was many times lower in the group without BMP than in the above-mentioned groups (p=0,001). We conclude that ethylene oxide gas sterilization reduces the bone-forming activity of native reindeer BMP by one third.

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Résumé Nous avons étudié les effets de la stérilisation à loxyde déthylène (Steri-Vac 4XL, température 29ºC, temps dexposition 4 h, concentration de loxyde déthylène 860 mg/l) sur losteoinductivité de la proteine morphogénétique osseuse (BMP) du renne partiellement purifié dans un modèle fait par une bourse du muscle de la patte postérieure de la souris NMRI virile. La BMP a été administré par des implants qui contiennent 3 mg dans un porteur de collagène. Des implants sans stérilisation et sans BMP ont servi de contrôle. La néo- formation osseuse a été évaluée sur la formation de calcium, radiographiquement et histologiquement trois semaines après implantation. Les implants sans BMP nétaient pas capables dinduire un nouvel os visible sur les radiographies. Avec la BMP stérilisé la surface moyenne dos nouveau était 35% (p=0.004) et la densité 32% (p=0.000) plus petite que dans le groupe BMP non—stérilisé. La production de calcium était 20% inférieure dans le groupe BMP stérilisé que dans le groupe BMP non—stérilisé, mais cette différence nétait pas significative (p=0.22). Elle était plusieurs fois plus faible dans le groupe sans BMP que dans les groupes susmentionnés (p=0,001). Nous concluons que la stérilisation à loxyde déthylène réduit dun tiers lactivité ostéoformatrice de la BMP de renne.

     

Reindeer bone extract can heal the critical-size rat femur defect

Bone extract from reindeer induces new ectopic bone formation (BF) in muscle pouches, but its feasibility in experimental bone lesions has not been evaluated. We investigated the effects of implants, containing 2, 5, 15, 20 or 50 mg of reindeer bone extract in a collagen carrier, on the healing of 8-mm femur defects in 78 rats. We used 30 μg of recombinant human bone morphogenetic protein-2 (rhBMP-2) in a collagen carrier, collagen and untreated defects as controls. Bone healing was evaluated with radiographs, peripheral quantitative computed tomography (pQCT), biomechanics and histology. In comparison with empty defects, the groups receiving bone extracts showed more BF at three weeks and had better bone union (BU), larger mean cross-sectional bone area at the defect site in groups receiving higher doses of extract, showed greater torsional stiffness of the bones and higher maximum breaking load of bones at six weeks. In comparison to all other groups, in the rhBMP-2 group, BF and BU were best at the three- and six-week follow-up, bone area was largest and mechanical test results were best. Although rhBMP-2 is superior for new bone regeneration, native reindeer bone extract is also effective in the six-week follow-up period.     

Mechanical Loading Stimulates BMP7, But Not BMP2, Production by Osteocytes

Bone mechanical adaptation is a cellular process that allows bones to adapt their mass and structure to mechanical loading. This process is governed by the osteocytes, which in response to mechanical loading produce signaling molecules that affect osteoblasts and osteoclasts. Bone morphogenic proteins (BMPs) are excellent candidates as signaling molecules, but it is unknown whether mechanically stimulated osteocytes affect bone adaptation through BMP production. Therefore, the aim of this study was to assess whether osteocytes produce BMPs in response to mechanical loading. In addition, since BMP7 has a vitamin D receptor (VDR) response element in the promoter region, we also investigated whether VDR is involved in the BMP7 response to mechanical loading. Human or VDR^−/− mouse primary bone cells were submitted in vitro to 1 h pulsating fluid flow (PFF) and postincubated without PFF (PI) for 1–24 h, and gene and protein expression of BMP2 and BMP7 were quantified. In human bone cells, PFF did not change BMP2 gene expression, but it upregulated BMP7 gene expression by 4.4- to 5.6-fold at 1–3 h PI and stimulated BMP7 protein expression by 2.4-fold at 6 h PI. PFF did not stimulate BMP7 gene expression in VDR^−/− mouse bone cells. These results show for the first time that mechanical loading upregulates BMP7, likely via the VDR, but not BMP2, gene and protein expression in osteocytes in vitro. Since BMP7 plays a major role in bone development and remodeling, these data might contribute to a better understanding of the mechanism leading to the mechanical adaptation of bone.     

Fracture resistance of gamma radiation sterilized cortical bone allografts.

Gamma radiation is widely used for sterilization of human cortical bone allografts. Previous studies have reported that cortical bone becomes brittle due to gamma radiation sterilization. This embrittlement raises concern about the performance of a radiation sterilized allograft in the presence of a stress concentration that might be surgically introduced or biologically induced. The purpose of this study was to investigate the effect of gamma radiation sterilization on the fracture resistance of human femoral cortical bone in the presence of a stress concentration. Fracture toughness tests of specimens sterilized at a dose of 27.5 kGy and control specimens were conducted transverse and longitudinal to the osteonal orientation of the bone tissue. The formation of damage was monitored with acoustic emission (AE) during testing and was histologically observed following testing. There was a significant decrease in fracture toughness due to irradiation in both crack growth directions. The work-to-fracture was also significantly reduced. It was observed that the ability of bone tissue to undergo damage in the form of microcracks and diffuse damage was significantly impaired due to radiation sterilization as evidenced by decreased AE activity and histological observations. The results of this study suggest that, for cortical bone irradiated at 27.5 kGy, it is easier to initiate and propagate a macrocrack from a stress concentration due to the inhibition of damage formation at and near the crack tip.     

Immunohistochemical localization of bone morphogenetic proteins (BMPs) 2, 4, 6, and 7 during induced heterotopic bone formation.

The distribution and staining intensity of bone morphogenetic proteins (BMPs) 2, 4, 6, and 7 were assessed by immunohistochemistry in ectopic bone induced in Nu/Nu mice by Saos-2 cell derived implants. Devitalized Saos-2 cells or their extracts can induce endochondral bone formation when implanted subcutaneously into Nu/Nu mice. BMP staining was mostly cytoplasmic. The most intense BMP staining was seen in hypertrophic and apoptotic chondrocytes, osteoprogenitor cells such as periosteal and perivascular cells, and osteoblasts. BMP staining in osteocytes and osteoclasts was variable, ranging from undetectable to intensely stained, and from minimal to moderately stained in megakaryocytes of the induced bone marrow. BMP-2, 4, 6, and 7 staining in Saos-2 implant-induced bone indicates the following: (1) Saos-2 cell products promote expression of BMPs by host osteoprogenitor cells, which in turn, leads to bone and marrow formation at ectopic sites; (2) strong BMP staining is seen in maturing chondrocytes, and thus may play a role in chondrocyte differentiation and/or apoptosis; (3) BMP expression in perivascular and periosteal cells indicates that osteoprogenitor cells also express BMP; (4) BMP release by osteoclasts may promote osteoblastic differentiation at sites of bone remodeling. These new data can be useful in understanding the role of BMPs in promoting clinical bone repair and in various pathologic conditions.     

Expression of CD105 and CD34 receptors controls BMP‐induced in vitro mineralization of mouse adipose‐derived stem cells but does not predict their in vivo bone‐forming potential

Adipose‐derived stem cells (ADSCs) can be excellent alternative to bone marrow derived stem cells for enhancing fracture repair since ADSCs can be isolated comparatively in large numbers from discarded lipoaspirates. However, osteogenic potential of ADSCs in vivo is very controversial. We hypothesized that adipose‐derived stem cells (ADSCs) that respond maximally to bone morphogenetic proteins (BMPs) in vitro would possess maximum bone‐forming potential. Four purified populations of mouse ADSCs: CD105⁺CD34⁺, CD105^?CD34^?, CD105⁺CD34^? and CD105^?CD34⁺ were obtained using fluorescence‐activated cell sorting (FACS) and their BMP‐responsiveness was determined in vitro. CD105⁺CD34^? population showed the strongest response to BMPs in terms of robust increase in mineralization. Expression of CD105 correlated with high BMP‐responsive phenotype and larger cell size while expression of CD34 correlated with low BMP‐responsive phenotype and smaller cell size. CD105⁺CD34^? population displayed higher gene expression of Alk1 or Alk6 receptors in comparison with other populations. However, CD105⁺CD34^? ADSCs failed to induce ectopic bone formation in vivo after they were transplanted into syngeneic mice, indicating that in vitro BMP‐responsiveness is not a good indicator to predict in vivo bone forming potential of ADSCs. Therefore greater precautions should be executed during selection of competent ADSCs for bone repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:625–632, 2015.

     

Enhanced Osteoclastogenesis Causes Osteopenia in Twisted Gastrulation‐Deficient Mice Through Increased BMP Signaling

The uncoupling of osteoblastic and osteoclastic activity is central to disorders such as osteoporosis, osteolytic malignancies, and periodontitis. Numerous studies have shown explicit functions for bone morphogenetic proteins (BMPs) in skeletogenesis. Their signaling activity has been shown in various contexts to be regulated by extracellular proteins, including Twisted gastrulation (TWSG1). However, experimental paradigms determining the effects of BMP regulators on bone remodeling are limited. In this study, we assessed the role of TWSG1 in postnatal bone homeostasis. Twsg1‐deficient (Twsg1^?/?) mice developed osteopenia that could not be explained by defective osteoblast function, because mineral apposition rate and differentiation markers were not significantly different compared with wildtype (WT) mice. Instead, we discovered a striking enhancement of osteoclastogenesis in Twsg1^?/? mice, leading to increased bone resorption with resultant osteopenia. Enhanced osteoclastogenesis in Twsg1^?/? mice was caused by increased cell fusion, differentiation, and function of osteoclasts. Furthermore, RANKL‐mediated osteoclastogenesis and phosphorylated Smad1/5/8 levels were enhanced when WT osteoclasts were treated with recombinant BMP2, suggesting direct regulation of osteoclast differentiation by BMPs. Increase in detectable levels of phosphorylated Smad 1/5/8 was noted in osteoclasts from Twsg1^?/? mice compared with WT mice. Furthermore, the enhanced osteoclastogenesis in Twsg1^?/? mice was reversed in vitro in a dose‐dependent manner with exposure to Noggin, a BMP antagonist, strongly suggesting that the enhanced osteoclastogenesis in Twsg1 mutants is attributable to increased BMP signaling. Thus, we present a novel and previously uncharacterized role for TWSG1 in inhibiting osteoclastogenesis through regulation of BMP activity.