Whole-genome sequencing identifies EN1 as a determinant of bone density and fracture

The extent to which low-frequency (minor allele frequency (MAF) between 1-5%) and rare (MAF Bone mineral density (BMD) is highly heritable, a major predictor of osteoporotic fractures, and has been previously associated with common genetic variants, as well as rare, population-specific, coding variants. Here we identify novel non-coding genetic variants with large effects on BMD (ntotal = 53,236) and fracture (ntotal = 508,253) in individuals of European ancestry from the general population. Associations for BMD were derived from whole-genome sequencing (n = 2,882 from UK10K (ref. 10); a population-based genome sequencing consortium), whole-exome sequencing (n = 3,549), deep imputation of genotyped samples using a combined UK10K/1000 Genomes reference panel (n = 26,534), and de novo replication genotyping (n = 20,271). We identified a low-frequency non-coding variant near a novel locus, EN1, with an effect size fourfold larger than the mean of previously reported common variants for lumbar spine BMD (rs11692564(T), MAF = 1.6%, replication effect size = +0.20 s.d., Pmeta = 2 x 10(-14)), which was also associated with a decreased risk of fracture (odds ratio = 0.85; P = 2 x 10(-11); ncases = 98,742 and ncontrols = 409,511). Using an En1(cre/flox) mouse model, we observed that conditional loss of En1 results in low bone mass, probably as a consequence of high bone turnover. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817(T), MAF = 1.2%, replication effect size = +0.41 s.d., Pmeta = 1 x 10(-11)). In general, there was an excess of association signals arising from deleterious coding and conserved non-coding variants. These findings provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of complex traits and disease in the general population.

Time for treating bone fracture using rhBMP-2: a randomised placebo controlled mouse fracture trial.

Although the mechanisms of osteoinduction by bone morphogenic proteins (BMPs) are increasingly understood, the most appropriate time to administer BMPs exogenously is yet to be clarified.The purpose of this study was to investigate when BMP may be administered to a fracture arena to maximise the enhancement of healing.Forty mice with externally fixed left femoral fractures were randomised into four groups: Group I, the control group was given a placebo of 30 ll saline at day 0; Groups II, III and IV were given 30 ll saline plus 2.5 lg rhBMP-2, at post-operative days 0, 4 or 8, respectively.Sequential radiographs were taken at days 0, 8, 16.On day 22 the mice were sacrificed and both femora were harvested for biomechanical assessment in 3-point bending and histological evaluation.Radiographic analysis indicated that healing of fractures in Groups II and III was significantly greater (p <0.05) than those in Groups I and IV, at both 16 and 22 days post-fracture. The highest median bone mineral content at the fracture site was evidenced in Group III and II.Furthermore, Group III also had the highest relative ultimate load values, followed by Groups II, IV and I.Greater percentage peak loads were observed between Group I and both Groups II and III (p <0.05). Histological examination confirmed that at 22 days post-fracture, only fractures in Groups II and III had united with woven bone, and Groups I and IV still had considerable amounts of fibrous tissue and cartilage at the fracture gap.Data presented herein indicates that there is a time after fracture when rhBMP administration is most effective, and this may be at the time of surgery as well as in the early fracture healing phases.

Flexible Multibody Simulation Approach in the Dynamic Analysis of Bone Strains Activity

The objective of this study is to show that bone strains due to dynamic mechanical loading during physical activity can be analysed using the flexible multibody simulation approach. Strains within the bone tissue play a major role in bone (re)modeling. Based on previous studies, it has been shown that dynamic loading seems to be more important for bone (re)modeling than static loading. The finite element method has been used previously to assess bone strains. However, the finite element method may be limited to static analysis of bone strains due to the expensive computation required for dynamic analysis, especially for a biomechanical system consisting of several bodies. Further, in vivo implementation of strain gauges on the surfaces of bone has been used previously in order to quantify the mechanical loading environment of the skeleton. However, in vivo strain measurement requires invasive methodology, which is challenging and limited to certain regions of superficial bones only, such as the anterior surface of the tibia. In this study, an alternative numerical approach to analyzing in vivo strains, based on the flexible multibody simulation approach, is proposed. In order to investigate the reliability of the proposed approach, three 3-dimensional musculoskeletal models where the right tibia is assumed to be flexible, are used as demonstration examples. The models are employed in a forward dynamics simulation in order to predict the tibial strains during walking on a level exercise. The flexible tibial model is developed using the actual geometry of the subject’s tibia, which is obtained from 3 dimensional reconstruction of Magnetic Resonance Images. Inverse dynamics simulation based on motion capture data obtained from walking at a constant velocity is used to calculate the desired contraction trajectory for each muscle. In the forward dynamics simulation, a proportional derivative servo controller is used to calculate each muscle force required to reproduce the motion, based on the desired muscle contraction trajectory obtained from the inverse dynamics simulation. Experimental measurements are used to verify the models and check the accuracy of the models in replicating the realistic mechanical loading environment measured from the walking test. The predicted strain results by the models show consistency with literature-based in vivo strain measurements. In conclusion, the non-invasive flexible multibody simulation approach may be used as a surrogate for experimental bone strain measurement, and thus be of use in detailed strain estimation of bones in different applications. Consequently, the information obtained from the present approach might be useful in clinical applications, including optimizing implant design and devising exercises to prevent bone fragility, accelerate fracture healing and reduce osteoporotic bone loss.

A light and scanning electron microscopy study of bone healing following inferior alveolar nerve lateralization: An experimental study in rabbits

Purpose: The purpose of this study was to evaluate the bone healing kinetics around commercially pure titanium implants following inferior alveolar nerve (IAN) lateralization in a rabbit model. Materials and Methods: Inferior alveolar nerve lateralization was performed in 16 adult female rabbits (Oryctolagus cuniculus). During the nerve lateralization procedure, 1 implant was placed through the mandibular canal, and the IAN was replaced in direct contact with the implant. During the 8-week healing period, various bone labels were administered for fluorescent microscopy analysis. The animals were euthanized by anesthesia overdose, and the mandibular blocks were exposed by sharp dissection. Nondecalcified samples were prepared for optical light and scanning electron microscopy (SEM) evaluation. Results: SEM evaluation showed bone modeling/remodeling between the IAN and implant surface. Fluorochrome area fraction labeling at different times during the healing period showed that bone apposition mainly occurred during the first 2 weeks after implantation. Conclusions: The results obtained showed that bone healing/deposition occurred between the alveolar nerves in contact with a commercially pure titanium implant. No interaction between the nerve and the implant was detected after the 8-week healing period. Appositional bone healing occurred around the nerve bundle structure, restoring the mandibular canal integrity and morphology.

Influence of ovariectomy on healing of autogenous bone block grafts in the mandible: A histomorphometric study in an aged rat model

Purpose: The aim of this study was to evaluate quantitatively and qualitatively the influence of estrogen deficiency on autogenous bone block grafts in aged variectomized rats. Materials and Methods: Fifty 12-month-old female Wistar rats were used in the study. They were divided into 2 groups, an ovariectomized group and a sham-operated group. After 30 days the animals received autogenous block bone grafts on the angle of the mandible, harvested from the calvaria. The animals were euthanized at 7, 14, or 28 days postoperatively. Results: Histologic analysis showed that at 7 days postsurgery, the interface between graft and recipient site in the sham-operated group appeared filled by a granulation tissue with angiogenic activity, whereas the ovariectomized group still exhibited a blood clot and a granulation tissue in organization. on the 14th postoperative day, the interface in the shamoperated group was partially filled by newly formed bone establishing a union between the graft and the recipient site. The interface in the ovariectomized group was typically filled by granulation tissue with discrete osteogenic activity in most specimens. on the 28th postoperative day, the graft in the sham-operated group appeared histologically integrated to the mandible. However, the interface in the ovariectomized group appeared partially filled by newly formed bone, with areas of interposed connective tissue. The statistical analysis revealed that bone neoformation was significantly greater in the sham-operated group (57.41% at 14 days and 68.35 at 28 days) in comparison with the ovariectomized group (40.82% at 14 days and 53.09 at 28 days) at the 5% level. Conclusion: The estrogen depletion caused by the ovariectomy hindered the healing process of autogenous block bone grafts placed in the mandibles of aged rats.

Effect of platelet-rich plasma on bone healing of autogenous bone grafts in critical-size defects

AimThis study histologically analysed the effect of autogenous platelet-rich plasma (PRP), prepared according to a new semiautomatic system, on healing of autogenous bone (AB) grafts placed in surgically created critical-size defects (CSD) in rabbit calvaria.Material and MethodsSixty rabbits were divided into three groups: C, AB and AB/PRP. A CSD was created in the calvarium of each animal. In Group C (control), the defect was filled by blood clot only. In Group AB (autogenous bone graft), the defect was filled with particulate autogenous bone. In Group AB/PRP (autogenous bone graft with platelet-rich plasma), it was filled with particulate autogenous bone combined with PRP. All groups were divided into subgroups (n=10) and euthanized at 4 or 12 weeks post-operatively. Histometric and histologic analyses were performed. Data were statistically analysed (anova, t-test, p < 0.05).ResultsGroup C presented significantly less bone formation compared with Group AB and AB/PRP in both periods of analysis (p < 0.001). At 4 weeks, Group AB/PRP showed a statistically greater amount of bone formation than Group AB (64.44 +/- 15.0% versus 46.88 +/- 14.15%; p=0.0181). At 12 weeks, no statistically significant differences were observed between Groups AB and AB/PRP (75.0 +/- 8.11% versus 77.90 +/- 8.13%; p > 0.05). It is notable that the amount of new bone formation in Group AB/PRP at 4 weeks was similar to that of Group AB at 12 weeks (p > 0.05).ConclusionWithin its limitation, the present study has indicated that (i) AB and AB/PRP significantly improved bone formation and (ii) a beneficial effect of PRP was limited to an initial healing period of 4 weeks.

Influence of Bioactive Glass and/or Acellular Dermal Matrix on Bone Healing of Surgically Created Defects in Rat Tibiae: A Histological and Histometric Study

The purpose of this study was to histologically analyze the influence of bioactive glass and/or acellular dermal matrix on bone healing in surgically created defects in the tibiae of 64 rats (Rattus norvegicus, albinus, Wistar). Materials and Methods: A 4-mm X 3-mm unicortical defect was created on the anterolateral surface of the tibia. Animals were divided into 4 groups: C, control; BG, the defect was filled with bioactive glass; ADM, the defect was covered with acellular dermal matrix; and BG/ADM, the defect was filled with bioactive glass and covered with acellular dermal matrix. Animals were sacrificed at 10 or 30 days postoperatively, and the specimens were removed for histologic processing. The formation of new bone in the cortical area of the defect was evaluated histomorphometrically. Results: At 10 and 30 days postoperatively, groups C (39.65% +/- 5.63% / 63.34% +/- 5.22%) and ADM (38.12% +/- 5.53 / 58.96% +/- 7.05%) presented a larger amount of bone formation compared to the other groups (P<.05). In the same periods, groups BG (13.10% +/- 6.29% / 29.5% +/- 5.56%) and BG/ADM (20.72% +/- 8.31% / 24.19% +/- 6.69%) exhibited statistically similar new bone formation. However, unlike the other groups, group BG/ADM did not present a significant increase in bone formation between the 2 time points. Conclusion: Based on these results, it can be concluded that all of the materials used in this study delayed bone healing in non-critical-size defects. INT J ORAL MAXILLOFAC IMPLANTS 2008;23:811-817

Immunolocalization of bone morphogenetic protein 2 during the early healing events after guided bone regeneration

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Radiographic and histomorphometric analysis of bone healing using autogenous graft associated with platelet-rich plasma obtained by 2 different methods

The aim of this study was to conduct radiographic and histomorphometric analysis of bone healing in the calvaria of rabbits, using an autogenous graft associated with PRP obtained by 2 different methods. Thirty rabbits were divided into control and experimental groups. Lesions were produced in the calvaria and filled with autogenous graft ( control) or autogenous graft and PRP obtained by the Anitua or modified Sonnleitner methods. The animals were humanely killed 15 days after surgery and the calvarias were radiographed. The radiographs were digitized to assess the radiographic density. By histologic images of the lesion, the bone matrix was quantified. There were no significant differences in the radiographic density and the bone matrix area between the groups. The association of PRP with autogenous bone did not improve the healing process, irrespective of the method used early during healing.

Mechanical Property Assessment of Bone Healing around a Titanium-Zirconium Alloy Dental Implant

Background: It has been reported that titanium-zirconium alloy with 13-17% zirconium (TiZr1317) implants show higher biomechanical stability and bone area percentage relative to commercially pure titanium (cpTi) grade 4 fixtures. Purpose: This study aimed to determine whether the higher stability for TiZr1317 implants is associated with higher mechanical properties of remodeling bone in the areas around the implants. Materials and Methods: This study utilized 36 implants (n=18: TiZr1317, n=18: cpTi), which were placed in the healed ridges of the mandibular premolar and first molar of 12 mini pigs (n=3 implants/animal). After 4 weeks in vivo, the samples were retrieved, and resin-embedded histologic sections of approximately 100μm in thickness were prepared. In order to determine the nanomechanical properties, nanoindentation (n=30 tests/specimen) was performed on the bone tissue of the sections under wet conditions with maximum load of 300μN (loading rate: 60μN/s). Results: The mean (±standard deviation) elastic modulus (E) and hardness (H) for the TiZr1317 group were 2.73±0.50GPa and 0.116±0.017GPa, respectively. For the cpTi group, values were 2.68±0.51GPa and 0.110±0.017GPa for E and H, respectively. Although slightly higher mechanical properties values were observed for the TiZr1317 implants relative to the cpTi for both elastic modulus and hardness, these differences were not significant (E=p>0.75; H=p>0.59). Conclusions: The titanium-zirconium alloy used in this study presented similar degrees of nanomechanical properties to that of the cpTi implants. © 2013 Wiley Periodicals, Inc.

Healing of fresh frozen bone allograft with or without platelet-rich plasma: A histologic and histometric study in rats

Purpose: This study histomorphometrically analyzed the effect of autogenous platelet-rich plasma (PRP) on healing of fresh frozen bone allograft (FFBA) in bony defects in rat calvaria. Materials and Methods: A 5mm-diameter defect was created in the calvarium of 30 rats. Animals were divided into three groups: C (defect was filled by blood clot only), FFBA (defect was filled with 0.01mL of FFBA), and FFBA/PRP (defect was filled with 0.01mL of FFBA combined with 100μL of PRP). All animals were euthanized at 30 days postoperatively. Histomorphometry and histology analyses were performed. Data were statistically analyzed (analysis of variance, Tukey, p<.05). Results: FFBA had a statistically smaller new bone area than groups FFBA/PRP and C. No statistically significant differences were observed between groups FFBA and FFBA/PRP with regard to remaining bone graft particle area. Conclusion: It can be concluded that (1) PRP improved the incorporation of FFBA, increasing the amount of new bone formed; (2) PRP has not influenced the resorption of nonviable particles of the FFBA; and (3) presence of remaining FFBA particles might have accounted for the smaller amount of new bone observed in group FFBA when compared with control group. © 2011 Wiley Periodicals, Inc.