Validation of Bone Conversion in Osteoconductive and Osteoinductive Bone Substitutes

Bone reconstruction can be performed with an autogeneic graft from various donor regions. Osteoconductive and osteoinductive bone substitutes originate from substances of diverse chemical and morphological types and can have a synthetic or a biological derivation. Alongside autogeneic bone transplants and allogenic and xenogeneic bone implants, alloplastic bone replacements of synthetic or semi-synthetic origin are being used for defect reconstruction. In an animal model in rabbits five bone substitutes and one autogeneic graft were surgically incorporated into identical bone defects (10times 10 mm in size) in six anatomically defined regions of the skull. With scintigraphic and histological methods, the metabolic dynamics of the bone is examined as it reacts to the transplantation of autogeneic bone or to implanted bone replacement material. The different autogeneic, xenogeneic and alloplastic bone replacement materials can be differentiated according to the functional quality of the new tissue and the dynamics of the bone conversion thus induced. In the comparison of mineralized, osteoconductive bone subsitutes (TCP, HA, calcium carbonate ceramics) with demineralized, osteoinductive implants (DBM new, DBM old) and autogeneic bone grafts, the bone inducing matrices show the largest quantity of new bone formation, making possible a volume-constant reconstruction. This revised version was published online in July 2006 with corrections to the Cover Date.     

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.     

Proteomic Analysis of Gingival Tissue and Alveolar Bone during Alveolar Bone Healing

Bone tissue regeneration is orchestrated by the surrounding supporting tissues and involves the build-up of osteogenic cells, which orchestrate remodeling/healing through the expression of numerous mediators and signaling molecules. Periodontal regeneration models have proven useful for studying the interaction and communication between alveolar bone and supporting soft tissue. We applied a quantitative proteomic approach to analyze and compare proteins with altered expression in gingival soft tissue and alveolar bone following tooth extraction. For target identification and validation, hard and soft tissue were extracted from mini-pigs at the indicated times after tooth extraction. From triplicate experiments, 56 proteins in soft tissue and 27 proteins in alveolar bone were found to be differentially expressed before and after tooth extraction. The expression of 21 of those proteins was altered in both soft tissue and bone. Comparison of the activated networks in soft tissue and alveolar bone highlighted their distinct responsibilities in bone and tissue healing. Moreover, we found that there is crosstalk between identified proteins in soft tissue and alveolar bone with respect to cellular assembly, organization, and communication. Among these proteins, we examined in detail the expression patterns and associated networks of ATP5B and fibronectin 1. ATP5B is involved in nucleic acid metabolism, small molecule biochemistry, and neurological disease, and fibronectin 1 is involved in cellular assembly, organization, and maintenance. Collectively, our findings indicate that bone regeneration is accompanied by a profound interaction among networks regulating cellular resources, and they provide novel insight into the molecular mechanisms involved in the healing of periodontal tissue after tooth extraction.Healthy dental gingival tissue and the alveolar bone that surrounds the teeth are essential for the proper function of teeth, as well as for a good appearance and good general health. Socket healing after tooth extraction is a useful experimental model for investigating the communication between gingival tissue and alveolar bone after tooth extraction. Preservation of the alveolar socket after tooth extraction requires the formation of a biological connection between the living and osseous tissue, which has to be created during the healing process. The success of such dental remodeling is dependent on the establishment of a soft tissue barrier that is able to shelter the underlying osseous structures and the osseo-integration of the soft tissue surrounding the alveolar bone. Understanding the processes governing soft and hard tissue healing and maintenance around the alveolar socket is paramount for oral health.Several studies have reported significant structural changes and bone reabsorption in fresh sockets following tooth extraction, with important dimensional changes in the surrounding alveolar bone (1–3). A reduction of alveolar bone may present problems after tooth extraction, especially in aged individuals in whom bone volume is important for both physiological and medical reasons. Although it has been shown that reduction defects in alveolar bone can be completely repaired using surgical techniques such as guided bone regeneration (4, 5), bone autograft, bone allograft, and xenograft (6, 7), these techniques are not broadly applicable (8). However, the introduction of biomimetic agents such as enamel matrix derivatives (9), platelet-rich plasma (10), platelet-derived growth factor (11, 12), and bone morphogenic proteins (BMPs)¹ (9) promises potentially better outcomes with bone regeneration treatments, although their efficacy remains controversial.The proteins present in bone are essential for all of the life processes ongoing in bone, and they are the most important final products of the homeostatic signaling pathways. Profiling those proteins is vital for a thorough understanding of bone biology. To date, proteome research on bone has been focused mainly on in vitro analysis of bone-forming cells (osteoblasts and osteoclasts) to determine which proteins are expressed under a given set of experimental conditions (13–16). Although important, such studies cannot identify the actual protein profile in oral alveolar bone. Recently, the extraction of proteins directly from skull bone for proteome analysis was reported (17, 18). The extracted proteins were first separated using two-dimensional gel electrophoresis, after which spots of interest were excised and the proteins were identified via mass spectrometry (MS). However, using two-dimensional gel electrophoresis to analyze extreme proteins (e.g. extremely basic or acidic, extremely small or large, extremely hydrophobic) is challenging. Shotgun proteomics, which is a method of high-throughput proteome analysis (19–21), avoids the intrinsic limitations of two-dimensional gel electrophoresis. Despite an interesting need for large-scale characterization of the bone proteome, one study has been reported to apply shotgun proteomics for proteome analysis of rat femur bone (22). However, they identified only 133 proteins, because they analyzed bone proteins using a one-step method without a demineralization stage. The other report showed only that bone proteins extracted from the skull bone of an adult beagle are carried using a demineralization step (23). There are no reports regarding the interaction between alveolar bone and soft tissue yet.The efficient extraction of bone proteins is a critical issue for proteome analysis (24). Because bone is largely mineralized, and therefore nearly solid, classical protein extraction methods used for soft tissues and cells may not be appropriate for bone. It is therefore necessary to develop methods to efficiently extract protein from bone. In earlier bone proteome analyses (17, 18, 22), the bones were first ground to powder, after which the proteins were extracted by means of incubating the powder in lysis buffer. However, mechanically breaking bones down into powder is laborious, especially for large animal bones. More important, large amounts of collagen and proteoglycans also are extracted, and this can impair the detection of low-abundance proteins and strongly affect isoelectric focusing (25). For the present study, we adopted an alternative method of demineralizing bone tissue and then investigated the efficiency of protein extraction from the demineralized bone tissue. This method was based on a recently reported sequential protein extraction protocol that was used to extract proteins from skull for comprehensive analysis of its proteome. Two-dimensional high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) was then applied to analyze the protein extracts, enabling the identification of 2479 proteins (23). We employed a similar method to extract and identify proteins in tooth alveolar bone.Given that a large number of proteins are likely involved in the healing of bone, as well as of soft tissues, another goal of the present study was to examine protein expression and putative signaling during bone healing after tooth extraction. Here, we used nano-UPLC-MS^E-based label-free quantitative proteomics to analyze alveolar bone and the adjacent soft tissue. The environment surrounding healing bone would be expected to affect the specific signaling networks involved in bone regeneration. We suggest that determining the protein networks in alveolar bone and gingival tissue will enable improvement of the soft tissue interface, aspects of the hard tissue, and dental appearance during and after therapy.     

微波高温灭活及自体髂骨异体骨粒复合骨水泥修复 治疗长骨骨巨细胞瘤骨缺损

目的:探讨微波高温灭活及自体髂骨、异体骨粒复合骨水泥修复骨巨细胞瘤病灶刮除后骨缺损的临床应用效果。方法:应用原位分离插入式微波天线高温灭活技术,自体髂骨、异体骨粒复合骨水泥修复21例长骨骨巨细胞瘤术后骨缺损,从手术技术、肿瘤复发情况、肢体关节功能等方面全面综合评价此方法临床应用效果。结果:21例患者均获得骨性愈合,无骨折及内固定断裂发生,2例复发,复发率9.8%;肢体关节功能优18例(85.7%)、良3例(14.3%)、中差0例。结论:微波高温能彻底杀灭肿瘤组织降低复发率,自体髂骨保证与近关节软骨下骨愈合,异体骨粒复合骨水泥能良好充填残余瘤腔、且具有良好的生物力学性能,以防发生关节软骨面塌陷。     

Bone development and repair

Although bone development during embryogenesis and bone repair after injury have a number of features which appear similar, they are distinctly different processes which involve separate controlling elements and cuing parameters. Repair of bone is influenced by bioactive factors which reside in bone itself; some of these factors are not present when embryonic mesenchymal cells first differentiate. For example, a bone protein which induces the conversion of mesenchymal cells into cartilage cells is not present in the embryo at the site where cartilage first differentiates into the anlagen for future bone. Ultimately, the sequence of cellular and molecular events which controls the success of bone repair is dependent on the prior embryologic and developmental packaging of bioactive factors into bone and the release of these factors at a bone break site. The success of the reparative events depends not only upon the release of these bone-derived factors but also on systemic factors and the identity and numbers of responding cells at the repair site.     

Evolutionary Patterns of Bone Histology and Bone Compactness in Xenarthran Mammal Long Bones

Bone microstructure reflects physiological characteristics and has been shown to contain phylogenetic and ecological signals. Although mammalian long bone histology is receiving increasing attention, systematic examination of the main clades has not yet been performed. Here we describe the long bone microstructure of Xenarthra based on thin sections representing twenty-two species. Additionally, patterns in bone compactness of humeri and femora are investigated. The primary bone tissue of xenarthran long bones is composed of a mixture of woven, parallel-fibered and lamellar bone. The vascular canals have a longitudinal, reticular or radial orientation and are mostly arranged in an irregular manner. Concentric rows of vascular canals and laminar organization of the tissue are only found in anteater bones. The long bones of adult specimens are marked by dense Haversian bone, a feature that has been noted for most groups of mammals. In the long bones of armadillos, secondary osteons have an oblique orientation within the three-dimensional bone tissue, thus resulting in their irregular shape when the bones are sectioned transversely. Secondary remodeling is generally more extensive in large taxa than in small taxa, and this could be caused by increased loading. Lines of arrested growth are assumed to be present in all specimens, but they are restricted to the outermost layer in bones of armadillos and are often masked by secondary remodeling in large taxa. Parameters of bone compactness show a pattern in the femur that separates Cingulata and Pilosa (Folivora and Vermilingua), with cingulates having a lower compactness than pilosans. In addition, cingulates show an allometric relationship between humeral and femoral bone compactness.     

非霍杰金氏淋巴瘤骨髓侵犯的骨髓涂片和骨髓病理分析

目的:探讨非霍杰金淋巴瘤(NHL)骨髓侵犯发生率与临床分期和病理分型的关系。方法:160例NHL患者均做骨髓涂片和骨髓活检,48例骨髓侵犯病例做免疫组织化学染色。结果:骨髓涂片和活检侵犯率分别为18.7%和30.0%,骨髓涂片噬血现象多见Ⅳ期。骨髓侵犯的NHL病理组织分型以B细胞为主,骨髓侵犯模式以弥散型为主。结论:骨髓活检可提高淋巴瘤骨髓侵犯的诊断率,噬血现象出现和临床分期有关。     

Dexamethasone Enhances Osteogenic Differentiation of Bone Marrow- and Muscle-Derived Stromal Cells and Augments Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

We evaluated whether dexamethasone augments the osteogenic capability of bone marrow-derived stromal cells (BMSCs) and muscle tissue-derived stromal cells (MuSCs), both of which are thought to contribute to ectopic bone formation induced by bone morphogenetic protein-2 (BMP-2), and determined the underlying mechanisms. Rat BMSCs and MuSCs were cultured in growth media with or without 10-7 M dexamethasone and then differentiated under osteogenic conditions with dexamethasone and BMP-2. The effects of dexamethasone on cell proliferation and osteogenic differentiation, and also on ectopic bone formation induced by BMP-2, were analyzed. Dexamethasone affected not only the proliferation rate but also the subpopulation composition of BMSCs and MuSCs, and subsequently augmented their osteogenic capacity during osteogenic differentiation. During osteogenic induction by BMP-2, dexamethasone also markedly affected cell proliferation in both BMSCs and MuSCs. In an in vivo ectopic bone formation model, bone formation in muscle-implanted scaffolds containing dexamethasone and BMP-2 was more than two fold higher than that in scaffolds containing BMP-2 alone. Our results suggest that dexamethasone potently enhances the osteogenic capability of BMP-2 and may thus decrease the quantity of BMP-2 required for clinical application, thereby reducing the complications caused by excessive doses of BMP-2.Highlights: 1. Dexamethasone induced selective proliferation of bone marrow- and muscle-derived cells with higher differentiation potential. 2. Dexamethasone enhanced the osteogenic capability of bone marrow- and muscle-derived cells by altering the subpopulation composition. 3. Dexamethasone augmented ectopic bone formation induced by bone morphogenetic protein-2.     

Remnant Woven Bone and Calcified Cartilage in Mouse Bone: Differences between Ages/Sex and Effects on Bone Strength

IntroductionMouse models are used frequently to study effects of bone diseases and genetic determinates of bone strength. Murine bones have an intracortical band of woven bone that is not present in human bones. This band is not obvious under brightfield imaging and not typically analyzed. Due to the band’s morphology and location it has been theorized to be remnant bone from early in life. Furthermore, lamellar and woven bone are well known to have differing mechanical strengths. The purpose of this study was to determine (i) if the band is from early life and (ii) if the woven bone or calcified cartilage contained within the band affect whole bone strength.

Woven Bone Origin Studies

In twelve to fourteen week old mice, doxycycline was used to label bone formed prior to 3 weeks old. Doxycycline labeling and woven bone patterns on contralateral femora matched well and encompassed an almost identical cross-sectional area. Also, we highlight for the first time in mice the presence of calcified cartilage exclusively within the band. However, calcified cartilage could not be identified on high resolution cone-beam microCT scans when examined visually or by thresholding methods.

Mechanical Strength Studies

Subsequently, three-point bending was used to analyze the effects of woven bone and calcified cartilage on whole bone mechanics in a cohort of male and female six and 13 week old Balb/C mice. Three-point bending outcomes were correlated with structural and compositional measures using multivariate linear regression. Woven bone composed a higher percent of young bones than older bones. However, calcified cartilage in older bones was twice that of younger bones, which was similar when normalized by area. Area and/or tissue mineral density accounted for >75% of variation for most strength outcomes. Percent calcified cartilage added significant predictive power to maximal force and bending stress. Calcified cartilage and woven bone could have more influence in genetic models where calcified cartilage percent is double our highest value.     

Zinc Supplementation Increased Bone Mineral Density,Improves Bone Histomorphology,and Prevents Bone Loss in Diabetic Rat

Biological Trace Element Research - Diabetic osteoporosis (DOP) is a complication of diabetes, with the characteristics of bone mineral density (BMD) reduction and bone structure destruction. Zinc...