Significant deterioration in nanomechanical quality occurs through incomplete extrafibrillar mineralization in rachitic bone: Evidence from in-situ synchrotron X-ray scattering and backscattered electron imaging

Bone diseases such as rickets and osteoporosis cause significant reduction in bone quantity and quality, which leads to mechanical abnormalities. However, the precise ultrastructural mechanism by which altered bone quality affects mechanical properties is not clearly understood. Here we demonstrate the functional link between altered bone quality (reduced mineralization) and abnormal fibrillar-level mechanics using a novel, real-time synchrotron X-ray nanomechanical imaging method to study a mouse model with rickets due to reduced extrafibrillar mineralization. A previously unreported N-ethyl-N-nitrosourea (ENU) mouse model for hypophosphatemic rickets (Hpr), as a result of missense Trp314Arg mutation of the phosphate regulating gene with homologies to endopeptidase on the X chromosome (Phex) and with features consistent with X-linked hypophosphatemic rickets (XLHR) in man, was investigated using in situ synchrotron small angle X-ray scattering to measure real-time changes in axial periodicity of the nanoscale mineralized fibrils in bone during tensile loading. These determine nanomechanical parameters including fibril elastic modulus and maximum fibril strain. Mineral content was estimated using backscattered electron imaging. A significant reduction of effective fibril modulus and enhancement of maximum fibril strain was found in Hpr mice. Effective fibril modulus and maximum fibril strain in the elastic region increased consistently with age in Hpr and wild-type mice. However, the mean mineral content was ∼21% lower in Hpr mice and was more heterogeneous in its distribution. Our results are consistent with a nanostructural mechanism in which incompletely mineralized fibrils show greater extensibility and lower stiffness, leading to macroscopic outcomes such as greater bone flexibility. Our study demonstrates the value of in situ X-ray nanomechanical imaging in linking the alterations in bone nanostructure to nanoscale mechanical deterioration in a metabolic bone disease. Copyright

Pannus invasion into cartilage and bone in rheumatoid arthritis

Rheumatoid arthritis is the most common of all types of arthritis and despite of intensive research etiology of the disease remains unclear. Distinctive features of rheumatic arthritis comprise continuous inflammation of synovium, in which synovial membrane expands on cartilage leading to pannus tissue formation. Pannus formation, appearance of proteolytic enzymes and osteoclast formation cause articular cartilage and bone destruction, which lead to erosions and permanent joint damage. Proteolytic pathways play major roles in the development of tissue lesions in rheumatoid arthritis. Degradation of extracellular matrix proteins is essential to pannus formation and invasion. Matrix metalloproteinases (MMP) form a large proteolytic enzyme family and in rheumatoid arthritis they contribute to pannus invasion by degrading extracellular matrix and to joint destruction by directly degrading the cartilage. MMP-1 and MMP-3 are shown to be increased during cell invasion and also involved in cartilage destruction. Increase of many cytokines has been observed in rheumatoid arthritis, especially TNF-α and IL-1β are studied in synovial tissue and are involved in rheumatoid inflammation and degradation of cartilage. Underlying bone resorption requires first demineralization of bone matrix with acid secreted by osteoclasts, which exposes the collagen-rich matrix for degradation. Cathepsin K is the best known enzyme involved in bone matrix degradation, however deficiency of this protein in pycnodysostosis patient did not prevent bone erosion and on the contrary pannus tissue invading to bone did not expressed much cathepsin K. These indicate that other proteinases are involved in bone degradation, perhaps also via their capability to replace the role of other enzymes especially in diseases like pycnodysostosis or during medication e.g. using cathepsin K inhibitors. Multinuclear osteoclasts are formed also in pannus tissue, which enable the invasion into underlying bone matrix. Pannus tissue express a receptor activator of nuclear factor kappa B ligand (RANKL), an essential factor for osteoclast differentiation and a disintegrin and a metalloproteinase 8 (ADAM8), an osteoclast-activating factors, involved in formation of osteoclast-like giant cells by promoting fusion of mononuclear precursor cells. The understanding of pannus invasion and degradation of extracellular matrix in rheumatic arthritis will open us new more specific methods to prevent this destructive joint disease.

Ligament-derived matrix stimulates a ligamentous phenotype in human adipose-derived stem cells.

Human adipose stem cells (hASCs) can differentiate into a variety of phenotypes. Native extracellular matrix (e.g., demineralized bone matrix or small intestinal submucosa) can influence the growth and differentiation of stem cells. The hypothesis of this study was that a novel ligament-derived matrix (LDM) would enhance expression of a ligamentous phenotype in hASCs compared to collagen gel alone. LDM prepared using phosphate-buffered saline or 0.1% peracetic acid was mixed with collagen gel (COL) and was evaluated for its ability to induce proliferation, differentiation, and extracellular matrix synthesis in hASCs over 28 days in culture at different seeding densities (0, 0.25 x 10(6), 1 x 10(6), or 2 x 10(6) hASC/mL). Biochemical and gene expression data were analyzed using analysis of variance. Fisher's least significant difference test was used to determine differences between treatments following analysis of variance. hASCs in either LDM or COL demonstrated changes in gene expression consistent with ligament development. hASCs cultured with LDM demonstrated more dsDNA content, sulfated-glycosaminoglycan accumulation, and type I and III collagen synthesis, and released more sulfated-glycosaminoglycan and collagen into the medium compared to hASCs in COL (p

Glucose-dependent insulinotropic polypeptide receptor deficiency leads to modifications of trabecular bone volume and quality in mice.

A role for the gastro-intestinal tract in controlling bone remodeling is suspected since serum levels of bone remodeling markers are affected rapidly after a meal. Glucose-dependent insulinotropic polypeptide (GIP) represents a suitable candidate in mediating this effect. The aim of the present study was to investigate the effect of total inhibition of GIP signaling on trabecular bone volume, microarchitecture and quality. We used GIP receptor (GIPR) knockout mice and investigated trabecular bone volume and microarchitecture by microCT and histomorphometry. GIPR-deficient animals at 16 weeks of age presented with a significant (20%) increase in trabecular bone mass accompanied by an increase (17%) in trabecular number. In addition, the number of osteoclasts and bone formation rate was significantly reduced and augmented, respectively in these animals when compared with wild-type littermates. These modifications of trabecular bone microarchitecture are linked to a remodeling in the expression pattern of adipokines in the GIPR-deficient mice. On the other hand, despite significant enhancement in bone volume, intrinsic mechanical properties of the bone matrix was reduced as well as the distribution of bone mineral density and the ratio of mature/immature collagen cross-links. Taken together, these results indicate an increase in trabecular bone volume in GIPR KO animals associated with a reduction in bone quality.

Osteocyte Density in the Peri-Implant Bone of Immediately Loaded and Submerged Dental Implants

Background: The role of osteocytes in bone structure and function remains partially unresolved. Their participation in mechanotransduction, i.e., the conversion of a physical stimulus into a cellular response, has been hypothesized. The present study was an evaluation of the osteocyte density in the peri-implant bone of immediately loaded and submerged dental implants. Methods: Fourteen male patients were included in the study; all of them were partially edentulous and needed a posterior mandibular restoration. Implants were inserted in these areas; half of the sample was loaded immediately (included in a fixed provisional prosthesis on the same day as implant surgery), whereas the other half was left to heal submerged. Fourteen implants (seven immediately loaded and seven unloaded) were retrieved with a trephine after a healing period of 8 weeks. The specimens were treated to obtain thin ground sections, and histomorphometry was used to evaluate the osteocyte index in the peri-implant bone. Results: A higher and statistically significant number of osteocytes was found in the peri-implant bone around immediately loaded implants (P=0.0081). A correlation between the percentage of bone-implant contact and osteocyte density was found for immediately loaded implants (P=0.0480) but not for submerged implants (P=0.2667). Conclusion: The higher number of osteocytes in the peri-implant bone around immediately loaded implants could be related to the functional adaptation required by the loading stimulus, which also explains the hypothesized involvement of the osteocytes in the maintenance of the bone matrix. J Periodontol 2009;80:499-504.

Maxillary Bone Defect Reconstruction Using Porous Polyethylene Implants

Purpose: The aim of this study was to evaluate the bone repair process in the maxillary sinus in monkeys treated with high-density porous polyethylene (Medpor)Methods: Four capuchin monkeys (Cebus apella) were submitted to bilateral horizontal osteotomies in the anterior wall of the maxillary sinus and divided into 2 groups: control group, left side with no implants, and porous polyethylene group, right side with Medpor. After a period of 145 days after implant placement, the maxillae were removed for histologic and histometric analyses.Results: Bone repair in osteotomized areas took place by connective tissue in 58.5% and 58.7% in the control group and the porous polyethylene group, respectively. In the contact surface with Medpor, bone repair occurred in 41.3%.Conclusions: Medpor was not reabsorbed within the period of this study and allowed bone repair surrounding it. The porous polyethylene constitutes a feasible alternative for bone defect reconstruction.

Effect of low-level laser therapy on bone repair: Histological study in rats

Background and Objectives: Bone remodeling is characterized as a cyclic and lengthy process. It is currently accepted that not only this dynamics is triggered by a biological process, but also biochemical, electrical, and mechanical stimuli are key factors for the maintenance of bone tissue. The hypothesis that low-level laser therapy (LLLT) may favor bone repair has been suggested. The purpose of this study was to evaluate the bone repair in defects created in rat lower jaws after stimulation with infrared LLLT directly on the injured tissue.Study Design/Materials and Methods: Bone defects were prepared on the mandibles of 30 Holtzman rats allocated in two groups (n = 15), which were divided in three evaluation period (15, 45, and 60 days), with five animals each. control group-no treatment of the defect; laser group-single laser irradiation with a GaAlAs semiconductor diode laser device (lambda = 780 nm; P = 35 mW t = 40 s; circle minus = 1.0 mm; D = 178 J/cm(2); E = 1.4 J) directly on the defect area. The rats were sacrificed at the preestablished periods and the mandibles were removed and processed for staining with hematoxylin and eosin, Masson's Trichrome and picrosirius techniques.Results: the histological results showed bone formation in both groups. However, the laser group exhibited an advanced tissue response compared to the control group, abbreviating the initial inflammatory reaction and promoting rapid new bone matrix formation at 15 and 45 days (P < 0. 05). on the other hand, there were no significant differences between the groups at 60 days.Conclusion: the use of infrared LLLT directly to the injured tissue showed a biostimulating effect on bone remodeling by stimulating the modulation of the initial inflammatory response and anticipating the resolution to normal conditions at the earlier periods. However, there were no differences between the groups at 60 days.

Use of bovine bone graft and bone membrane in defects surgically created in the cranial vault of rabbits. Histologic comparative analysis

Purpose: This study was proposed to analyze histologically the process of repairing bone defects created surgically in the cranial vaults of rabbits. Materials and Methods: Thirty adult male rabbits (Oryctolagus cunilicus) received, under general anesthesia, bilateral parietal osteotomies by means of a 6mm-diameter trephine. The bony defects were divided into 4 groups. In group 1 the defect did not receive any treatment; in group 2 the defect was filled with lyophilized bovine bone (Biograft); in group 3 it was filled with bovine bone and covered with a bone matrix membrane (Bioplate); in group 4 it was covered with a bone matrix membrane. Animals were sacrificed in 3 equal groups at 15, 30, and 60 days. The specimens were subjected to routine laboratory procedures to evaluate the degree of bone repair. Results: After 60 days, new bone formation in group 2 was not satisfactory when compared to that of group 3. Large amounts of new bone formation in maturation were seen in group 3. In the defects covered with a membrane the results were similar to those of group 1 (ie, the cavity was filled with fibrous connective tissue). The implanted bone and membranes were totally resorbed. Discussion and Conclusions: the use of a membrane served as a barrier against the migration of cells from the adjacent tissue and the bone graft/membrane preserved the cavity space, resulting in an enhanced osteogenic effect.

Assessment of bovine biomaterials containing bone morphogenetic proteins bound to absorbable hydroxyapatite in rabbit segmental bone defects

OBJETIVO: Avaliar a capacidade osteo-regenerativa de dois biomateriais utilizando um modelo de defeito segmentar efetuado nas diáfises do rádio de coelhos. MÉTODOS: O defeito direito foi preenchido com pool de proteínas morfogenéticas ósseas (pBMPs) e hidroxiapatita em pó ultrafina absorvível (HA) combinada com matriz óssea inorgânica desmineralizada e colágeno, derivados do osso bovino (Grupo A). O defeito esquerdo foi preenchido com matriz óssea desmineralizada bovina com pBMPs e hidroxiapatita em pó ultrafina absorvível (Grupo B). em ambos os defeitos utilizou-se membrana reabsorvível de cortical bovina desmineralizada para reter os biomateriais no defeito ósseo e guiar a regeneração tecidual. Os coelhos foram submetidos à eutanásia aos 30, 90 e 150 dias após a cirurgia. Foram efetuados exames radiográficos, tomográficos e histológicos em todos os espécimes. RESULTADOS: Aos 30 dias de pós-cirúrgico, o osso cortical desmineralizado foi totalmente reabsorvido em ambos os grupos. A HA tinha reabsorvido nos defeitos do Grupo A, mas persistiu nos do Grupo B. Uma reação de corpo estranho foi evidente com ambos os produtos, porém mais pronunciada no Grupo B. Aos 90 dias os defeitos do grupo B tinham mais formação óssea que os do Grupo A. Entretanto, aos 150 dias após a cirurgia, nenhum tratamento havia promovido o completo reparo do defeito. CONCLUSÃO: Os biomateriais testados contribuíram pouco ou quase nada para a reconstituição do defeito segmentar.

Combined TUNEL and TRAP methods suggest that apoptotic bone cells are inside vacuoles of alveolar bone osteoclasts in young rats

Although it is generally accepted that osteoclasts breakdown and resorb bone matrix, the possibility that they may also be able to engulf apoptotic osteoblasts/ lining cells and/or osteocytes remains controversial. Apoptosis of osteoblasts/ lining cells and/or osteocytes and interactions between these cells and osteoclasts are extremely rapid events that are difficult to observe in viva. A suitable in viva model for studying these events is the alveolar bone of young rats because it is continuously. Thus, sections of aldehyde fixed alveolar undergoing intense resorption/remodeling bone of young rats were stained by the combined terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and the tartrate-resistant acid phosphatase (TRAP) method for the simultaneous visualization of apoptotic cells and osteoclasts in the same section. The combined TUNEL and TRAP reactions, in the same section, greatly facilitated visualization of relationship between osteoclasts and apoptotic bone cells during alveolar bone remodeling. Our results showed that several TRAP-positive osteoclasts exhibited large vacuoles containing TUNEL positive apoptotic structures, probably derived from osteoblasts/lining cells and/or osteocytes. These results support the idea that alveolar bone osteoclasts are able to internalize dying apoptotic bone cells.

Effect of calcitonin on bone regeneration in male rats: a histomorphometric analysis

The aim of this study was to evaluate the effectiveness of calcitonin in promoting bone growth in surgical bone defects in rat mandibles. Fifty male rats were divided into two groups: bone defect (control) and bone defect with calcitonin (experimental). A circular bone defect 4 mm in diameter was made in the mandibular bone of the rats in the angle region, and covered with a polytetrafluoroethylene barrier. The experimental group received 2 IU/kg of synthetic salmon calcitonin intramuscularly three times a week, with treatment starting immediately after surgery. The animals were killed 3, 7, 14, 21 and 28 days after the surgical procedure. The bone defects were examined histologically and by histomorphometric analysis. The Student t-test was applied to the histomorphometric data, with the level of significance set at 5%. The animals of the experimental group showed a lower level of bone formation at almost all time points than the control group, but no difference between groups was observed 28 days after surgery. The volume of newly formed bone matrix was significantly greater in the control than the experimental group at 7, 14 and 21 days, as determined by both morphologic and histomorphometric analysis. Bone repair in the calcitonin-treated animals was delayed in comparison to controls, indicating the need for further studies on male rats.

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.

Homogenous implant in rat tibias of matrix preserved in 98% glycerin: histomorphologic study.

As a chemical medium for preservation of tissues, glycerin has shown good results because it maintains the cellular integrity despite the tissue dehydration it causes. Taking advantage of the osteoinducing properties of the osseous matrix and glycerin as a proper medium for tissue preservation, osseous matrix was implanted in rat tibias. Twenty-four rats were used, each receiving two surgical wounds. In one of the wounds an osseous matrix preserved in 98% glycerin was implanted and the other received a matrix without preservatives. Six animals were sacrificed on days 10, 20, 30 and 60 post-implant. After routine histological processing, the specimens were stained in hematoxylin-eosin and Masson's trichrome. The results showed that the matrixes preserved in glycerin presented faster resorption with replacement by newly formed tissue.

Histomorphometric analysis of homogenous demineralized dentin matrix as osteopromotive material in rabbit mandibles

Purpose: The aim of this work was to evaluate the effectiveness of homogenous demineralized dentin matrix (HDDM) slices in surgical bone defects created in the mandibles of rabbits and occluded with a polytetrafluoroethylene (PTFE) membrane in the promotion of bone growth. Materials and Methods: Surgical bone defects were created in 36 adult rabbits and divided into 4 groups: bone defect (control), bone defect with PTFE membrane, bone defect with HDDM, and bone defect with both HDDM and a PTFE membrane (HDDM + PTFE). The rabbits were sacrificed after 30, 60, and 90 days, and the bone defects were examined histologically and by histomorphometric analysis (analysis of variance and the Tukey test). Results: The volume of newly formed bone matrix was significantly greater in the HDDM and HDDM + PTFE groups than in the control and PTFE groups. The discrete inflammatory reaction found in the HDDM and HDDM + PTFE groups did not prevent the osteopromotive activity of the dentin matrix. Discussion: HDDM slices were biocompatible and were resorbed during the bone remodeling process. They stimulated the newly formed bone until 30 days after implantation. Conclusion: Bone repair was accelerated in the bone defects treated with HDDM in comparison to the control group.

Densitometric analysis of the autogenous demineralized dentin matrix on the dental socket wound healing process in humans

The aim of this study was to evaluate the effects of the autogenous demineralized dentin matrix (ADDM) on the third molar socket wound healing process in humans, using the guided bone regeneration technique and a polytetrafluoroethylene barrier (PTFE). Twenty-seven dental sockets were divided into three groups: dental socket (Control), dental socket with PTFE barrier (PTFE), and dental socket with ADDM slices associated to PTFE banier (ADDM + PTFE). The dental sockets were submitted to radiographic bone densitometry analysis and statistical analysis on the 15th, 30th, 60th and 90th days using analysis of variance (ANOVA) and Tukey's test (p ≤ 0.05). The radiographic analysis of the ADDM + PTFE group showed greater homogeneity of bone radiopacity than the Control group and the PTFE group, during all the observation times. The dentin matrix gradually disappeared from the dental socket during the course of the repair process, suggesting its resorption during the bone remodeling process. It was concluded that the radiographic bone density of the dental sockets treated with ADDM was similar to that of the surrounding normal bone on the 90th day. The ADDM was biocompatible with the bone tissue of the surgical wounds of human dental sockets. The radiographic analysis revealed that the repair process was discreetly faster in the ADDM + PTFE group than in the Control and PTFE groups, although the difference was not statistically significant. In addition, the radiographic image of the ADDM + PTFE group suggested that its bone architecture was better than that of the Control and PFTE groups.