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Objective To better understand the risks of bisphosphonates in order to develop guidance for appropriate clinical usage, to compared femoral fracture healing at different time points and to explore the effects of Residronate on fracture healing. Methods Osteoporosis model was achieved by ovariectomy surgery, followed by surgical incision of left femoral shaft 4 weeks after ovariectomy surgery. Three days after fracture surgery, risedronateor saline was fed by intragastric administration. X ray examination was used to check the callus formation, Bone Mineral Density (BMD), Bone Mineral Content (BMC), biomechanical, imaging and micromorphological of bone tissue as well as the trabecular bone parameters were all examined. The femoral pathology tissue of each rat was used to analyze trabecular bone parameters, including trabecular bone volume/tissue volume (Tb. BV/TV), bone surface to tissue volume ratio (BS/TV), trabecular bone mineral density (Tb. BMD), trabecular bone number (Tb. N), trabecular bone thickness (Tb. Th) and small bone Trabecular bone space (Tb. Sp). Results Via X‐ray and pathologically, risedronate treatment promoted the callus forming at the fracture site during the following 6 weeks after osteoporotic fracture by X‐ray (P, Using a rat osteoporotic model and controls we compared femoral fracture healing at different time points to explore the effects of Residronate on fracture healing at different time points measured: (i) via x‐ray and pathologically; (ii) through evaluation of bone mineral density and bone mineral content; and (iii) through load testing. Our results demonstrate that Risedronate could promote fracture healing at the first 3 weeks but delayed healing at the following 3 weeks. During the first 3 weeks, risedronate could obviously increase the BMD, decrease the BMC and delay the decrease of BMC of the fracture site. During the healing process of osteoporotic fracture rats, risedronate can improve the structural strength and mechanical index of newborn callus. It strongly recommends that longer than 7 weeks usage of third generation bisphosphonate of risedronate does not contribute to osteoporotic fracture.

We report here on the histological and structural characteristics of the gas bladder, the vertebral morphology, and the bladder-vertebra relationships of the butterfly fish, Pantodon buchholzi. The bladder opens at the boundary between the pharynx and the esophagus by a middle slit. A pneumatic duct is absent. The bladder shows a dorsolateral wall that adapts to the anfractuosities of the coelomic cavity and a ventral wall in contact with the abdominal organs. The vertebral bodies are formed by an hourglass shaped autocentrum, and by an arcocentrum reduced to several longitudinal ridges. The transverse processes adopt the structure of a cage whose walls are formed by bone trabeculae of variable size and distribution pattern. The dorsolateral wall of the bladder is a membrane that covers the kidney, adapts to the irregular shape of the vertebrae, and invades the transverse processes at several points before extending laterally. However, invasion of the vertebral bodies, the presence of a labyrinth, or the formation of respiratory parenchyma were not observed. The luminal surface of this wall is a thin respiratory barrier containing a single epithelial cell type. In addition, the wall contains numerous eosinophils that may be implicated in immune defense. The bladder ventral wall is a membrane rich in collagen, vessels, smooth muscle, and nerves that lacks a respiratory barrier. Its luminal surface contains ciliated and nonciliated cells. The two cell types appear implicated in surfactant production.

Objective To evaluate the effect of sodium alendronate on orthodontic tooth movement in rats using microtomographic analysis. Settings and sample population Thirty male Wistar rats (Rattus norvegicus) were divided into three groups of 10 rats and administered saline (control), 1 mg/kg sodium alendronate or 6 mg/kg sodium alendronate, respectively. Materials and methods The drug was administered once per week for 5 weeks by gavage. Orthodontic movement was induced during the last 2 weeks of medication administration by inserting a closed nickel-titanium spring between the left upper first molar and central incisors. The opposite side served as the control. Tooth movement and bone trabeculation in the furcation region were evaluated by microtomographic analysis in the first (moved) and third (static) molars. The data were subjected to one-way or two-way ANOVA and Bonferroni test (P Results The microtomographic images of the group that received 6 mg/kg sodium alendronate demonstrated significantly less tooth movement (P = .048), less space between the trabeculae (P = .031) and greater number of bone trabeculae (P = .033) compared to the other groups. There were no statistically significant differences in bone volume and mean trabecular thickness between the three groups. The static teeth did not show the same alterations (P > .05). Conclusion Sodium alendronate treatment reduced tooth movement in rats.

Background/aim The nervous system controls bone mass via both the central (CNS) and the peripheral (PNS) nervous systems. Intriguingly, neuropeptide Y (NPY) signaling occurs in both. Less is known on how the PNS stimulated NPY signaling controls bone metabolism. The objective of this study was to evaluate whether NPY or NPY1 receptor antagonist changes local bone mineral density (BMD) when injected into a Wistar rat tibia. Materials and methods Tibial intramedullary area of 24 wild type male Wistar rats (average weight = 350 ± 50 g, average age = 4 ± 0.5 months) were injected with NPY (1 × 10-5 M and 1 × 10-6 M) and NPY1 receptor antagonist (1 × 10-4 M) dissolved in hyaluronic acid (HA) separately. Tibiae were collected after one and two weeks. BMD was measured with dual-energy X-ray absorptiometry (DXA) and micro quantitative computer tomography (QCT). Histological changes were analyzed with light microscopy, Goldner's Masson trichrome (MT), and hematoxylin-eosin staining. Results According to DXA, the mean BMD of NPY dose 1 (1 × 10-5 M) was significantly lower than that of the control (HA applied) group and not significantly but still lower than that of the NPY dose 2 and NPY1 antagonist applied groups. QCT results indicated the same pattern statistically insignificantly in the trabecular area but not in the cortex of the bones. Histologically, only NPY1 antagonist applied tibiae revealed young spongiosis bone trabeculae formed in the borderline of the cortical bones. HA was remarkably biocompatible and late degrading in the tissues. Conclusion Local administration of NPY and NPY1 antagonists may hold regulating potential of BMD and bone formation. NPY1 antagonist caused new bone formation in trabecular bone when applied locally. NPY dissolved in HA however can be used to suppress bone formation.

Aim The purpose of this research was to survey the effect of low-level laser irradiation time on socket healing in rats. Materials and methods This randomized nonblinded animal study was done on 24 male rats that were divided into four groups. First maxillary molars of rats were extracted through general anesthesia, and laser was used in all four groups: first group with zero radiation time, second group with 3 minutes, third group with 5 minutes, and fourth group with 10 minutes of radiation by the diode laser (power: 100 mW, wavelength: 980 nm). Half of the rats (three rats) were sacrificed on the 3rd day and another half of rats were sacrificed on the 7th day. Then, the presence of angiogenesis, bone trabeculae, fibroblasts, neutrophil cells, macrophage cells, and lymphocyte cells was assessed. Data were analyzed by SPSS (version 21) using parametric tests. Results Among 24 rats, on the 3rd day, the percentage of macrophage and bone trabecula increased significantly in the 5 minute group (p = 0.041 and p 0.05). Conclusion Low-level laser radiation can accelerate the process of tooth socket healing, which was particularly noticeable in the 5 minute radiation over 3 days. Clinical significance Using a low-level laser can be helpful in accelerating the healing of the tooth socket and reduce the complications after tooth extraction.

Bone tissue engineering strategy involves the 3D scaffolds and appropriate cell types promoting the replacement of the damaged area. In this work, we aimed to develop a fast and reliable clinically relevant protocol for engineering viable bone grafts, using cryopreserved adipose tissue-derived mesenchymal stromal cells (MSCs) and composite 3D collagen-nano-hydroxyapatite (nanoHA) scaffolds. Xeno- and DMSO-free cryopreserved MSCs were perfusion-seeded into the biomimetic collagen/nanoHA scaffolds manufactured by cryotropic gelation and their osteoregenerative potential was assessed in vitro and in vivo. Cryopreserved MSCs retained the ability to homogenously repopulate the whole volume of the scaffolds during 7 days of post-thaw culture. Moreover, the scaffold provided a suitable microenvironment for induced osteogenic differentiation of cells, confirmed by alkaline phosphatase activity and mineralization. Implantation of collagen-nanoHA cryogels with cryopreserved MSCs accelerated woven bone tissue formation, maturation of bone trabeculae, and vascularization of femur defects in immunosuppressed rats compared to cell-free collagen-nanoHA scaffolds. The established combination of xeno-free cell culture and cryopreservation techniques together with an appropriate scaffold design and cell repopulation approach accelerated the generation of viable bone grafts.

A new alloplastic high-permeable material based on tricalcium phosphate with Kelvin architectonics created by stereolithographic 3D-printing was studied in vivo. A monocortical defect of the femur was modeled in rats and the material was implanted into the defect area. In 24 weeks, the animals were euthanized and histological examination of the defect area was performed. One femur fracture with fixator migration was recorded after implantation of the studied material and the reference chronOS synthetic material. The studied material demonstrated better osteoconductive properties then traditional osteoplastic material, which was seen from greater number of bone trabeculae and their area in the defect area.

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder caused by heterozygous mutations in RUNX2. Affected individuals exhibit delayed maturation or hypoplasia in various bones, mainly including those formed by intramembranous ossification. Although several reports described deformation of the sphenoid bone in CCD patients, details of the associated changes have not been well documented. Most parts of the sphenoid bone are formed by endochondral ossification; however, the medial pterygoid process is formed by intramembranous ossification associated with secondary cartilage. We first investigated histological changes in the medial pterygoid process during different developmental stages in Runx2+/+ and Runx2+/- mice, finding that mesenchymal cell condensation of the anlage of this structure was delayed in Runx2+/- mice as compared with that in Runx2+/+ mice. Additionally, in Runx2+/+ mice, Osterix-positive osteoblastic cells appeared at the upper region of the anlage of the medial pterygoid process, and bone trabeculae appeared to associate with subsequent secondary cartilage formation. By contrast, few Osterix-positive osteoblastic cells appeared at the upper region of the anlage of the medial pterygoid process, and no bone trabeculae appeared thereafter in Runx2+/- mice. At more advanced embryonic stages, endochondral ossification occurred at the lower part of the medial pterygoid process in both Runx2+/+ and Runx2+/- mice. After birth, well-developed bone trabeculae occupied two-thirds of the cranial side of the medial pterygoid process, and cartilage appeared beneath these bones in Runx2+/+ mice, whereas thin trabecular bone appeared at the center of the cartilage of the medial pterygoid process in Runx2+/- mice. In adult mice, the body and medial pterygoid processes of the sphenoid bone comprised mature bones in both Runx2+/+ and Runx2+/- mice, although the axial length of the medial pterygoid processes was apparently lower in Runx2+/-mice as compared with that in Runx2+/+mice based on histological and micro-computed tomography (CT) examinations. Moreover, medical-CT examination revealed that in CCD patients, the medial pterygoid process of sphenoid bone was significantly shorter relative to that in healthy young adults. These results demonstrated that the medial pterygoid process of the sphenoid bone specifically exhibited hypoplasia in CCD.

Objectives: To investigate the effects of inferior alveolar nerve on new bone formation in rabbit mandibular distraction osteogenesis. Methods: 20 New Zealand White rabbits underwent bilateral distraction osteogenesis with a rate of 1 mm/day. The inferior alveolar nerve of one side was resected under the surgical microscope, with the inferior alveolar vascular intact. The contralateral side received sham operation. The rabbits were sacrificed at consolidation time of 28 days. The regenerate callus underwent radiograph examination, dual-energy X-ray absorptiometry, haematoxylin and eosin staining and histomorphometric analysis. A paired t-test was performed using SPSS 16.0 software package. Results: The BMD of the new bone in the distraction gap on the denervation side of mandibular was significantly lower (P

Purpose: This study assessed the regeneration potential of mesenchymal stem cells (MSC) from adipose tissue associated with platelet-rich plasma (PRP) in bone regeneration. Methods: Thirty Wistar rats (Rattus norvegicus albinos) were divided into five groups (according to the grafting material and time to euthanasia): (1) autograft - 14 days (control), (2) autograft - 28 days (control), (3) MSC + PRP - 14 days, (4) MSC + PRP + papaverine - 14 days and (5) MSC + PRP + papaverine - 28 days. After euthanasia, the graft was removed and histological slides were prepared. They were assessed by a blinded pathologist using a previously published histological scale as parameter. Results: There was some degree of neoformed bone trabeculae (NBT) in 93.3% of the samples, as well as osteoblastic activity (OA). The autograft groups (14 and 28 days) had higher levels in the formation of bone trabeculae. Nonparametric data were analyzed using the Wilcoxon-Mann-Whitney test and proved not to be statistically significant at p < 0.05. Conclusions: Experimental parietal bone reconstruction, combining MSC, PRP and papaverine presented regeneration in all groups with no significant difference among them.

Large bone defects pose an unsolved challenge for orthopedic surgeons. Our group has previously reported the construction of a barrier membrane made of ammoniomethacrylate copolymer USP (AMCA), which supports the adhesion, proliferation, and osteoblastic differentiation of human mesenchymal stem cells (hMSCs). In this study, we report the use of AMCA membranes to seclude critical segmental defect (~1.0 cm) created in the middle third of rabbit radius and test the efficiency of bone regeneration. Bone regeneration was assessed by radiography, biweekly for 8 weeks. The results were verified by histology and micro-CT at the end of the follow-up. The AMCA membranes were found superior to no treatment in terms of new bone formation in the defect, bone volume, callus surface area normalized to total volume, and the number of bone trabeculae, after eight weeks. Additional factors were then assessed, and these included the addition of simvastatin to the membrane, coating the membrane with human MSC, and a combination of those. The addition of simvastatin to the membranes demonstrated a stronger effect at a similar radiological follow-up. We conclude that AMCA barrier membranes per se and simvastatin delivered in a controlled manner improve bone regeneration outcome.

The aim of the study was to develop a technology for repairing an osteomyelitic bone defect using autologous adipose tissue mesenchymal stromal cells (MSCs) bound to a collagen matrix and to test the efficacy of this technique. Materials and Methods. The study was carried out with 17 rabbits. A bone defect was created using a milling cutter applied to the proximal third of the leg. The wound (8.0×4.0 mm and a depth of 4.0 mm) involved the periosteum, cortical layer, and cancellous substance. Staphylococcus aureus strain was used as an infectious agent. After the development of chronic osteomyelitis, the animals underwent osteonecrectomy. In the study group, autologous MSCs in Collatamp EG collagen carrier were placed into the bone defect. MSCs were obtained from adipose tissue and cultured in the matrix for 5 days. In control, the defect was filled with the collagen matrix without cells. Results. On day 14 upon the initiation of chronic osteomyelitis, bacteriological examination of the discharge from the fistula showed the presence of mixed bacterial flora (Staphylococcus aureus and Escherichia coli) in all operated animals. Results of X-ray, laboratory, and histological tests confirmed the formation of a focus of chronic osteomyelitis. Two months after the treatment (collagen with or without MSCs) began, all animals of the study group showed mature bone tissue regenerated in the affected zone. In the control group, proliferation of osteoblasts on the surface of the bone trabeculae was also observed; however, mature osteoid tissue was more often detected in the study group (35.0 vs 20.0% in control). In the study group (MSCs + collagen matrix), there was a decrease in bone marrow fibrosis (50.0 vs 100.0% in control) and cartilage formation (30.0 and 66.7%, respectively). After full treatment, newly formed bone trabeculae were detected more often (100.0 vs 60.0% in control); they were more mature and filled the defect area more efficiently. Conclusion. Our results indicate that the use of a collagen matrix with autologous MSCs is a promising plastic material for repairing osteomyelitic defects following necrectomy. The MSCs were able to increase the density of the filling material in the bone cavity, significantly accelerate the formation of bone beams around the matrix, and increase the tissue volume around the implant. The presence of MSCs significantly decreased the interference of a connective tissue component with osteogenesis and chondrogenesis.

Made available in DSpace on 2021-06-25T10:46:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Universidade Estadual Paulista Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The treatment of polytrauma patients represents a great challenge in the maxillofacial and orthopedic surgery fields. Therefore, this study tested the hypothesis that the use of a bioactive coating (by plasma electrolytic oxidation, PEO) on titanium microplates could improve the fracture healing of low bone mineral density (BMD) rats. Thirty female rats underwent bilateral ovariectomy surgery (OVX), and 35 rats underwent fake surgery (SHAM). Three months later, animals were subjected to femoral fracture simulation and were fixed with either non-coated (CONV) or coated (PEO) titanium miniplates. Eight weeks postoperatively, microplate/bone complexes were analyzed through computed microtomography, histometric, confocal microscopy, molecular, and biomechanical analysis. Bioactive elements (Ca and P) were incorporated on the PEO microplate and the surface was modified in a volcano-like structure. In the microCT analysis the OVX/PEO group had greater values for Tb.Th (bone trabecular thickness), Tb.Sp (separation of bone trabeculae) and Tb.N (number of trabeculae) parameters compared to the OVX/CONV group. According to histometric analysis, the OVX/PEO group showed significantly higher new bone formation than the OVX/CONV group (P < 0.05). For the fluorochrome area, the OVX groups (PEO and CONV) showed greater values for calcein precipitation (old bone) than alizarin red (new bone). Molecular results showed greater values for proteins related to the final phase of bone formation (P < 0.05) in the OVX/PEO group. The OVX/PEO group showed higher bone/miniplate system resilience compared to the others (P < 0.05). It was concluded that PEO coating optimizes bone healing on simulated femoral fractures in low bone mineral density rats. This sheds new light in the treatment of osteoporotic patients with bone fractures. Division of Oral and Maxillofacial Surgery Department of Diagnosis and Surgery Aracatuba School of Dentistry São Paulo State University (UNESP) Bone Res Lab School of Dentistry of Ribeirao Preto University of São Paulo (USP) Department of Prosthodontics and Periodontology Piracicaba Dental School University of Campinas (UNICAMP) Instituto de Biociências de Botucatu (IBB) São Paulo State University (UNESP) Department of Basic Science Aracatuba School of Dentistry São Paulo State University (UNESP) Programa de Pós-Graduação Multicêntrico em Ciências Fisiológicas SBFis São Paulo State University (UNESP) Laboratory of Technological Plasmas (LaPTec) Science and Technology Institute of Sorocaba (ICTS) São Paulo State University (UNESP) Division of Oral and Maxillofacial Surgery Department of Diagnosis and Surgery Aracatuba School of Dentistry São Paulo State University (UNESP) Instituto de Biociências de Botucatu (IBB) São Paulo State University (UNESP) Department of Basic Science Aracatuba School of Dentistry São Paulo State University (UNESP) Programa de Pós-Graduação Multicêntrico em Ciências Fisiológicas SBFis São Paulo State University (UNESP) Laboratory of Technological Plasmas (LaPTec) Science and Technology Institute of Sorocaba (ICTS) São Paulo State University (UNESP) Universidade Estadual Paulista: 01.12.0530.00-FINEP/PROINFRA 01/2011 FAPESP: 2016/20297-6 FAPESP: 2018/00522-0

Post-extraction healing of the socket may take up to 24 weeks to complete. This systematic review aims to evaluate whether photobiomodulation accelerates bone healing in those sockets. A search strategy was developed in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. PubMed, Cochrane Library, and Scopus electronic databases were searched for in vivo studies with restrictions on the year (

Background Complementary and alternative medicine approaches are gaining ground in everyday practice. Propolis is a bee product that has been suggested to have bone regenerative effects. This study was conducted to compare the effectiveness of Egyptian propolis with nanohydroxyapatite graft on furcation defect regeneration in mongrel dogs. Methods A split mouth design was utilized in six clinically healthy mongrel dogs. A total of 24 Class II furcation defects were surgically created in mandibular third and fourth premolars. The defects on the right side were filled with nanohydroxyapatite graft and covered with collagen membrane, while on the left side the defects were filled with propolis and also covered with collagen membrane. The dogs were sacrificed after 1 and 3 months. Segments containing the defects were prepared for histological evaluation. Results The histological evaluation after one month revealed newly formed bone in both treatment groups. However, after 3 months, the bone trabeculae appeared thinner in the collagen/nanohydroxyapatite group than in the collagen/propolis group. The histomorphometric evaluation showed a significant increase in bone height as well as bone surface area for the collagen/propolis group compared to collagen/nanohydroxyapatite group. Conclusions Both Egyptian propolis and nanohydroxyapatite graft material showed favorable periodontal regenerative effect. Propolis showed increased cellular proliferative ability that could be beneficial in reducing the healing period needed after periodontal therapy.

Background and objective: Computational homology is an emerging mathematical tool for characterizing shapes of data. In this work, we present a methodology using computational homology for obtaining quantitative measurements of the connectivity in bone morphometry. We introduce the Betti number ratios as novel morphological descriptor for the classification of bone fine structures in three dimensions. Methods A total of 51 Japanese white rabbits were used to investigate the connectivity of bone trabeculae after the administration of alendronate in a tendon graft model in rabbits. They were divided into a control group C and an experimental group A. Knee joints specimens were harvested for examination of their bone trabecular structure by micro-CT. Applying the computational homology software to the reconstructed 3D image data, we extract the morphological feature of a steric bone structure as the Betti numbers set (β0, β1, β2). The zeroth Betti number β0 indicates the number of the connected components corresponding to isolated bone fragments. The first and second Betti numbers, β1 and β2, indicate the numbers of open pores and closed pores of bone trabeculae, corresponding to a 2D empty space enclosed by a 1D curve and a 3D empty space enclosed by a 2D surface, respectively. Results We define the Betti number ratios β1/β0 and β2/β0 to better distinguish the two groups A and C in the scatter plots. Testing the discriminant function line for 29 data points of A (22 data points of C), the 17 points (resp. 18 points) are correctly classified into group A (resp. C). The accuracy rate is 35/51. The classification results in terms of the Betti number ratios are consistent with the histomorphometric measurements observed by medical doctors. Conclusions: This study is the first application of computational homology to bone morphometry in three dimensions. We show the mathematical basis of the Betti numbers index which are useful in a statistical description of the topological features of sponge-like structures. The potential benefits associated with our method include both improved quantification and reproducibility for the stereology.

The healing capacity of bone marrow mesenchymal stem cells (BMMSCs) has been evaluated in various studies. This study aimed to evaluate the effect of BMMSCs on the healing of temporomandibular joints (TMJs) with induced rheumatoid arthritis. Fifty healthy male Sprague Dawley rats were divided into three groups: group I (n = 10), negative control; group II (n = 20), positive control (induction of arthritis by adjuvant followed by intravenous injection of 0.1 ml of PBS); and group III (n = 20), intervention (as for group II but injected intravenously with 1 × 106 cells ml-1 of BMMSCs suspended in PBS). Half of the rats in each group were euthanized 3 wk after the start of the experiment and the other half was euthanized after 5 wk. Group I revealed normal TMJ features. Group II showed thickening of disc, thinning of cartilage, disordered bone trabeculae, and decreased in mean % area staining positive of collagen fibers at 3 wk, while at 5 wk these effects were more aggravated. Group III showed nearly normal thickness of disc and condylar cartilage, nearly normal arrangement of bone trabeculae and regenerated collagen fibers at 3 wk, while after 5 wk the TMJ features were almost normal. Two-way anova revealed statistically significant differences between groups. Thus, treatment of induced rheumatoid arthritis with BMMSCs shows promising results that need to be further investigated in humans.

BACKGROUND Sofosbuvir is a nucleotide compound that has proved to be among the most potent orally available antiviral treatments. Infrequent but serious adverse events have been reported with the use of oral nucleos(t)ide analogues. OBJECTIVES Investigating sofosbuvir-induced alterations in the rat mandibular alveolar process. MATERIAL AND METHODS In the study, 30 adult male albino rats were used and divided randomly into following groups: group 1, group 2 and group 3 (10 rats per group). Group 1 served as a control, group 2 received sofosbuvir through oral gavage at a dose of 40 mg/kg/day for 6 weeks, and group 3 was similar to group 2 but received sofosbuvir for 12 weeks. The animals were sacrificed at the end of the experiment. The mandibles were dissected and examined histologically as well as by scanning electron microscope (SEM) and energy dispersive X-ray unit (EDX). RESULTS Histologically, group 1 showed normal alveolar process. In group 2, histopathological changes occurred as bone trabeculae demonstrated obvious Howship's lacuna of osteoclasts. In group 3, bone trabeculae exhibited multiple degenerated areas as well as apparent vacuolization. Scanning electron microscopic examination revealed smooth alveolar bone architecture in group 1. On the other hand, groups 2 and 3 demonstrated irregular bone architecture with formation of multiple pores. EDX analysis demonstrated the highest calcium concentration in the control group, while the lowest was found in group 3. Statistical analysis of the EDX results revealed a statistically significant difference among the studied groups as the p-value was

Three-dimensional (3D) matrices scaffolds play a noteworthy role in promoting cell generation and propagation. In this study, scaffolds prepared from chitosan/polyvinyl alcohol loaded with/without an osteoporotic drug (risedronate) and nano-bioactive glass (nBG) have been developed to promote healing of bone defects. The scaffolds were characterized by scanning electron microscopy (SEM), porosity test as well as mechanical strength. The pattern of drug release and ability to promote the proliferation of Saos-2osteosarcoma cells had also been reported. Osteogenic potential of the scaffolds was evaluated by testing their effect on healing critical-sized dog's mandibular bone defects. Increasing chitosan and nBG in the porous scaffolds induced decrease in drug release, increased the scaffold's strength and supported their cell proliferation, alkaline phosphatase (ALP) activities, as well as increased calcium deposition. Histological and histomorphometric results demonstrated newly formed bone trabeculae inside critical-sized mandibular defects when treated with scaffolds. Trabecular thickness, bone volume/tissue volume and the percentage of mature collagen fibers increased in groups treated with scaffolds loaded with 10% nBG and risedronate or loaded with 30% nBG with/without risedronate compared with those treated with non-loaded scaffolds and empty control groups. These findings confirmed the potential osteogenic activity of chitosan/polyvinyl alcohol-based scaffolds loaded with risedronate and nBG.

It has been reported that carbon nanotubes (CNTs) serve as nucleation sites for the deposition of bone matrix and cell proliferation. Here, we evaluated the effects of multi-walled CNTs (MWCNTs) on bone repair of rat tibiae. Furthermore, because sodium hyaluronate (HY) accelerates bone restoration, we associated CNTs with HY (HY-MWCNTs) in an attempt to boost bone repair. The bone defect was created by a 1.6-mm-diameter drill. After 7 and 14 days, tibiae were processed for histological and morphometric analyses. Immunohistochemistry was used to evaluate the expression of vascular endothelial growth factor (VEGF) in bone defects. Expression of osteocalcin (OCN), bone morphogenetic protein-2 (BMP-2), and collagen I (Col I) was assessed by real-time PCR. Histomorphometric analysis showed a similar increase in the percentage of bone trabeculae in tibia bone defects treated with HY and HY-MWCNTs, and both groups presented more organized and thicker bone trabeculae than nontreated defects. Tibiae treated with MWCNTs or HY- MWCNTs showed a higher expression of VEGF. Treatment with MWCNTs or HY-MWCNTs increased the expression of molecules involved in the bone repair process, such as OCN and BMP-2. Also, HY- and MWCNT-treated tibiae had an increased expression of Col I. Thus, it is tempting to conclude that CNTs associated or not with other materials such as HY emerged as a promising biomaterial for bone tissue engineering.

Introduction This study aimed to verify the effects of cigarette smoke exposure in bone mineralization and fibrillar matrix composition as well as in bone healing after tibial fracture induction. Methods C57Bl/6 Mice were assigned according to exposure and surgery: C room air; F room air and tibia open osteotomy; CS cigarette smoke; FCS cigarette smoke and tibia open osteotomy. In order to study fracture healing we performed, under anesthesia, a bone injury through a tibial shaft osteotomy. Bone samples were obtained to evaluate bone histomorphometry, trabecular morphology and volume, trabecular collagen types composition and presence of inflammatory cytokines and growth factors. Results CS exposure significantly reduced the thickness of bone trabeculae associated with decrease in mineralizing surface and mineral deposition rate, leading a lower bone formation rate and longer mineralization time. Resorption surface and osteoclastic surface were greater in the CS group, attesting increased resorptive action. There was a decrease in type I collagen deposition and genes expression in the CS and FCS groups compared to C group and in contrast there was an increase in type V collagen deposition and genes expression in the CS, FC and FSC groups compared to C group. Also, CS exposure induced a decrease in bone forming cytokines and an increase in inflammatory associated cytokines, and these changes were intensified under fracture conditions. Conclusion Cigarette smoke exposure alters bone matrix composition and worsens bone mineralization, leading to bone fragility by increasing collagen V synthesis and deposition and impairing collagen I fibril forming and assembling. And these deleterious effects contributed to the worsening in fracture healing after tibia osteotomy.

Purpose The purpose of the present study was to evaluate the efficacy of simvastatin administration as an osteoinductive agent combined with bovine bone material (BBM) for augmentation of human maxillary sinuses. Materials and Methods In the present randomized clinical trial with a split-mouth design, 24 maxillary sinuses in 12 patients were augmented using BBM alone or BBM combined with simvastatin. Biopsy samples were taken 9 months after maxillary sinus floor augmentation for histologic and histomorphometric analyses. A total of 44 implants were placed in the augmented bone. Results The results of the microscopic assessment of most samples revealed no inflammation or only mild chronic inflammation. Lamellation was detectable in old bone trabeculae under polarized light microscopy but was not observed in newly formed bone. Osteocytes were found with a lower frequency in the lacunae of newly formed bone compared with normal bone. No significant differences were found in the amount of newly formed bone and the amount of residual particles between the 2 groups. Conclusions Despite the greater mean percentage of newly formed bone in the test group, the histomorphometric analysis results did not show a significant positive effect for the use of simvastatin in maxillary sinus augmentation.

Detailed knowledge of age-related changes in the structure and mineralization of bones is important for interpreting osseous changes in wild mammals caused by exposure to environmental contaminants. This study analyzed mandibular size, microarchitecture and volumetric bone mineral density (vBMD) in harbor seals (n = 93, age range 0.5 months to 25 years) from the German North Sea. Bone microarchitecture and vBMD were assessed using high-resolution peripheral quantitative computed tomography (HR-pQCT). Significant differences were observed between the analyzed age classes (i) young juveniles (0.5-10 months), (ii) yearlings (12-23 months), and (iii) adults (12-25 years) for several of the variables, indicating an overall increase in cortical and trabecular area, cortical thickness and total and cortical vBMD with age. Furthermore, for juvenile animals (≤ 23 months), significant positive correlations with age were observed for mandible length and perimeter, cortical area, cortical thickness, trabecular separation, and total and cortical vBMD. The findings demonstrate a rapid increase in overall size, cortical dimensions and the degree of mineralization of the harbor seal mandible during the first two years after birth. Negative correlations with age existed for trabecular number and thickness as well as for trabecular bone volume fraction in the juveniles. The findings suggest a reduction in trabecular bone volume fraction with age, due to the bone trabeculae becoming thinner, less numerous and more widely spaced. Given the strong age dependence of most analyzed parameters, it is recommended to standardize samples with respect to age in future studies comparing microarchitecture and mineralization of harbor seal mandibles from different populations or different collection periods.

International audience; Loss or reduction of teeth has occurred independently in all major clades of mammals [1]. This process is associated with specialized diets, such as myrmecophagy and filter feeding [2, 3], and led to an extensive rearrangement of the mandibular anatomy. The mandibular canal enables lower jaw innervation through the passage of the inferior alveolar nerve (IAN) [4, 5]. In order to innervate teeth, the IAN projects ascending branches directly through tooth roots [5, 6], bone trabeculae [6], or bone canaliculi (i.e., dorsal canaliculi) [7]. Here, we used micro-computed tomography (μ-CT) scans of mandibles, from eight myrmecophagous species with reduced dentition and 21 non-myrmecophages, to investigate the evolutionary fate of dental innervation structures following convergent tooth regression in mammals. Our observations provide strong evidence for a link between the presence of tooth loci and the development of dorsal canaliculi. Interestingly, toothless anteaters present dorsal canaliculi and preserve intact tooth innervation, while equally toothless pangolins do not. We show that the internal mandibular morphology of anteaters has a closer resemblance to that of baleen whales [7] than to pangolins. This is despite masticatory apparatus resemblances that have made anteaters and pangolins a textbook example of convergent evolution. Our results suggest that early tooth loci innervation [8] is required for maintaining the dorsal innervation of the mandible and underlines the dorsal canaliculi sensorial role in the context of mediolateral mandibular movements. This study presents a unique example of convergent redeployment of the tooth developmental pathway to a strictly sensorial function following tooth regression in anteaters and baleen whales.

Several methods have been developed to regenerate lost alveolar bone. Platelet-rich fibrin (PRF) is a useful adjunct for new bone formation in dentistry. To elucidate the effect of advanced PRF (A-PRF) on bone formation, we inserted A-PRF clots in sockets after tooth extraction. Premolars were extracted from beagle dogs, and A-PRF was applied to the socket. New bone formation was assessed using histological and immunofluorescence examinations, and the bone formation ratio was evaluated 14 and 30 days postoperatively. Histological examination revealed newly formed bone filling the sockets up to the center in the A-PRF group at 14 days postoperatively, while thick and regular bone trabeculae were arranged in porous bone after 30 days. Higher expressions of osteocalcin and osteopontin were observed in newly formed bone in the A-PRF group, compared to the control group. The bone formation ratio was also higher in the A-PRF group than in the control group. Thus, A-PRF application may result in enhanced new bone formation and may aid in accelerating bone formation. A-PRF was more rapid than a self-limiting process during induction of bone formation by enhancing osteoblast activity and may be useful for bone formation in clinical medicine.

The objectives of this study were to assess the effects of hyaluronic acid (HY), multi-walled carbon nanotubes (MWCNT), and MWCNT functionalized with HY (HY-MWCNT) on the resolution of neutrophilic inflammation in the pleural cavity of LPS-challenged mice and to assess the influence of these materials in the inflammatory process of bone repair of tooth sockets of rats. C57Bl/6 mice were intra-pleurally injected with HY, MWCNT, HY-MWCNT, phosphate-buffered saline (PBS), or LPS. The animals were euthanized after 8 and 24 h, and cells were harvested for total and differential cell counting. The tooth sockets of Wistar rats were filled with HY, MWCNT, HY-MWCNT, or blood clot (control). After 1, 3, and 7 days, histological and morphometric analyses evaluated the number of cell nuclei and blood vessels, and bone trabeculae formation in the sockets. Myeloperoxidase (MPO) activity quantified neutrophil accumulation in the sockets. HY, MWCNT, and HY-MWCNT increased neutrophilic recruitment at 8 h and reduced the inflammatory process at 24 h in the pleural cavity. Histological and morphometric analyses and MPO activity showed no significant differences in the recruitment of inflammatory cells in the tooth sockets. HY increased the number of blood vessels, and HY and HY-MWCNT increased bone trabeculae formation at 7 days of tooth extraction. HY, MWCNT, and HY-MWCNT resolved the neutrophilic inflammation in the pleural cavity of the mice. However, these materials did not modulate the inflammatory process in the early stages of bone repair of the tooth sockets, thereby excluding this action as a possible mechanism by which these biomaterials accelerate bone repair. HY-MWCNT is capable of accelerating bone repair/regeneration without affecting the inflammatory phase during the bone healing process.

Background Existing preclinical murine models often fail to predict effects of anti-cancer drugs. In order to minimize interspecies-differences between murine hosts and human bone tumors of in vivo xenograft platforms, we tissue-engineered a novel orthotopic humanized bone model. Methods Orthotopic humanized tissue engineered bone constructs (ohTEBC) were fabricated by 3D printing of medical-grade polycaprolactone scaffolds, which were seeded with human osteoblasts and embedded within polyethylene glycol-based hydrogels containing human umbilical vein endothelial cells (HUVECs). Constructs were then implanted at the femur of NODscid and NSG mice. NSG mice were then bone marrow transplanted with human CD34 + cells. Human osteosarcoma (OS) growth was induced within the ohTEBCs by direct injection of Luc-SAOS-2 cells. Tissues were harvested for bone matrix and marrow morphology analysis as well as tumor biology investigations. Tumor marker expression was analyzed in the humanized OS and correlated with the expression in 68 OS patients utilizing tissue micro arrays (TMA). Results After harvesting the femurs micro computed tomography and immunohistochemical staining showed an organ, which had all features of human bone. Around the original mouse femur new bone trabeculae have formed surrounded by a bone cortex. Staining for human specific (hs) collagen type-I (hs Col-I) showed human extracellular bone matrix production. The presence of nuclei staining positive for human nuclear mitotic apparatus protein 1 (hs NuMa) proved the osteocytes residing within the bone matrix were of human origin. Flow cytometry verified the presence of human hematopoietic cells. After injection of Luc-SAOS-2 cells a primary tumor and lung metastasis developed. After euthanization histological analysis showed pathognomic features of osteoblastic OS. Furthermore, the tumor utilized the previously implanted HUVECS for angiogenesis. Tumor marker expression was similar to human patients. Moreover, the recently discovered musculoskeletal gene C12orf29 was expressed in the most common subtypes of OS patient samples. Conclusion OhTEBCs represent a suitable orthotopic microenvironment for humanized OS growth and offers a new translational direction, as the femur is the most common location of OS. The newly developed and validated preclinical model allows controlled and predictive marker studies of primary bone tumors and other bone malignancies.

Testosterone has profound immune-modulatory actions, which may be important for the sexual dimorphism in immune-related disorders, such as autoimmune diseases. A well-known effect of androgens is inhibition of bone marrow B lymphopoiesis; however, a plausible target cell for this effect has not yet been presented. The aim of this study was to determine the target cell for androgen-mediated regulation of bone marrow B lymphopoiesis in males. We confirm higher number of bone marrow B cells in male mice with global inactivation of the androgen receptor (AR) and these global AR knockout (G-ARKO) mice had increased number of B cell precursors from the pro-B stage. Because osteoblast-lineage cells are known to support B lymphopoiesis at the pro-B stage, we investigated the effect on B lymphopoiesis in osteoblast-lineage cell-specific ARKO (O-ARKO) mice; O-ARKO mice had increased number of B cells in the bone marrow, and the number of B cell precursors was increased from the pro-B stage, demonstrating that O-ARKO mimics the bone marrow B lymphopoiesis pattern of G-ARKO mice. By contrast, O-ARKO mice displayed only minor changes in B cell numbers in the splenic compartment compared with G-ARKO. Further, O-ARKO mice had moderately reduced number of bone trabeculae in the vertebrae, whereas cortical bone was unaffected. In conclusion, androgens exert inhibitory effects on bone marrow B lymphopoiesis in males by targeting the AR in osteoblast-lineage cells. The identification of the likely target cell for androgen-mediated regulation of bone marrow B lymphopoiesis will contribute to elucidation of the mechanisms by which androgens modulate immune-related disorders.

This study evaluates the influence of platelet-rich plasma derived from bone marrow aspirate (PRP-BMA) on the healing of periodontal fenestration defects in rats.Periodontal fenestration defects were surgically created in the mandibles of 40 rats. The animals were randomly divided into two groups, control and PRP-BMA, in which defects were filled with blood clot or PRP-bma, respectively. Animals were euthanized at either 10 or 30 days post-surgery. Histologic, histometric, and immunohistochemical analyses were performed. Percentage of new bone area (NBA), area of bone trabeculae (ABT), new cementum (NC), and extension of remaining defect were histometrically evaluated. Proliferating cell nuclear antigen (PCNA), bone sialoprotein (BSP), osteocalcin (OCN), and tartrate-resistant acid phosphatase (TRAP) immunohistochemical staining were performed. Immunolabeled cells were quantified. Data were statistically analyzed (analysis of variance; Tukey, P0.05).At 10 days, control and PRP-BMA groups presented similar amounts of NBA and ABT; NC formation was not observed. At 30 days, control and PRP-BMA groups presented similar amounts of NBA and ABT; the PRP-BMA group showed NC formation with collagen fibers inserted obliquely or perpendicularly to the root surface. NC formation was not observed in any control group specimen. PRP- BMA presented higher numbers of PCNA-positive and BSP-positive cells than control at 10 and 30 days post-surgery. No significant differences in the number of either OCN-positive or TRAP-positive cells were observed between groups at 10 or 30 days.PRP-BMA promoted NC formation with a functional periodontal ligament when applied at experimental periodontal fenestration defects.

Magnesium phosphate minerals have captured increasing attention during the past years as suitable alternatives for calcium phosphate bone replacement materials. Here, we investigated the degradation and bone regeneration capacity of experimental struvite (MgNH4PO4·6H2O) forming magnesium phosphate cements in two different orthotopic ovine implantation models. Cements formed at powder to liquid ratios (PLR) of 2.0 and 3.0 g ml−1 were implanted into trabecular bone using a non-load-bearing femoral drill-hole model and a load-bearing tibial defect model. After 4, 7 and 10 months the implants were retrieved and cement degradation and new bone formation was analyzed by micro-computed tomography (µCT) and histomorphometry. The results showed cement degradation in concert with new bone formation at both defect locations. Both cements were almost completely degraded after 10 months. The struvite cement formed with a PLR of 2.0 g ml−1 exhibited a slightly accelerated degradation kinetics compared to the cement with a PLR of 3.0 g ml−1. Tartrat-resistant acid phosphatase (TRAP) staining indicated osteoclastic resorption at the cement surface. Energy dispersive X-ray analysis (EDX) revealed that small residual cement particles were mostly accumulated in the bone marrow in between newly formed bone trabeculae. Mechanical loading did not significantly increase bone formation associated with cement degradation. Concluding, struvite-forming cements might be promising bone replacement materials due to their good degradation which is coupled with new bone formation. Statement of Significance Recently, the interest in magnesium phosphate cements (MPC) for bone substitution increased, as they exhibit high initial strength, comparably elevated degradation potential and the release of valuable magnesium ions. However, only few in vivo studies, mostly including non-load-bearing defects in small animals, have been performed to analyze the degradation and regeneration capability of MPC derived compounds. The present study examined the in vivo behavior of magnesiumammoniumphosphate hexahydrate (struvite) implants with different porosity in both mechanically loaded and non-loaded defects of merino sheep. For the first time, the effect of mechanical stimuli on the biological outcome of this clinically relevant replacement material is shown and directly compared to the conventional unloaded defect situation in a large animal model.

The purpose of this study was to assess and evaluate new bone formation in rabbit marginal mandibular defects using expanded bone marrow-derived osteoprogenitor cells seeded in three-dimensional scaffolds of polycaprolactone/tricalcium phosphate (PCL/TCP). Bone marrow was harvested from the rabbit ilium and rabbit bone marrow-derived osteoprogenitor cells were isolated and expanded in standard culture medium and osteogenic medium supplement. The cells were then seeded into the PCL/TCP scaffolds and the cell/scaffold constructions were implanted into prepared defects in rabbit mandibles. PCL/TCP scaffold alone and autogenous bone graft from the mandible were also implanted into the other prepared defects. The specimens were evaluated at 4 and 8 weeks after the implantation using clinical, radiographic, and histological techniques. The results of the experimental group demonstrated more newly formed bone on the surface and in the pores of the PCL/TCP scaffolds. In addition, the osteoblasts, osteocytes, and new bone trabeculae were identified throughout the defects that were implanted with the cell/scaffold constructions. The PCL/TCP alone group was filled mostly with fibrous cells particularly in the middle region with less bone formation. These results would suggest that the derived osteotoprogenitor cells have the potential to form bone tissue when seeded onto PCL/TCP scaffolds.

Many studies have assessed the effects of either low-level laser therapy (LLLT) or low-intensity pulsed ultrasound (LIPUS) on bone repair; however, an evaluation of the combination of these modalities (LLLT + LIPUS) has not yet been considered. The aim of this study was to demonstrate the effects of LLLT + LIPUS on bone repair. Male Wistar rats (n = 128; four groups of 32) were used; the animals underwent a partial tibial bone osteotomy. One group had the osteotomized limb treated with LLLT, the second group with LIPUS, and the third group with the combined treatment protocols of the LLLT and LIPUS groups; the fourth group received no further treatment (control). Each group was divided into two subgroups for assessment at two different time-points, 14 and 21 days. After the completion of treatment rats were sacrificed and the tibias submitted to a three-point bending test or to histomorphometric analysis. Histological evaluation showed increased bone trabeculae, increased vascularization, and decreased inflammation in the LLLT + LIPUS group. Mechanical evaluation revealed increased biomechanical properties including maximum force, maximum stress, and stiffness, in the LLLT + LIPUS group. Combined LLLT + LIPUS treatment enhanced bone healing both histologically and mechanically, shortening the length of the treatment period, when compared to treatment with LLLT or LIPUS alone.

STUDY DESIGN Expansive pedicle screw (EPS) and polymethylmethacrylate-augmented pedicle screw (PMMA-PS) were inserted in sheep vertebrae in vitro and were evaluated by performing biomechanical tests, radiographic examinations and histological observations. OBJECTIVE The objective of the study was to compare the biomechanical and interfacial performances of EPS and PMMA-PS in sheep lumbar vertebrae in vitro. SUMMARY OF BACKGROUND DATA It is a great challenge for orthopedic surgeons performing transpedicular fixation in the osteoporotic spine. It was reported that either the EPS or PMMA-PS could increase the screw stability. However, there are no studies comparing the 2 kinds of screws especially in primary spinal instrumentation. METHODS A total of 60 sheep lumbar vertebrae were randomly divided into 3 groups. A pilot hole was made in advance in all samples using the same method. Thereafter, the conventional pedicle screw (CPS) was inserted directly into the pilot hole in the CPS group; the hole in PMMA-PS group was first filled with polymethylmethacrylate (PMMA; 1.0 mL) and then inserted with CPS; and the EPS was inserted directly into the vertebrae in EPS group. After a period of 24 hours, biomechanical tests were performed to evaluate screw stability, and x-ray examination, micro-computerized tomography analysis, and histologic observation were performed to evaluate the interface between screw and bone. RESULTS Compared with the stability of CPS, those of EPS and PMMA-PS were significantly enhanced. However, no significant differences were detected between the stabilities of EPS and PMMA-PS. The PMMA surrounding the screw blocked direct contact between bone and screw and formed a "screw-PMMA-bone" interface in the PMMA-PS group. There was a "screw-bone" interface in both CPS and EPS groups. Nevertheless, the expanded anterior part of EPS formed a claw-like structure pressing the surrounding bone trabeculae, which made the local bone tissue more compacted and denser than that in the CPS group. CONCLUSIONS EPS can enhance the screw stability as markedly as the traditional PMMA-PS in primary surgery, and EPS can form a better immediate interface between screw and bone compared with PMMA-PS. EPS also can effectively avoid thermal injury, leakage, and compression caused by PMMA. A great feasibility was proved in this study to perform comparisons between the 2 kinds of pedicle screws in osteoporotic sheep vertebrae in vivo in the further research. In conclusion, we propose that EPS has a great application potential in augmentation of screw stability in the clinic.

Bone growth on and into implants exhibiting substantial surface porosity is a promising strategy in order to improve the long-term stable fixation of bone implants. However, the reliability in clinical applications remains a point of discussion. Most attention has been dedicated to the role of macroporosity, leading to the general consensus of a minimal pore size of 50–100 μm in order to allow bone ingrowth. In this in vivo study, we assessed the feasibility of early bone ingrowth into a predominantly microporous Ti coating with an average thickness of 150 μm and the hypothesis of improving the bone response through surface modification of the porous coating. Implants were placed in the cortical bone of rabbit tibiae for periods of 2 and 4 weeks and evaluated histologically and histomorphometrically using light microscopy and scanning electron microscopy. Bone with osteocytes encased in the mineralized matrix was found throughout the porous Ti coating up to the coating/substrate interface, highlighting that osseointegration of microporosities (

PURPOSE To examine the biologic response to titanium implant surfaces treated with a neodymium:yttrium-aluminum-garnet laser. MATERIALS AND METHODS Sixty mini-implants made of grade 2 titanium were placed in the femora of 30 Wistar rats. Thirty implants had a machined surface and the other 30 had surfaces that were roughened by laser treatment. The animals were subdivided into three groups according to bone repair periods of 15, 30, and 60 days. The samples were observed under light and electron scanning microscopes and analyzed with the Student t test. RESULTS Formation of new bone trabeculae toward the surface was apparent for the laser-treated implants at 15 days. Thin layers of bone matrix in intimate contact with the surface in the area of the central screw threads were observed, indicating high biocompatibility. Similar results were seen with machined implants after 30 days. A significant difference in bone formation was observed between the implant types at 15 days. CONCLUSION Bone-to-implant contact was better on the surfaces subjected to laser treatment than on the machined titanium implants. The development of new laser treatments, which promote alterations in the surface energy as well as in the macro- and microstructures of titanium, may lead to improved bone-to-implant contact and thus better outcomes.

Introduction During root formation, Smad-4 plays a key role during the epithelial–mesenchymal interactions and the Hertwig's epithelial root sheath (HERS) apical proliferation. The root formation and eruption of rat molars is impeded by alendronate treatment due to the inhibition of bone resortion by this drug. The present study aimed to examine the structures affected in the developing root and immunodetect the presence of Smad-4 in rats treated with alendronate. Methods Newborn Wistar rats were daily injected 2.5 mg/kg alendronate (ALN) during 9, 12 and 30 days. The controls (CON) were injected with saline. The maxillae were fixed and embedded in paraffin or Spurr resin. Paraffin sections were incubated in Smad-4 antibody that was labelled with DAB. The ultrathin sections were examined in a transmission electron microscope. Results In ALN, a short portion of root dentine was formed; the epithelial diaphragm (ED) and the dental follicle (DF) were disorganized by the contact of bone trabeculae. The (CON) molar roots developed normally. Smad-4 labelling was detected in the cytoplasm of fibroblasts and cementoblasts adjacent to the cementum in CON; in ALN group, few ED cells presented weak immunolabelling. Ultrastructurally, the ED and DF appeared disrupted due to the presence of thin bone trabeculae between its cells. It resulted in the lack of apical proliferation of HERS and, consequently, arrest of root formation. Conclusion The immunodetection of Smad-4 in the DF cells of ALN specimens indicates that the signalling for the differentiation of these cells into cementum-forming fibroblasts and cementoblasts occurs, despite the impairment of root elongation.

Objective In this work we evaluated the bone-forming potential of BMP4, TGFβ1 and BMP4/TGFβ1 mixed by performing histological and morphometric analysis. We also evaluated the immunolabelling of fibronectin (FN) and collagen type III (Col III), two determinant proteins for the early phase of bone repair. Design Histological, histomorphometric and immunohistochemistry analysis were used to evaluate new bone and blood vessels formation as well as fibronectin and collagen type III expression. 112 male Wistar rats weighing 250–300 g had their maxillary second molar extracted. Sockets filled with blood clot (BC) or treated with L (empty liposome), P (PBS), BP (BMP-4 in PBS) and TP (TGF-β1 in PBS), as well as with BL (BMP-4 in liposome) and TL (TGF-β1 in liposome) administered isolated or in association (BTL) were obtained. The animals were sacrificed at 3, 7, 14 and 21 days after surgery. Results An increased percentage of bone trabeculae, and a higher number of blood vessels were observed in groups BL or TL administered isolated or in association when compared to groups BC, L, P, BP and TP. Fibronectin and collagen type III analysis revealed enhanced expression firstly detected at 3 days followed by a peak at 7 days. Lower levels of immunoreactivity were observed in the sockets filled with blood clot, and treated with L, P, BP and TP when compared with sockets from groups BL, TL and BTL. Conclusion The present study indicates growth factors carried by liposomes, either in isolated or associated forms, as successful enhancers of the healing process in rat tooth sockets. We also conclude that the expression of fibronectin and collagen type III increases during the early phases of bone repair.

Since bone defects can lead to various disabilities, in recent years, many increasing attempts have been made in bone tissue engineering. In this regard, scaffolds have attracted a lot of attention as three dimensional substrates for cell attachment which improve successful tissue engineering. The aim of the present study was to provide an interconnected porous scaffold to facilitate cell infiltration. To do so, cancellous bone from bovine femur was dissected in fragments and decellularized by physicochemical methods, including snap freeze/thaw, rinsing in hot water and treatment with different solutions of sodium dodecyl sulfate (SDS). Histological analysis and 4',6-diamidino-2-phenylindole staining revealed that the best results were obtained after treatment with 2.5%, 5%, and 8% SDS for 8, 3, or 1 h respectively, which significantly removed bone cells with intact trabeculae geometry. Further characterization of decellularized scaffolds by the compression tests also revealed no significant difference between elastic modulus values of the three different SDS treatments. Moreover, studying the ratio of bone trabeculae to bone surfaces (BT/BS) as assessed by Clemex vision software 3.5 showed that treatment with 2.5% SDS for 8 h resulted in a BT/BS score in the range of native bone and therefore this treatment was used for further experiments. Histological studies and scanning electron microscopy revealed rat mesenchymal stem cells integration, adhesion, and maintenance during the 2 and 7 d of culture in vitro. In conclusion, the present results support the effective role of SDS in cancellous bovine bone decellularization and also propensity of treated samples in providing a suitable three-dimentional environment to support the maintenance and growth of mesenchymal stem cells.

Purpose To investigate the effect of low-intensity ultrasound on mandibular osteoradionecrosis. Materials and Methods Using a canine model, radiotherapy was delivered to the mandible at doses of 25 and 28 Gy. The microstructure of the mandible and changes in microvascular density in the low-intensity ultrasound treatment (group A) and nontreatment (group B) groups were determined and compared with those of the control group. Results At a single dose of 28 Gy, the canines developed epilation of the mandibular skin, ulcers, and small pieces of oral sequestered material. The microvascular density in group A was significantly greater than that in group B (P < .05). Most osteocytes in group B had disappeared, with atrophy of the cancellous bone trabeculae. In contrast, in group A, a substantial amount of bone had been formed, with increasing amounts of bone trabeculae and a large number of osteoblasts. Conclusions Low-intensity ultrasound effectively improves the healing of irradiated bone.

Surgical hemostasis is critical in reducing the likelihood of excessive bleeding and blood transfusion. In treating some cases, commonly used hemostatic agent showed limited efficacy and prolonged degradation and clearance, causing an inhibition of bone healing. Starch absorbable polysaccharide (SAPH) is a novel hemostatic agent made from a plant starch, which can be completely absorbed and achieve better hemostatic effects than many commonly used hemostatic agents. However, whether SAPH can induce a promotion of bone healing remains unknown. In this study, we used a model of rabbit parietal bone defect and a mouse osteoblast cell line MC3T3-E1 to evaluate the effects of SAPH on bone healing. We found that SAPH significantly decreased bone healing scores, reduced defective area of parietal bone, and increased the areas of bone trabeculae and cavitas medullaris. In addition, SAPH enhanced MC3T3-E1 osteoblasts proliferation, up-regulated the expressions of alkaline phosphatase (ALP) and osteocalcin and increased the level of bone morphogenetic protein 2 (BMP-2) in MC3T3-E1 osteoblasts. These SAPH-induced benefits in MC3T3-E1 osteoblasts were significantly abolished by the application of BMP-2-siRNA. These findings suggested that SAPH enhances bone healing, promotes the proliferation, differentiation and maturation of osteoblast by up-regulating BMP-2 expression in osteoblastic cells.

Introduction Bone transport (BT) for segmentary bone defects is a well-known technique as it enables correction with new bone formation, which is similar to the previous bone. Despite the high number of experimental studies of distraction osteogenesis in bone lengthening, the types of ossification and histological changes that occur in the regenerate of the bone transport process remain controversial. Objective The aim of this study is to provide the complete evolution of tissues and the types of ossification in the regenerate during the different phases of bone formation after BT until the end of the remodelling period. Methods A histological study was performed using ten adult sheep that were submitted to BT. The types of ossification as well as the evolution of different tissues in the regenerate were determined using histomorphometry and inmunohistochemical studies. The evolution of trabeculae thickness, osteoblast and osteoclast densities, relationship between collagen types and changes in vascularization were also studied. Results Ossification was primarily intramembranous, with some focus of endochondral ossification in isolated animals. The cell counts showed a progression of cellular activity from the periphery to the centre, presenting the same progression as the growth of bone trabeculae, whose trabeculae thickness was quadrupled at the end of remodelling. Inmunohistochemical studies confirmed the prevalence of type I collagen and the ratio of the Type I/Type II collagen ratio was found to be 2.48. The percentages of the vascularized areas were proximally higher than distally in all animals, but distal zone obtained higher rates than the central region. Conclusions Bone transport regenerate exhibits a centripetal ossification model and a mixed pattern with predominance of intramembranous over endochondral ossification. The data obtained resemble partially to those found in models of bone lengthening applied to large animals. This study provides a detailed structural characterization of the newly formed tissue, which may help to explain the development of the regenerate of bone transport in humans. It will also serve for future mechanobiological models that may aid research on the effect of loading or distractor stiffness in clinical results.

Excessive mechanical loads on the temporomandibular joint (TMJ) can cause mandibular cartilage degradation and subchondral bone erosion, but the treatment of these conditions remains challenging. Salubrinal, which target eukaryotic translation initiation factor 2 alpha, has been shown to have multiple beneficial effects on skeletal tissue. Here, we examined the effect of a Salubrinal injection on the mandibular cartilage and subchondral bone of the TMJ under various compressive stresses. We conducted in vivo analyses in rat models using various compressive stresses (40 g and 80 g), and we observed time-related degeneration and pathological changes in the cartilage and subchondral bone of the TMJ at days 1, 3 and 7 through histological measurements, subcellular observation, and changes in proliferation and apoptosis. After the Salubrinal injection, the thickness of the cartilage recovered, and the pathological change was alleviated. In the Salubrinal/light (Sal/light) compressive stress group, the drug altered the proliferation and apoptosis of chondrocytes most significantly at day 1. In the Salubrinal/heavy (Sal/heavy) compressive stress group, the drug increased the proliferation of chondrocytes most significantly at day 1 and reduced the apoptosis of chondrocytes most significantly at day 7. Salubrinal also increased the area of the bone trabeculae and suppressed inflammatory responses and pathological change in the subchondral bone of the TMJ. Together, these results indicate that the administration of Salubrinal reduces apoptosis and strengthens the proliferation of chondrocyte to varying degrees at days 1, 3 and 7 under various compressive mechanical stresses, both of which contribute to the recovery of cartilage thickness and the alleviation of pathological change. Salubrinal also suppresses inflammatory responses and pathological change in the subchondral bone of the TMJ.

Sympathetic nerve system has been proved to have important regulative effects to bone mass. However, the role of sympathetic nerve system in distraction osteogenesis (DO) is unclear. Here we show that the sympathetic nerve system plays an important role in mandibular DO. Thirty male Sprague-Dawley rats were divided into 2 groups at random. Right-side mandibular DO was performed on the 15 rats in control group (group A). Bilateral transection of cervical sympathetic trunk and right-side mandibular DO were performed on the 15 rats in the experimental group (group B). After operation, quantitative general observations, micro-computed tomography bone morphology analysis, and hematoxylin-eosin staining osseous tissue on new osteotylus in distraction gap were performed at consolidation time of 1, 14, and 28 days. SPSS 12.0 software package was used for statistical analysis. At 1 and 14 days of consolidation time, there was more continuous bone formation in the experimental group than that of the control group as determined by gross observation. Bone formation parameters including bone mineral density, bone volume-total volume ratio, bone trabeculae number as determined by micro-CT, and histological study of the test group were significantly higher than those of the control group (P < 0.05). No significant difference was noted between the 2 groups on consolidation time of 28 days. Our study suggested that the sympathetic innervation loss could improve mandibular DO and new bone formation, and the sympathetic nerve system might negatively regulate the process of DO.

Injuries to growth plates may initiate the formation of reversible or irreversible bone-bridges, may leading to bone length discrepancy or axis deviation. As vascular invasion is essential for the formation of bone tissue, the aim of our study was to investigate the kinetic expression of Vascular Endothelial Growth Factor (VEGF) and its receptors R1 and R2 and the ingrowth of vessels in the formation of bone bridges in a rat physeal injury model. Quantitative Real-Time Polymerase Chain Reaction was performed for VEGF and its receptors. Samples from the proximal physis of the tibial bone were immunohistochemically evaluated for the expression of VEGF and its R1 and R2 receptors and Laminin. Morphologically, physeal bone bridge formation was validated by means of Magnetic Resonance Imaging. Kinetic expression of VEGF and VEGF-R1 mRNA documented a tendency towards an increase in expression on day 7. Histological analyses showed a hematoma containing bone debris on day 1 which was replaced with bony trabeculae by day 14, forming a bone bridge by day 28 which was preceded and accompanied by angiogenesis and consistent with MRI data. VEGF and VEGF-R2 was expressed on the debris within the hematoma and bone trabeculae from days 1 to 28. VEGF-R1 expression was only noted until day 14. The findings of our study suggest that physeal bone bridge formation is in part triggered by VEGF expression and associated with angiogenesis, which was shown to precede bone bridge formation and may be further stimulated through VEGF-positive bone debris.

Sternal synthesis after median sternotomy, a conventional access practice in thoracic and cardiac surgery, is at the basis of severe complications, often impairing the clinical outcome of surgical interventions. In this work, we propose the use of an acellular biomaterial scaffold, to be interposed across the fracture rime during closure operations, directly exposing the biomaterial to bone marrow, in order to expedite healing process. A rabbit model of median sternotomy was performed and an electrospun scaffold composed of a hydroxyapatite-loaded absorbable biopolymer (poly-L: -lactide), shaped into a fibrillar structure, was used. CT follow-up confirmed a complete healing in the scaffold-treated group 1 week before the control. Histological evaluation demonstrated presence of newly formed bone trabeculae among scaffold fibers showing a higher degree of maturity with respect to the control untreated group. The proposed approach is able to both guide a more rapid healing and modulate inflammatory response across the wound site, resulting in improved healing and tissue remodeling with respect to conventional closure technique.

For a better understanding of traumatic bone fractures, knowledge of the mechanical behavior of bone at high strain rates is required. Importantly, it needs to be clarified how quasistatic mechanical testing experiments relate to real bone fracture. This merits investigating the mechanical behavior of bone with an increase in strain rate. Various studies examined how cortical and trabecular bone behave at varying strain rates, but no one has yet looked at this question using individual trabeculae. In this study, three-point bending tests were carried out on bovine single trabeculae excised from a proximal femur to test the trabecular material's strain rate sensitivity. An experimental setup was designed, capable of measuring local strains at the surface of such small specimens, using digital image correlation. Microdamage was detected using the bone whitening effect. Samples were tested through two orders of magnitude, at strain rates varying between 0.01 and 3.39 s(-1). No linear relationship was observed between the strain rate and the Young's modulus (1.13-16.46 GPa), the amount of microdamage, the maximum tensile strain at failure (14.22-61.65%) and at microdamage initiation (1.95-12.29%). The results obtained in this study conflict with previous studies reporting various trends for macroscopic cortical and trabecular bone samples with an increase in strain rate. This discrepancy might be explained by the bone type, the small sample geometry, the limited range of strain rates tested here, the type of loading and the method of microdamage detection. Based on the results of this study, the strain rate can be ignored when modeling trabecular bone.

Whereas recent work has demonstrated the role of oxygen tension in the regulation of skeletal cell function and viability, the microenvironmental oxemic status of bone cells remains unknown. In this study, we have employed the Krogh cylinder model of oxygen diffusion to predict the oxygen distribution profiles in cortical and cancellous bone. Under the assumption of saturation-type Michaelis-Menten kinetics, our numerical modeling has indicated that, under steady-state conditions, there would be oxygen gradients across mature osteons and trabeculae. In Haversian bone, the calculated oxygen tension decrement ranges from 15 to 60%. For trabecular bone, a much shallower gradient is predicted. We note that, in Haversian bone, the gradient is largely dependent on osteocyte oxygen utilization and tissue oxygen diffusivity; in trabecular bone, the gradient is dependent on oxygen utilization by cells lining the bone surface. The Krogh model also predicts dramatic differences in oxygen availability during bone development. Thus, during osteon formation, the modeling equations predict a steep oxygen gradient at the initial stage of development, with the gradient becoming lesser as osteonal layers are added. In contrast, during trabeculum formation, the oxygen gradient is steepest when the diameter of the trabeculum is maximal. Based on these results, it is concluded that significant oxygen gradients exist within cortical and cancellous bone and that the oxygen tension may regulate the physical dimensions of both osteons and bone trabeculae.

The lactogenic hormone prolactin (PRL) directly regulates osteoblast functions in vitro and modulates bone remodeling in nulliparous rats, but its osteoregulatory roles in pregnant and lactating rats with physiological hyperprolactinemia remained unclear. Herein, bone changes were investigated in rats treated with bromocriptine (Bromo), an inhibitor of pituitary PRL release, or Bromo+PRL at different reproductive phases, from mid-pregnancy to late lactation. PRL receptors were strongly expressed in osteoblasts lining bone trabeculae, indicating bone as a target of PRL actions. By using dual energy X-ray absorptiometry, we found a significant increase in bone mineral density in the femora and vertebrae of pregnant rats. Such pregnancy-induced bone gain was, however, PRL independent and may have resulted from the increased cortical thickness. Bone trabeculae were modestly changed during pregnancy as evaluated by bone histomorphometry. On the other hand, lactating rats, especially in late lactation, showed massive bone loss in bone trabeculae but not in cortical shells. Further study in Bromo- and Bromo+PRL-treated rats suggested that PRL contributed to decreases in trabecular bone volume and number and increases in trabecular separation and eroded surface, as well as a paradoxical increase in bone formation rate in late lactation. Uncoupling of trabecular bone formation and resorption was evident in lactating rats, with the latter being predominant. In conclusion, pregnancy mainly induced cortical bone gain, whereas lactation led to trabecular bone loss in both long bones and vertebrae. Although PRL was not responsible for the pregnancy-induced bone gain, it was an important regulator of bone modeling during lactation.

β-Tricalcium phosphate (β-TCP) is a suitable biomaterial in oral and maxillofacial surgery since it can induce a rapid proliferation of woven bone. Granules, prepared by the polyurethane foam method, were implanted in critical size defects performed in the femoral condyles of New Zealand rabbits. Animals were studied after 8 and 28 days. Ketoprofen (a non-steroidal anti-inflammatory drug (NSAID)) was given for 8 and 28 days to evaluate its effects on the healing of the graft. Before euthanasia, the rabbits received an intravenous injection of fluorescent microbeads. Bones were analyzed by microcomputed tomography. β-TCP granules induced metaplastic bone trabeculae as early as 8 days post-surgery. At 28 days, the amount of bone was increased and the biomaterial volume decreased due to simultaneous macrophagic resorption. The amount of macrophages labeled with microbeads was less in the grafted area than in the vicinal intact marrow spaces. Ketoprofen had no effect on the amount of bone formed and on the number of labeled macrophages. The influence of small doses of NSAID, given in a short duration period, did not present deleterious effects on bone graft healing.

Scanning acoustic microscopy (SAM) provides high-resolution mapping of acoustic impedance related to tissue stiffness. This study investigates changes in tissue acoustic impedance resulting from mechanical loading in trabecular bone cores cultured in 3-D bioreactor. Trabecular bone cores were extracted from bovine sternum (n = 15) and ulna metaphysis (n = 15). From each bone, the samples were divided in three groups. The basal control (BC) group was fixed post-extraction, the control (C) and loaded (L) groups were maintained as viable in a controlled culture-loading cell over three weeks. Samples of L group underwent a dynamic compressive strain, whereas C samples were left free from loading. After three weeks, L and C samples were embedded in polymethylmethacrylate and all samples were explored with a 200-MHz SAM. For each specimen, the acoustic impedance distribution was obtained over flat and polished section of bone blocks prepared parallel to the loading axis. Our results showed that in basal controls, the acoustic impedance varied with bone anatomical location and was 15% higher in weight-bearing ulna compared with nonweight-bearing sternum. The comparison between loaded and nonloaded groups showed that sternum-only exhibited significant change in acoustic impedance (L vs. C sternum: +9%). This result suggests that when the applied load is comparable with the stress naturally experienced by a weight-bearing bone (ulna), the tissue material properties (manifested by acoustic impedance) remained unchanged. In conclusion, SAM is a potentially relevant tool for the assessment of subtle changes in intrinsic microelastic properties of bone induced by adaptive remodeling process in response to mechanical loading.