Obtention and characterization of collagen and chitosan based cements for bone regerneration. Part 1: extraction and characterization of collagen

Several cements are used as biomaterials. Biopolymers such as chitosan and collagen exhibit excellent biocompatibility and can be used in the remodeling of bone tissue. The cement must have high mechanical strength and compatibility with original tissue. In this context, the objective of this study was to extract, characterize and cross-link collagen from bovine tendon, forlater associate it with chitosan and calcium phosphate to obtain cements for bone regeneration. Glutaraldehyde was used as cross-linker in 0.1, 0.5, 1.0 and 10% concentration. Infrared analysis confirmed the presence of functional groups characteristic of collagen, whereas the capacity of water absorption decreased with the increasing of cross-linking degree. Denaturation temperatures of collagen samples were obtained by Differential Scanning Calorimetry and Scanning Electron Microscopy showed the fiber structure characteristics of collagen, which were more organized for high degree of cross-linking samples.

Bone stress and strain after use of a miniplate for molar protraction and uprighting: a 3-dimensional finite element analysis

The aim of this study was to use the finite element method to evaluate the distribution of stresses and strains on the local bone tissue adjacent to the miniplate used for anchorage of orthodontic forces. Methods: A 3-dimensional model composed of a hemimandible and teeth was constructed using dental computed tomographic images, in which we assembled a miniplate with fixation screws. The uprighting and mesial movements of the mandibular second molar that was anchored with the miniplate were simulated. The miniplate was loaded with horizontal forces of 2, 5, and 15 N. A moment of 11.77 N.mm was also applied. The stress and strain distributions were analyzed, and their correlations with the bone remodeling criteria and miniplate stability were assessed. Results: When orthodontic loads were applied, peak bone strain remained within the range of bone homeostasis (100-1500 mu m strain) with a balance between bone formation and resorption. The maximum deformation was found to be 1035 mu m strain with a force of 5 N. At a force of 15 N, bone resorption was observed in the region of the screws. Conclusions: We observed more stress concentration around the screws than in the cancellous bone. The levels of stress and strain increased when the force was increased but remained within physiologic levels. The anchorage system of miniplate and screws could withstand the orthodontic forces, which did not affect the stability of the miniplate.

Repair of Bone Defects Filled with New Calcium Aluminate Cement (EndoBinder)

The aim of this study was to evaluate and compare the repair of bone defects filled with calcium aluminate cement (EndoBinder), mineral trioxide aggregate (MTA), and calcium hydroxide. Methods After mixing, the cements were inserted into bone defects (3.3 mm) mechanically created in the right and left tibias of 30 rats (Rattus norvegicus, Wistar). In the control group, the bone defects were filled with blood clot of the animal itself. After time intervals of 7, 30, and 90 days had elapsed, bone tissue biopsies (n = 5) were surgically obtained and submitted to laboratory processing. The response of bone tissue in contact with the materials was microscopically analyzed. The percentage of neoformed bone tissue in the defect was determined by means of planimetry counting points superimposed on the histologic image. Results Significant increase in the percentage of neoformed bone tissue was observed throughout the experimental periods in all groups (P < .05). For the cements EndoBinder and MTA (30 and 90 days), these percentage values were statistically higher than those of the control group (P < .05); however, they were similar to those of calcium hydroxide (P > .05). Conclusions EndoBinder and MTA allowed complete repair of bone defects created in rat tibias.

A new heterologous fibrin sealant as scaffold to recombinant human bone morphogenetic protein-2 (rhBMP-2) and natural latex proteins for the repair of tibial bone defects

Tissue engineering has special interest in bone tissue aiming at future medical applications Studies have focused on recombinant human bone morphogenetic protein-2 (rhBMP-2) and natural latex proteins due to the osteogenic properties of rhBMP-2 and the angiogenic characteristic of fraction 1 protein (P-1) extracted from the rubber tree Hevea brasiliensis. Furthermore, heterologous fibrin sealant (FS) has been shown as a promising alternative in regenerative therapies. The aim of this study was to evaluate these substances for the repair of bone defects in rats. A bone defect measuring 3 mm in diameter was created in the proximal metaphysis of the left tibia of 60 rats and was implanted with rhBMP-2 or P-1 in combination with a new heterologous FS derived from snake venom. The animals were divided into six groups: control (unfilled bone defect), rhBMP-2 (defect filled with 5 mu g rhBMP-2), P-1 (defect filled with 5 mu g P-1), FS (defect filled with 8 mu g FS), FS/rhBMP-2 (defect filled with 8 mu g FS and 5 mu g rhBMP-2), FS/P-1 (defect filled with 8 mu g FS and 5 mu g P-1). The animals were sacrificed 2 and 6 weeks after surgery. The newly formed bone projected from the margins of the original bone and exhibited trabecular morphology and a disorganized arrangement of osteocyte lacunae. Immunohistochemical analysis showed intense expression of osteocalcin in all groups. Histometric analysis revealed a significant difference in all groups after 2 weeks (p < 0.05), except for the rhBMP-2 and FS/rhBMP-2 groups (p > 0.05). A statistically significant difference (p < 0.05) was observed in all groups after 6 weeks in relation to the volume of newly formed bone in the surgical area. In conclusion, the new heterologous fibrin sealant was found to be biocompatible and the combination with rhBMP-2 showed the highest osteogenic and osteoconductive capacity for bone healing. These findings suggest a promising application of this combination in the regeneration surgery.

Is bone transplantation the gold standard for repair of alveolar bone defects?

New strategies to fulfill craniofacial bone defects have gained attention in recent years due to the morbidity of autologous bone graft harvesting. We aimed to evaluate the in vivo efficacy of bone tissue engineering strategy using mesenchymal stem cells associated with two matrices (bovine bone mineral and α-tricalcium phosphate), compared to an autologous bone transfer. A total of 28 adult, male, non-immunosuppressed Wistar rats underwent a critical-sized osseous defect of 5 mm diameter in the alveolar region. Animals were divided into five groups. Group 1 (n = 7) defects were repaired with autogenous bone grafts; Group 2 (n = 5) defects were repaired with bovine bone mineral free of cells; Group 3 (n = 5) defects were repaired with bovine bone mineral loaded with mesenchymal stem cells; Group 4 (n = 5) defects were repaired with α-tricalcium phosphate free of cells; and Group 5 (n = 6) defects were repaired with α-tricalcium phosphate loaded with mesenchymal stem cells. Groups 2-5 were compared to Group 1, the reference group. Healing response was evaluated by histomorphometry and computerized tomography. Histomorphometrically, Group 1 showed 60.27% ± 16.13% of bone in the defect. Groups 2 and 3 showed 23.02% ± 8.6% (p = 0.01) and 38.35% ± 19.59% (p = 0.06) of bone in the defect, respectively. Groups 4 and 5 showed 51.48% ± 11.7% (p = 0.30) and 61.80% ± 2.14% (p = 0.88) of bone in the defect, respectively. Animals whose bone defects were repaired with α-tricalcium phosphate and mesenchymal stem cells presented the highest bone volume filling the defects; both were not statistically different from autogenous bone.

Differential Expression of ADAM23, CDKN2A (P16), MMP14 and VIM Associated with Giant Cell Tumor of Bone

Though benign, giant cell tumor of bone (GCTB) can become aggressive and can exhibit a high mitotic rate, necrosis and rarely vascular invasion and metastasis. GCTB has unique histologic characteristics, a high rate of multinucleated cells, a variable and unpredictable growth potential and uncertain biological behavior. In this study, we sought to identify genes differentially expressed in GCTB, thus building a molecular profile of this tumor. We performed quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry and analyses of methylation to identify genes that are putatively associated with GCTB. The expression of the ADAM23 and CDKN2A genes was decreased in GCTB samples compared to normal bone tissue, measured by qPCR. Additionally, a high hypermethylation frequency of the promoter regions of ADAM23 and CDKN2A in GCTB was observed. The expression of the MAP2K3, MMP14, TIMP2 and VIM genes was significantly higher in GCTB than in normal bone tissue, a fact that was confirmed by qPCR and immunohistochemistry. The set of genes identified here furthers our understanding of the molecular basis of GCTB.

Phenotypic, genetic and environmental parameters for traits related to femur bone integrity and body weight at 42 days of age in a broiler population

Intense selection among broilers, especially for performance and carcass traits, currently favors locomotion problems and bone resistance. Conducting studies relating to development and growth of bone tissue in broilers is necessary to minimize losses. Thus, genetic parameters were estimated for a broiler population's phenotypic traits such as BW at 42 d of age (BW42), chilled femur weight (CFW) and its yield (CFY), and femur measurements: calcium, DM, magnesium, phosphorus, and zinc content; breaking strength; rigidity; length; and thickness. Variance components were estimated through multitrait analyses using the restricted maximum likelihood method. The model included a fixed group effect (sex and hatch) and additive and residual genetic random effects. The heritability estimates we obtained ranged from 0.10 ± 0.05 to 0.50 ± 0.08 for chilled femur yield and BW42, respectively, and indicated that the traits can respond to the selection process, except for CFY, which presented low-magnitude heritability coefficients. Genetic correlation estimates between breaking strength, rigidity, and traits related to mineral content indicated that selection that aims to improve the breaking strength resistance of the femur is highly correlated with mineral content. Given the genetic correlation estimates between BW42 and minerals, it is suggested that in this population, selection for BW42 can be performed with greater intensity without affecting femoral integrity.

The Fresh Frozen Bone Allograft (FFBA) to reconstruc the atrophic maxilla: evaluation of the clinical and histological viability at six and nine months in the maxillary sinus

This work aimed to analyze clinically and histologically the allogen bone graft behavior at 6 and 9 months. A leukoderm, female, 55 years old patient sought dental care for oral rehabilitation with dental implants and implant supported fixed prosthesis in the maxilla. Bilateral sinus lifting procedure were performed in a same patient, the analysis were made after 6 and 9 months, respectively. At 6 months, there was lack of a better bone remodeling in the region, associated to the presence of fibrous connective tissue within the collected tissue, showing us that this is not the best stage of tissue maturation to place dental implants. The 9 months period showed an improvement, with a more organized bone tissue surrounding particles of homogenous bone, what possibly had increased implant stability at the time of surgery. There is no doubt about the increase of clinical applications of FFBA, but there are no studies available regarding the standardization of time to load implants inserted in allografts. So papers with long-term monitoring are necessary to eliminate questions that still remain to be answered.

Reconstruction of the anterior wall of the frontal sinus using bovine bone matrix: histological study with monkeys

The purpose of this study was to evaluate the repair process in the reconstruction of the anterior wall of the frontal sinus of monkeys with bovine bone matrix. Four adult Cebus apella monkeys underwent an ostectomy of the anterior wall of the frontal sinus. The frontal sinus mucosa and the nasofrontal duct were not manipulated. Reconstruction occurred with implants of bovine bone matrix laminae measuring 2.0 x 2.5 cm and 0.4 mm thick, stabilized under pressure in the lateral wall of the frontal sinus. The monkeys were sacrificed over a period of 150 days and routine laboratory procedures were followed for hematoxylin-eosin staining and histologic evaluation of the specimens. Neoformed bone tissue was observed in contact with the frontal sinus mucosa and the bovine bone matrix. The frontal sinus mucosa remained whole without fibrous tissue or cystic formations. There was no occurrence of cellularization as well as revascularization of the bovine bone matrix, though it has permitted bone conduction on this surface. It was possible to conclude that the demineralized bovine bone matrix was biotolerable, being incorporated into the bone without the presence of inflammatory cells with characteristics of inertness and antigenicity and behaved as an osteoconductive material.

Bone defect repair on the alveolar wall of the maxillary sinus using collagen membranes and temporal fascia: an experimental study in monkeys

Few studies has been done using guided bone regeneration in maxillary sinus defects. AIM: To assess the bone repair process in surgical defects on the alveolar wall of the monkey maxillary sinus, which communicates with the sinus cavity, by using collagen membranes: Gen-derm - Genius Baumer, Pro-tape - Proline and autologous temporal fascia. MATERIALS AND METHODS: In this prospective and experimental study, orosinusal communications were performed in four tufted capuchin monkeys (Cebus apella) and histologic analysis was carried out 180 days after. RESULTS: In the defects without a cover (control), bone proliferation predominated in two animals and fibrous connective tissue predominated in the other two. In defects repaired with a temporal fascia flap, fibrous connective tissue predominated in three animals and bone proliferation predominated in one. In the defects repaired with Gen-derm or Pro-tape collagen membranes there was complete bone proliferation in three animals and fibrous connective tissue in one. CONCLUSIONS: Surgical defect can be repaired with both bone tissue and fibrous connective tissue in all study groups; collagen membranes was more beneficial in the bone repair process than temporal fascia or absence of a barrier.

Immunohistochemical, tomographic, and histological study on onlay bone graft remodeling. Part III: allografts

Objective In the last decades aroused the interest for bone tissue bank as an alternative to autogenous grafting, avoiding donor sites morbidity, surgical time, and costs reduction. The purpose of the study was to compare allografts (ALg) with autografts (AUg) using histology, immunochemistry, and tomographic analysis. Material and methods Fifty-six New Zealand White rabbits were submitted to surgical procedures. Twenty animals were donors and 36 were actually submitted to onlay grafting with ALg (experimental group) and AUg (control group) randomly placed bilaterally in the mandible. Six animals of each group were sacrificed at 3, 5, 7, 10, 20, and 60 postoperative days. Immunolabeling was accomplished with osteoprotegerin (OPG); receptor activator of nuclear factor-k ligand (RANKL); alkaline phosphatase (ALP); osteopontin (OPN); vascular endothelial growth factor (VEGF); tartrate-resistant acid phosphatase (TRAP); collagen type I (COL I); and osteocalcin (OC). Density and volume of the grafts was evaluated on tomography obtained at the surgery and sacrifice. Results The ALg and AUg exhibited similar patterns of density and volume throughout the experiments. The intra-group data showed statistical differences at days 7 and 60 in comparison with other time points (P = 0.001), in both groups. A slight graft expansion from fixation until day 20 (P = 0.532) was observed in the AUg group and then resorbed significantly at the day 60 (P = 0.015). ALg volume remained stable until day 7 and decreased at day 10 (P = 0.045). The light microscopy analysis showed more efficient incorporation of AUg onto the recipient bed if compared with the ALg group. The immunohistochemical labeling picked: at days 10 and 20 with OPG in the AUg group and at day 7 with TRAP in the ALg group (P = 0.001 and P = 0.002, respectively). Conclusions ALg and AUg were not differing in patterns of volume and density during entire experiment. Histological data exhibit more efficient AUg incorporation into recipient bed compared with the ALg group. Immunohistochemistry outcomes demonstrated similar pattern for both ALg and AUg groups, except for an increasing resorption activity in the ALg group mediated by TRAP and in the AUg group by higher OPG labeling. However, this latter observation does not seem to influence clinical outcomes.

Preparation and evaluation of the electrospun chitosan/PEO fibers for potential applications in cartilage tissue engineering

Fibrous materials have morphological similarities to natural cartilage extracellular matrix and have been considered as candidate for bone tissue engineering scaffolds. In this study, we have evaluated a novel electrospun chitosan mat composed of oriented sub-micron fibers for its tensile property and biocompatibility with chondrocytes (cell attachment, proliferation and viability). Scanning electronic microscope images showed the fibers in the electrospun chitosan mats were indeed aligned and there was a slight cross-linking between the parent fibers. The electrospun mats have significantly higher elastic modulus (2.25 MPa) than the cast films (1.19 MPa). Viability of cells on the electrospun mat was 69% of the cells on tissue-culture polystyrene (TCP control) after three days in culture, which was slightly higher than that on the cast films (63% of the TCP control). Cells on the electrospun mat grew slowly the first week but the growth rate increased after that. By day 10, cell number on the electrospun mat was almost 82% that of TCP control, which was higher than that of cast films (56% of TCP). The electrospun chitosan mats have a higher Young’s modulus (P <0.01) than cast films and provide good chondrocyte biocompatibility. The electrospun chitosan mats, thus, have the potential to be further processed into three-dimensional scaffolds for cartilage tissue repair.

Bovine Bone Matrix Action Associated With Morphogenetic Protein in Bone Defects in Rats Submitted to Alcoholism

Extended excessive alcohol use causes changes in bone tissue, thus affecting osteogenesis. The objective of this study was to evaluate if demineralized bone matrix (Gen-ox (R)) associated with bone morphogenetic protein (Gen-pro (R)) changes bone neoformation in rats submitted to experimental alcoholism. Forty male rats (Rattus norvegicus) were separated into 2 groups of 20 animals each: Group E1, which received ethyl alcohol at 25% and had the surgical cavity filled in only with blood clot; and Group E2. which received ethyl alcohol at 25% and had the surgical cavity filled in with demineralized bovine cortical bone associated with bone morphogenetic protein. The animals were submitted to a three-week period of gradual adaptation to alcohol, and then continued receiving alcohol at 25% for 90 days, when the surgical cavity was made. After the surgery, the animals continued consuming alcohol until reaching the sacrifice periods of 10, 20, 40, and 60 days, when the tibias were removed for histological processing. Results showed that surgical cavity repair and bone marrow reorganization occurred faster in Group E1 than in Group E2. At the end of the experiment, it was observed that animals in Group E2 had thick bony trabeculae surrounding the implanted material particles and a small area of connective tissue in the surface region. In conclusion, the implanted material did not accelerate bone neoformation, rather it served as a structure for osteogenesis.

Biological evaluation of the bone healing process after application of two potentially osteogenic proteins: an animal experimental model

doi: 10.1111/j.1741-2358.2011.00526.x Biological evaluation of the bone healing process after application of two potentially osteogenic proteins: an animal experimental model Objective: The aim of this work was to analyse qualitatively and quantitatively the newly formed bone after insertion of rhBMP-2 and protein extracted from Hevea brasiliensis (P-1), associated or not with a carrier in critical bone defects created in Wistar rat calvarial bone, using histological and histomorphometrical analyses. Materials and methods: Eighty-four male Wistar rats were used, divided into two groups, according to the period of time until the sacrifice (2 and 6 weeks). Each one of these groups was subdivided into six groups with seven animals each, according to the treatments: (1) 5 mu g of pure rhBMP-2, (2) 5 mu g of rhBMP-2/monoolein gel, (3) pure monoolein gel, (4) 5 mu g of pure P-1, (5) 5 mu g of P-1/monoolein gel and (6) critical bone defect controls. The animals were euthanised and the calvarial bone tissue removed for histological and histomorphometrical analyses. Result and conclusion: The results showed an improvement in the bone healing process using the rhBMP-2 protein, associated or not with a material carrier in relation to the other groups, and this process demonstrated to be time dependent.

FINITE ELEMENT ANALYSIS OF MAXILLARY BONE STRESS CAUSED BY ARAMANY CLASS IV OBTURATOR PROSTHESES

Statement of problem. The retention of an Aramany Class IV removable partial dental prosthesis can be compromised by a lack of support. The biomechanics of this obturator prosthesis result in an unusual stress distribution on the residual maxillary bone. Purpose. This study evaluated the biomechanics of an Aramany Class IV obturator prosthesis with finite element analysis and a digital 3-dimensional (3-D) model developed from a computed tomography scan; bone stress was evaluated according to the load placed on the prosthesis. Material and methods. A 3-D model of an Aramany Class IV maxillary resection and prosthesis was constructed. This model was used to develop a finite element mesh. A 120 N load was applied to the occlusal and incisal platforms corresponding to the prosthetic teeth. Qualitative analysis was based on the scale of maximum principal stress; values obtained through quantitative analysis were expressed in MPa. Results. Under posterior load, tensile and compressive stresses were observed; the tensile stress was greater than the compressive stress, regardless of the bone region, and the greatest compressive stress was observed on the anterior palate near the midline. Under an anterior load, tensile stress was observed in all of the evaluated bone regions; the tensile stress was greater than the compressive stress, regardless of the bone region. Conclusions. The Aramany Class IV obturator prosthesis tended to rotate toward the surgical resection when subjected to posterior or anterior loads. The amount of tensile and compressive stress caused by the Aramany Class IV obturator prosthesis did not exceed the physiological limits of the maxillary bone tissue. (J Prosthet Dent 2012;107:336-342)