Evaluating Nuclear Factor NF-κB Activation following Bone Trauma: A Pilot Study in a Wistar Rats Model.

The present study investigated the moment of peak NF-kB activation and its dissipation in the cortical bone in the femur of Wistar rat stimulated by surgical trauma. Sixty-five Wistar rats were divided into 13 groups (n = 5 per group): eight experimental groups (expG 1-8) divided based on the euthanasia time point (zero, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h) and five sham control groups (conG 1-5) killed at zero, 1 h, 2 h, 4 h and 6 h, respectively. A 1.8-mm-diameter defect was generated 0.5 mm from the femur proximal joint using a round bur to induce the surgical trauma. Overall, the activation peak of NF-κB in the cortical bone was 6 h (expG5 group) independent of the evaluated position; this peak was significantly different compared to those in the other groups (p < 0.05). The surgical trauma resulted in a spread of immune markings throughout the cortical bone with an accentuation in the knee region. The present study provides the first evidence that the NF-κB activation peak was established after 6 hours in the cortical bone of Wistar rats. The signs from a surgical trauma can span the entire cortical bone and are not limited to the damaged region.

Cytocompatibility of porous biphasic calcium phosphate granules with human mesenchymal cells by a multiparametric assay.

This work aims to evaluate the cytocompatibility of injectable and moldable restorative biomaterials based on granules of dense or porous biphasic calcium phosphates (BCPs) with human primary mesenchymal cells, in order to validate them as tools for stem cell-induced bone regeneration. Porous hydroxyapatite (HA) and HA/beta-tricalcium phosphate (ß-TCP) (60:40) granules were obtained by the addition of wax spheres and pressing at 20 MPa, while dense materials were compacted by pressing at 100 MPa, followed by thermal treatment (1100°C), grinding, and sieving. Extracts were prepared by 24-h incubation of granules on culture media, with subsequent exposition of human primary mesenchymal cells. Three different cell viability parameters were evaluated on the same samples. Scanning electron microscopy analysis of the granules revealed distinct dense and porous surfaces. After cell exposition to extracts, no significant differences on mitochondrial activity (2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) or cell density (Crystal Violet Dye Elution) were observed among groups. However, Neutral Red assay revealed that dense materials extracts induced lower levels of total viable cells to porous HA/ß-TCP (P < 0.01). Calcium ion content was also significantly lower on the extracts of dense samples. Porogenic treatments on BCP composites do not affect cytocompatibility, as measured by three different parameters, indicating that these ceramics are well suited for further studies on future bioengineering applications.

Identification of the nuclear factor kappa-beta (NF-kB) in cortical of mice Wistar using Technovit 7200 VCR®.

OBJECTIVE: this study aimed to develop a nondecalcified bone sample processing technique enabling immunohistochemical labeling of proteins by kappa-beta nuclear factor (NF-kB) utilizing the Technovit 7200 VCR® in adult male Wistar rats. STUDY METHOD: A 1.8 mm diameter defect was performed 0.5 mm from the femur proximal joint by means of a round bur. Experimental groups were divided according to fixing solution prior to histologic processing: Group 1--ethanol 70%; Group 2--10% buffered formalin; and Group 3--Glycerol diluted in 70% ethanol at a 70/30 ratio + 10% buffered formalin. The post-surgical periods ranged from 01 to 24 hours. Control groups included a non-surgical procedure group (NSPG) and surgical procedures where bone exposure was performed (SPBE) without drilling. Prostate carcinoma was the positive control (PC) and samples subjected to incomplete immunohistochemistry protocol were the negative control (NC). Following euthanization, all samples were kept at 4°C for 7 days, and were dehydrated in a series of alcohols at -20°C. The polymer embedding procedure was performed at ethanol/polymer ratios of 70%-30%, 50%-50%, 30%-70%, 100%, and 100% for 72 hours at -20°C. Polymerization followed the manufacturer's recommendation. The samples were grounded and polished to 10-15 m thickness, and were deacrylated. The sections were rehydrated and were submitted to the primary polyclonal antibody anti-NF-kB on a 1:75 dilution for 12 hours at room temperature. RESULTS: Microscopy showed that the Group 2 presented positive reaction to NF-kB, diffuse reactions for NSPG and SPBE, and no reaction for the NC group. CONCLUSION: The results obtained support the feasibility of the developed immunohistochemistry technique.

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

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

Effect of meloxicam and diclofenac sodium on peri-implant bone healing in rats.

BACKGROUND: This study evaluated the effects of diclofenac sodium and meloxicam on peri-implant bone healing. METHODS: Thirty male rats were divided into three groups: the control group (CG) received no drug; the diclofenac sodium group (DSG) received 1.07 mg/kg twice a day for 5 days; and the meloxicam group (MG) received 0.2 mg/kg daily for 5 days. A screw-shaped titanium implant was placed in the tibia. Fluorochromes, oxytetracycline (OxT), calcein (CA), and alizarin (AL), were injected at 7, 14, and 21 days, respectively, after implantation, and the animals were sacrificed 28 days after implant placement. The percentages of OxT-, CA-, and AL-labeled bone as well as the percentages of bone-to-implant contact (BIC), cortical bone area (CBA), and trabecular bone area (TBA) within the implant threads were evaluated. RESULTS: Bone healing was delayed in the DSG during the first 14 days after implant placement (OxT-labeled bone: DSG: 5.3% +/- 7.3% versus CG: 13.2% +/- 9.8%, P = 0.002, and versus MG:14.4% +/- 13.1%, P = 0.05). The percentages of BIC (DSG: 49.6% +/- 21.9%; MG: 67.1% +/- 22.8%; and CG: 68.1% +/- 22.8%) and CBA (DSG: 63.7% +/- 21.2%; MG: 82.7% +/- 12.4%; CG: 84.9% +/- 10.6%) were lower in the DSG compared to the MG and CG (P <0.001). The percentage of TBA was significantly greater in the DSG compared to the MG and CG (DSG: 36.3% +/- 21.2% versus MG: 17.3% +/- 12.7% and versus CG: 15.1% +/- 10.6%; P <0.001). CONCLUSION: Diclofenac sodium seemed to delay peri-implant bone healing and to decrease BIC, whereas meloxicam had no negative effect on peri-implant bone healing.

Tissue response around silicon nitride implants in rabbits.

The chemical and dimensional stability associated with suitable fracture toughness and propitious tribological characteristics make silicon nitride-based ceramics potential candidates for biomedical applications, mainly as orthopedic implants. Considering this combination of properties, silicon nitride components were investigated in relation to their biocompatibility. For this study, two cylindrical implants were installed in each tibia of five rabbits and were kept in the animals for 8 weeks. During the healing time, tissue tracers were administrated in the animals so as to evaluate the bone growth around the implants. Eight weeks after the surgery, the animals were euthanized and histological analyses were performed. No adverse reactions were observed close to the implant. The osteogenesis process occurred during the entire period defined by the tracers. However, this process occurred more intensely 4 weeks after the surgery. In addition, the histological analyses showed that bone growth occurred preferentially in the cortical areas. Different kinds of tissue were identified on the implant surface, characterized by lamellar bone tissue containing osteocytes and osteons, by a noncalcified matrix containing osteoblasts, or by the presence of collagen III, which may change to collagen I or remain as a fibrous tissue. The results demonstrated that silicon nitride obtained according to the procedure proposed in this research is a biocompatible material.

Hydroxyapatite grafting promotes new bone formation and osseointegration of smooth titanium implants.

Titanium is the ideal metal for intra-osseous dental implants. It permits the natural formation of an oxide layer on its surface and thereby it prevents the release of potentially toxic molecules. New formation of bone around implants, partially placed into the bone marrow cavity, is a gradual process that runs from the endosteum to the surface of the implant. Deposition of hydroxyapatite crystals on collagen type I fibrils is initiated by acidic proteins and leads to bone mineralization. This study analyzed the effects of hydroxyapatite upon peri-implant bone formation after insertion of smooth titanium implants. Screw-shaped smooth titanium implants of 3.75 mm thickness and 8.5 mm length were inserted into the metaphysis of rabbit tibia, either together with bovine hydroxyapatite into the right tibia or in controls without hydroxyapatite into the left tibia. Polyfluorochrome tracers (alizarin complex, calcein, tetracycline) were injected subcutaneously at different time intervals after implantation to evaluate the time frame of bone new formation over a period of 8 weeks. All samples were processed for histology and analyzed by fluorescence and polarizing microscopy. Our results showed a higher quantity of mature type I collagen fibers around implants and an acceleration of bone formation in the presence of hydroxyapatite. Mainly immature organic matrix was formed at the surface of implants in controls. The presence of hydroxyapatite seems to promote the maturation of collagen fibers surrounding the titanium implants and to support osteoconduction. Moreover, new formation of bone was faster in all samples where implants were inserted together with hydroxyapatite.

Titanium implant osseointegration with calcium pyrophosphate in rabbits.

The objective of this study was to characterize calcium pyrophosphate material, evaluate its in vitro cytotoxicity, and assess its ability to induce bone formation. X-ray diffraction (XRD) was used to determine crystallinity and phases present in material. Serial dilutions of extracts, from 10-day dissolution tests in modified Eagle's medium, were exposed for 24 h to mouse fibroblasts and cytotoxicity assessed via viable staining. In vivo performance was determined by placing Ti screws with and without calcium pyrophosphate agglutinated with marrow adipose tissue in the tibiae of eight rabbits. New bone formation around test and control implants was evaluated histomorphometrically by using three fluorochrome labels: alizarin, calcein, and tetracycline. After 8 postoperative weeks, the animals were killed and specimens were retrieved and processed for fluorescence and light microscopic analysis. Calcium pyrophosphate showed no cytotoxicity and the XRD showed that the main phase of the analyzed sample corresponded to beta-calcium pyrophosphate. The largest fluorochrome labeling area occurred during the fourth and fifth postoperative weeks, in both control and experimental groups. Histologically, the bone neoformation occurred in regions where the calcium pyrophosphate was resorbed. The morphometric analysis showed implants placed with calcium pyrophosphate resulted in smaller polyfluorochrome labeling area (p < 0.05).

Effects of alcohol consumption on osseointegration of titanium implants in rabbits.

Alcohol consumption affects bone metabolism by impairing osteoblast proliferation and by increasing osteoclastic activity. The purpose of this study was to evaluate bone formation in alcohol-fed rabbits following the insertion of dental titanium implants. Animals were fed with 20% ethanol sugarcane brandy pre- and postoperatively (group 1), preoperatively only (group 2), and with water as control (group 3). During the postoperative period, rabbits received doses of polyfluorochrome labels (i.e., alizarin, calcein, and tetracycline). Rabbits were killed 8 weeks after the implant insertion. The polyfluorochrome-labeled bone areas in rabbits with alcohol consumption in pre- and postoperative (group 1) and preoperatively only (group 2) were significantly less (P <.05) than in the control group (group 3). The percentage of direct bone-to-implant contact was significantly less in pre- and postoperative (49.5%) and preoperative-only (49.2%) groups than in the control group (64.7%) (P <.05). Alcoholic rabbits demonstrated significantly less bone density and direct bone-to-implant contact.

Bone healing after the inferior alveolar nerve lateralization: a histologic study in rabbits (Oryctolagus cuniculus).

PURPOSE: The purpose of this article is to evaluate the bone healing around 2 different dental implant surfaces after the lateralization surgery of the inferior alveolar nerve (IAN) during an 8-week healing period in rabbits, and to check if there is any interaction between the implants and the nerve. MATERIALS AND METHODS: The IAN lateralization was performed in an experimental rabbit model. Eight adult female rabbits (Oryctolagus cuniculus) underwent the surgical procedure, and 1 implant was placed on each side of the mandible while the nerve was lateralized. On both sides, the nerve was repositioned directly in contact with the implant surface. With the intention of obtaining comparative results, smooth titanium implants were installed on the right side, and blasted Al2O3 ones were placed on the left. During the healing period, bone tracers were administered subcutaneously to check different periods of bone ingrowth. RESULTS: Histologic section, regardless of the studied surface, showed bone remodeling around the nerve, without contact between the nerve and the implanted surface. The bone blocks containing implants were histomorphometrically examined through computerized analysis, and the results obtained showed that the bone remodeling around the nerve occurred during the first weeks of healing. Through analysis of variance, the blasted Al2O3 implants showed at least 2.5-fold greater bone neoformation compared with the smooth titanium implants. CONCLUSIONS: Our results showed that there was no significant difference in the healing process concerning the nerve between the 2 studied surfaces. No interaction was detected between the nerve and the implants.

Histological analysis of bone/heteroplastic implant interfaces in dog tibia.

This work presents histological analysis of interfaces between bone and heteroplastic implants in dog tibias. The study was performed in four tibias (of four mongrel dogs) into which cylindrical implants were inserted. One ceramic (titania) implant and three grit-blasted titanium implants (with sandblasted and acid-corroded surfaces) were chosen for histological analysis of the implant surface/bone tissue interface. The implants remained in the tibias for eight months and none were loaded during this period. The animals were subsequently sacrificed and the samples were processed for analysis. Light microscope analysis revealed a large amount of osteoid tissue and proximity of osteoblasts and osteocytes to the implant surfaces. In addition, little or no fibrous tissue was observed between the bone and implant surfaces. The titanium implants presented better osseointegration than did the ceramic implant.

Bone regeneration in rabbit sinus lifting associated with bovine BMP.

Autogenous bone is considered the optimal grafting material for sinus lifting, although its harvesting causes great patient discomfort. Various approaches have been taken in order to obtain sinus lifting with preexisting tissue. However, because of the unsuitability of such tissue, additional materials have been required. Alternatively, biomaterials from humans or other animals are used. In this study, the efficacy of using morphogenetic bovine bone protein (BMPb) to augment the maxillary sinus floor was examined. Four grafting materials were employed: lyophilized bovine bone powder, absorbable collagen flakes, natural hydroxylapatite, and synthetic hydroxylapatite. Two groups of rabbits were studied. In one group, graft material only was used. In the other, graft material was combined with 0.5 mg BMPb. During 8 weeks of observation, polyfluorochrome tracers were injected in subcutaneous tissue to evaluate new bone- deposition periods. Following sacrifice, the samples were examined under fluorescent and light microscopes. Results indicated 33.34% more newly formed bone in BMPb animals than in controls. Graft-material resorption increased, but natural HA showed no significant alterations. The results show that the use of BMPb, although providing osteoinduction, might not promote sufficient bone formation. Nonetheless, this material could provide an alternative to autogenous grafts, thereby avoiding patient discomfort.

The effects of bovine BMP associated to HA in maxillary sinus lifting in rabbits.

This study examines the efficacy of using bone morphogenetic protein of bovine origin associated with other biomaterials in maxillary sinus floor augmentation procedures in rabbits (Oryctolagus cuniculus). Various approaches have been attempted to obtain sinus lifting with pre-existing tissue, but all of them have been considered inadequate, because such tissue offers very low bio-quality, requiring additional materials to stimulate bone neo-formation. For this purpose, autogenous bone is considered to be the best grafting material, but harvesting it results in great discomfort for the patient. Biomaterials from human beings or other animals are used as a substitute. In this study, four different grafting materials were used: lyophilized bovine bone powder, absorbable collagen flakes, natural hydroxyapatite (nHA) and synthetic hydroxyapatite (sHA), in nine animals divided into two groups: (A) control group (left sinus)--using just graft material, and (B) BMP group (right sinus)--using graft material with 0.5 mg bovine bone morphogenetic protein (BMPb). The observation periods were of 8 and 12 weeks duration and sequential bone neo-formation polyfluorochrome tracers (alizarin complex, calcein, and tetracycline) were subcutaneously injected, to evaluate the periods of new bone deposition. After the animals were sacrificed, the material was obtained and examined under a fluorescent microscope and also activated by UV light and the conclusion reached that the newly formed bone increase was of 33.34% when compared to the control group at 8 weeks At 12 weeks, the bone deposition in the "BMP" group was not significant while in the "control" group there was continuous growth. This difference showed that the BMP stimulated bone formation during the early periods of healing (8 weeks), although it altered the normal cycle of bone deposition over the longer period (12 weeks). The graft material showed increasing reabsorption, but the natural HA did not show significant alterations. The results of this new animal model indicated that the BMPb used, although facilitating osteoinduction, might not be sufficient to promote qualitative and quantitative bone neo-formation, which could guarantee better prognoses. The BMPb material studied may possibly become an alternative to autogenous grafts causing less discomfort for the patient.