Rat Parietal Bone Research Articles

Porous 3D implants of degradable poly-3-hydroxybutyrate used to enhance regeneration of rat cranial defect.

The study describes preparation and testing of porous 3D implants of natural degradable polymer of 3-hydroxybutyric acid P(3HB) for regeneration of bone tissue defects. The ability of the P(3HB) implants to favor attachment and facilitate proliferation and directed differentiation of mesenchymal stem cells (MSCs) was studied in the culture of MSCs isolated from bone marrow and adipose tissue. Tissue-engineered hybrid systems (grafts) constructed using P(3HB) and P(3HB) in combination with osteoblasts were used in experiments on laboratory animals (n = 48) with bone defect model. The defect model (5 mm in diameter) was created in the rat parietal bone, and filling of the defect by the new bone tissue was monitored in the groups of animals with P(3HB) implants, with commercial material, and without implants (negative control). Computed tomography (CT) and histologic examination showed that after 120 days, in the group with the osteoblast-seeded P(3HB) implants, the defect was completely closed; in the group with the cell-free P(3HB) implants, the remaining defect was no more than 10% of the initial one (0.5 mm); in both the negative and positive controls, the size of the defect was about 1.0-1.2 mm. These results suggest that P(3HB) has good potential as osteoplastic material for reconstructive osteogenesis. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 566-577, 2017.

The effect of piezosurgery with cvd tip in osteotomy in rat parietal bone: histologic and histomorphometric analysis

Objective: The aim of this study was to compare the response of bone tissue in osteotomy under piezosurgery using a CVD tip in rat parietal bone in contrast with the standard protocol using low-speed tungsten burs. Material and Methods: A bone defect model was created in parietal bone of 20 male Wistar rats using CVD tip and drill. Five animals were sacrificed after 3, 7, 14 and 28 days and the bone containing the defects was submitted to the histologic and histomorphometric analysis. The results of histomorphometry were statistically analyzed using RM ANOVA with a significance level of ? = 0.05. The results were still subjected to Tukey's multiple comparison test. Results: The results showed statistically significant difference (p <0.05) between the two types of treatment in the analyzed periods. Piezosurgery promoted slower and more precise cut with less bone loss and less bleeding during surgery, promoting conditions for a faster repair compared to the traditional method of osteotomy, fewer inflammatory cells, faster bone formation, cleaner surgical wound in the experimental group in all periods. Conclusion: It was concluded that the use of CVD tip in piezosurgery was proven to be valid for osteotomy, with more precise cuts, less tissue damage, fewer pronounced inflammatory response and faster bone formation in the early periods when compared with low-speed tungsten burs.

Comparative Study of hydroxyapatite prepared from seashells and eggshells as a bone graft material

The aims of this study were to determine the physical properties of hydroxyapatite from seashells (sHA) and from eggshells (eHA), to analyze elements within sHA and eHA, and to compare the bone regeneration ability between sHA and eHA in a rat parietal bone defect model. The sHA and eHA particles had a similar morphology in scanning electron microscope images. From the Fourier-transform infrared absorbance spectra and X-ray diffraction results, both types of hydroxyapatite (HA) had the characteristics of pure HA. Inductively coupled plasma atomic emission spectroscopy results suggested that the sHA had higher levels of sodium and strontium than the eHA, whereas the eHA had higher levels of magnesium than the sHA. In μ-CT results, the mean bone mineral density of the sHA was significantly higher than the control at 4 weeks after the operation (p = 0.012). The mean bone volume of the eHA was significantly higher than the control at 8 weeks after the operation (p = 0.012). In the histological images at 4 weeks after the operation, foreign body multinucleated giant cells were observed around the agglomerated sHA particles, while there were fewer inflammatory reactions around the agglomerated eHA particles. The eHA group showed better results in bone formation than did the sHA group in this study.

Use of a gelatin hydrogel membrane containing β-tricalcium phosphate for guided bone regeneration enhances rapid bone formation.

The purpose of this study was to evaluate a thin gelatin hydrogel membrane containing β-tricalcium phosphate (G-TCP) for use in guided bone regeneration, a technique that we developed. G-TCP membranes were fabricated from gelatin and β-TCP powder, freezedried, and cross-linked by heating. The resulting G-TCP membranes were as thin as collagen membranes, with high mechanical integrity. Proliferation and differentiation of rat bone marrow stromal cells (BMSCs) on G-TCP and collagen membranes were examined. On both membranes, BMSCs proliferated well and expressed alkaline phosphatase. However, more bone-like mineralized tissue formed on G-TCP membranes than on collagen membranes at 4 weeks. The effects of G-TCP and collagen membranes on bone regeneration in rat parietal bone defects were histologically examined. Bone bridges with mature uniform bone were observed under G-TCP membranes as early as 2 weeks. These results indicate that G-TCP is a GBR membrane that is comparable or superior to collagen membrane.

Effects of low intensity pulsed ultrasound stimulation on bone regeneration in rat parietal bone defect model

Purpose: Low intensity pulsed ultrasound stimulation (LIPUS) has been clinically applied to promote bone fracture healing in the orthopedic field. Thus, it is likely that LIPUS also stimulates bone regeneration in bone defects in the cranial-maxillofacial area. However, this has not been clearly proved. Furthermore, optimal time point and period of the application after the surgery has not been reported. The purpose of the present study was to evaluate the effect of LIPUS on bone regeneration in the rat parietal bone defects especially focusing on time and period of the application. Materials and Methods: Eighteen Wistar rats (14 weeks old) were divided into 6 groups: 5 experimental groups and a control group. Bone defect of 5 mm diameter was prepared on each side of the parietal bone and customized gelatin membranes were placed over the bone defects. LIPUS (160 mW/cm2, 15 min/day) was applied to the defect area with an active transducer externally in the experimental groups according to the schedules of the applications: Group 1 (day 6 - 12), group 2 (day 13 - 19), group 3 (day 20 - 26), group 4 (day 6 - 19) and group 5 (day 6 - 26). All the animals were sacrificed at 28 days. The defects were analyzed with micro CT and then histologically. Results: In Group 1, new bone formation was significantly promoted and the newly-formed bone was thick and matured compared to the one of the control group. In other experimental groups there were tendencies of stimulation of new bone formation; however, they were not statistically significant. Discussion and Conclusion: The present study demonstrated that amount of new bone formation in the bone defect depended on the time and period of LIPUS application. It has been suggested that application of LIPUS at an early healing period, the second week after the surgery, effectively accelerated new bone formation.

Ultrasound Insertion Loss of Rat Parietal Bone Appears to Be Proportional to Animal Mass at Submegahertz Frequencies

Transcranial ultrasound therapy is an increasing area of research for noninvasive treatments in the brain including targeted drug delivery. Measurements of ultrasound transmission through rat parietal bone at five frequencies (0.268 MHz, 0.841 MHz, 1.409 MHz, 1.972 MHz and 2.53 MHz) were performed at 88 locations in 22 ex vivo rat skullcaps (Wistar) using a fiber-optic hydrophone system. At submegahertz frequencies, the skull insertion loss was found to be proportional to animal mass, while at higher frequencies this trend was lost. Maps of the transverse pressure profile of the transducer before and after skull insertion showed increased distortion effects at higher frequencies. Parietal bone thickness was measured and was found to increase with increasing body mass. Additional measurements were made through mouse and rabbit skulls at 2.53 MHz. At this frequency, aberration effects through mouse skull were negligible, while large distortions were observed through rat and rabbit skull. Preclinical transcranial ultrasound studies in rats may be improved by scaling applied powers according to body mass to produce more consistent in situ pressures.

Immunohistochemical study of rat parietal bone defect repair with β-tricalcium phosphate and carbonate apatite

In order to examine effects of β-tricalcium phosphate (TCP) or carbonate apatite particles (CAP) in bone repair, we studied bone healing processes histopathologically and immunohistochemically in rat parietal bone defects filled with TCP or CAP. Without TCP or CAP filling, the bone repair was carried out by osteoblasts derived from the edge of the bone defect but did not occur at the center area of the defect. However, with TCP or CAP filling, conductive bone formation occurred along the surfaces of both particles, indicating that those ceramics definitely induced osteoblastic differentiation, which was confirmed by immunohistochemical expressions of such osteoblastic differentiation markers as BMP2/4, Runx2 and Osterix. The expressions of BMP2/4 preceding those of the other osteoblastic markers seemed to be essential for the conductive bone formation by calcium phosphate ceramics. Osteoblastic cells on these particles were derived from resting osteoblasts on the remaining bone surface. Type I collagen, dentin matrix protein 1, and osteocalcin were also expressed in osteoblastic cells as well as in the bone matrix, suggesting that these molecules participated in both bone formation and regulation of osteoblastic differentiation.

Effect of richlocaine on proliferative activity of osteoblasts and intracellular calcium content in rats.

We studied the effect of local anesthetic richlocaine on proliferation and intracellular calcium content in cultured osteoblasts from rat parietal bone. In a concentration of 1 mg/ml this drug produced a cytotoxic effect on osteoblasts. In concentrations of 0.01 and 0.001 mg/ml richlocaine in the absence and presence of subtoxic dose of sodium cyanide (0.2 mM) increased the number of osteoblasts by 15.4 and 36.6 or 13.8 and 38.6%, respectively. In a concentration of 1 mg/ml, richlocaine increased the content of cytosolic calcium in osteoblasts by 105%. These effects of richlocaine in low concentrations (0.01 and 0.001 mg/ml) can be related to stimulation of metabolic processes in osteoblasts.

Angiogenesis after sintered bone implantation in rat parietal bone.

We studied the effect of bone substitutes on revascularization and the restart of blood supply after sintered bone implantation in comparison with synthetic hydroxyapatite implantation and fresh autogenous bone transplantation (control) in rat parietal bones. Methods for the study included the microvascular corrosion cast method and immunohistochemical techniques were also used. The revascularization of the control group was the same as that for usual wound healing in the observations of the microvascular corrosion casts. The sintered bone implantation group was quite similar to that of the control group. In the synthetic hydroxyapatite group, immature newly-formed blood vessels existed even on the 21st day after implantation and the physiological process of angiogenesis was interrupted. Immunohistochemically, vascular endothelial growth factor (VEGF), which activates angiogenesis, appeared at the early stages of both the control group and the sintered bone implantation group. VEGF reduced parallel with the appearance of the transforming growth factor factor-beta-1 (TGF-beta-1), which obstructs angiogenesis, and the angiogenesis passed gradually into the mature stage. In the hydroxyapatite implantation group, TGF-beta-1 appeared at the early stage of the implants. The appearance of VEGF lagged and it existed around the pores of hydroxyapatite even on the 21st day of the implantation. Proliferation and wandering of endothelial cells continued without any maturing of the vessels. These findings suggest that the structure and the components of the implant material affect angiogenesis after implantation as well as new bone formation.

Effects of enamel matrix derivative on mineralized tissue formation during bone wound healing in rat parietal bone defects.

Enamel matrix derivative (EMD: Emdogain) has been reported to stimulate the biosynthesis and regeneration of trabecular bone. To address whether the biological action of EMD is dependent on the local environment of osseous tissue, circular perforations were made in parietal bones and immediately filled with either EMD or its carrier, propylene glycol alginate (PGA), as control. On post-operative days 4-60, the dissected bones were examined by various histological techniques. New bone matrix, which was immunoreactive for bone sialoprotein (BSP), was formed from the periosteum at the peripheral area of perforations. Different from the findings reported in injured long bones, mineralized tissue was produced in the regenerating connective tissue within bone defects. This mineralized tissue was hardly immunostained for BSP, contained few collagen fibres, and lacked osteocytic lacunae and layers of osteoblasts and osteoid. Energy-dispersive X-ray analysis showed that Ca and P weight % and Ca/P molar ratio of this mineralized tissue were similar to or slightly higher than those in the pre-existing parietal bones. In addition, most multinucleated cells located in mineralized tissue lacked a ruffled border structure and showed weak immunoreaction for the lysosomal cysteine proteinase, cathepsin K, whereas those located in the bone matrix exhibited ruffled borders and strong cathepsin K expression. However, multinucleated cells located in both tissues were strongly stained for tartrate-resistant acid phosphatase. The volume fraction of such mineralized tissue appeared to be higher in EMD-applied bones than in PGA-applied controls. The mineralized tissue-forming stromal cells within bone defects appeared to show greater accumulation in EMD-applied bones than in PGA-applied controls. Our results suggest that the bioactive effects of EMD on bone wound healing and mineralized tissue formation depend, at least in part, on the local osseous environment where EMD has been applied.

Effect of Crohn's disease on bone metabolism in vitro: a role for interleukin-6.

Circulating proinflammatory cytokines may be involved in osteopenia associated with Crohn's disease (CD). Therefore, the effect of interleukin (IL)-6, IL-1beta, and tumor necrosis factor (TNF) a contained in Crohn's serum on bone formation was examined in a bone organ culture system. Initially, serum levels of IL-6, IL-1beta, and TNF-a were determined by ELISA in newly diagnosed, untreated children with CD and healthy age-matched controls. Serum IL-6 levels were significantly higher in patients with CD than in controls (23.9 +/- 2.8 pg/ml vs. 0.7 pg/ml +/- 0.2; p < 0.001), whereas IL-1beta and TNF-alpha serum levels were not. In the organ culture studies, 20-day-old fetal rat parietal bones were incubated for 96 h with CD or control serum, serum preincubated with a neutralizing antibody to each cytokine or a nonimmune immunoglobulin control, and with IL-6. Bone formation measured by assaying calcium content and dry weight was significantly decreased in bones exposed to Crohn's serum. Light microscopy of the bones treated with CD serum revealed a discontinuous, uneven mineralized bone matrix and disorganized osteoblasts with altered morphology. Incubation with an antibody that neutralized IL-6 activity prevented the change in osteoblast and bone morphology. TNF-a and IL-1beta antibodies had no apparent effects. Collagen synthesis and DNA content were not affected by CD serum. Also, addition of IL-6 to the culture medium decreased mineralization. These results suggest that IL-6 is a mediator of the effects of Crohn's serum on in vitro mineralization and may be a contributing factor to the osteopenia associated with CD.

Implanted octacalcium phosphate is more resorbable than β-tricalcium phosphate and hydroxyapatite

Our previous studies have suggested that synthetic octacalcium phosphate (OCP) could be resorbed and replaced by newly formed bone if implanted in rat skull defects. We hypothesized that the implanted OCP is more resorbable than other commonly used bone graft substitutes of calcium phosphate compounds, such as hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP). To test the hypothesis, the present study was designed to compare histomorphometrically resorption of the implanted OCP, HA, and β-TCP, which were kept in the experimental cranial defect of rats for a long term. A full thickness of standardized trephine defect was made in the rat parietal bone, and the same volume of granules of OCP, HA, and β-TCP were implanted into the defect. Five specimens of each group were fixed 6 months after implantation. The percentage of remaining implants (r-Imp%) and newly formed bone (n-Bone%) in the defect was analyzed histomorphometrically. The statistical analysis showed that the r-Imp% of OCP was significantly lower than that of HA and β-TCP. In contrast, the n-Bone% of OCP was significantly higher than that of HA and β-TCP. The present study has shown that the implanted OCP in the rat cranial defect is more resorbable than the implanted β-TCP and HA, whereas the implanted OCP enhances bone formation more than the implanted β-TCP and HA. © 2001 Wiley Periodicals, Inc. J Biomed Mater Res 59: 29–34, 2002

Implanted octacalcium phosphate is more resorbable than beta-tricalcium phosphate and hydroxyapatite.

Our previous studies have suggested that synthetic octacalcium phosphate (OCP) could be resorbed and replaced by newly formed bone if implanted in rat skull defects. We hypothesized that the implanted OCP is more resorbable than other commonly used bone graft substitutes of calcium phosphate compounds, such as hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP). To test the hypothesis, the present study was designed to compare histomorphometrically resorption of the implanted OCP, HA, and beta-TCP, which were kept in the experimental cranial defect of rats for a long term. A full thickness of standardized trephine defect was made in the rat parietal bone, and the same volume of granules of OCP, HA, and beta-TCP were implanted into the defect. Five specimens of each group were fixed 6 months after implantation. The percentage of remaining implants (r-Imp%) and newly formed bone (n-Bone%) in the defect was analyzed histomorphometrically. The statistical analysis showed that the r-Imp% of OCP was significantly lower than that of HA and beta-TCP. In contrast, the n-Bone% of OCP was significantly higher than that of HA and beta-TCP. The present study has shown that the implanted OCP in the rat cranial defect is more resorbable than the implanted beta-TCP and HA, whereas the implanted OCP enhances bone formation more than the implanted beta-TCP and HA.

Osteoblastic differentiation of periosteum‐derived cells is promoted by the physical contact with the bone matrix in vivo

The periosteum contains osteoprogenitors that differentiate to osteoblasts in bone growth or repair. Our previous studies suggested the hypothesis that the physical contact of the periosteum with the bone matrix is requisite for the differentiation of osteoblasts. To test the hypothesis, the present study was designed to investigate how the contact between the periosteum and the bone matrix influences the osteoblastic differentiation of periosteal cells with establishing a new experimental model in vivo. Differentiation of osteoblasts was assessed by gene expression of type I collagen, osteocalcin and bone sialoprotein using in situ hybridization. A barrier was designed to prevent periosteal cells from contacting the bone matrix using the membrane filter. The membrane filter was inserted surgically between the surface of rat parietal bone and the periosteum after being punched out with pin holes. Periosteal cells were allowed to contact with the bone surface only through the pin holes. The pin hole was filled with cells derived from the periosteum 1 week after inserting the filter. Differentiation of osteoblasts in week 2 and noticeable bone formation in week 3 were identified on the bone surface only under the pin hole but not under the filter. The present study demonstrated that the physical contact with the bone matrix promotes osteoblastic differentiation of periosteum-derived cells in vivo.

Implantation of octacalcium phosphate combined with transforming growth factor-beta1 enhances bone repair as well as resorption of the implant in rat skull defects.

In our previous study, we reported that synthetic octacalcium phosphate (OCP) enhances bone repair if implanted in rat skull defects. We hypothesized that OCP can be used as an effective carrier for transforming growth factor-beta1 (TGF-beta1) to promote bone repair. We designed the present study to investigate histomorphometrically whether combination with recombinant human TGF-beta1 could promote bone repair caused by OCP per se (Control/OCP). A full-thickness standardized trephine defect was made in the rat parietal bone and OCP combined with recombinant human TGF-beta1 (TGF-beta1/OCP) or Control/OCP was implanted into the defect. Four rats from each group were fixed at 2, 4, and 8 weeks after implantation. Histomorphometrical analysis of the percentage of newly formed bone (n-Bone %) and remaining implants (r-Imp %) in the defect was performed. The statistical analysis showed the n-Bone % of TGF-beta1/OCP was significantly higher than that of the Control/OCP in week 4, whereas the r-Imp % of TGF-beta1/OCP was significantly lower than that of the Control/OCP. The present study demonstrated that OCP can be used as an effective carrier for TGF-beta1 and their combination enhances bone repair as well as resorption of the carrier OCP in the early stage of bone formation.

Site Dependence of Bone Formation Induced by rhBMP-2: Comparing Rat Parietal Bone and Heterotopic Back Region

Site Dependence of Bone Formation Induced by rhBMP-2: Comparing Rat Parietal Bone and Heterotopic Back Region

Effect of irradiation on autogenous bone transplantation in rat parietal bone.

To determine the appropriate time for bone reconstruction after irradiation, the healing process after autogenous iliac bone transplantation in the irradiated parietal bone was examined by scanning electron microscopy and light microscopy. Bone transplantation was carried out at the second and the fourth weeks after Cobalt-sixty (60Co) irradiation with calculated dose and fractionation. Animals without irradiation were used as control. The results show the appearance of mesenchymal cells and blood vessels around the transplantation to be extremely few one week after transplantation which was carried out at the second week after irradiation. These inhibitions were still seen two weeks after transplantation. Four weeks after transplantation, there were no differences in the bone formation among the experimental groups. Bone formation in the transplantation at the fourth week after irradiation was similar to that of the control group. Microvascularization in the transplantation at the second week after irradiation was inhibited one week after transplantation. The delay in bone healing was responsible for the retardation of revascularization and caused microcirculatory failures as well as the damage of osteogenic cells. It is quite clear that damaged cells and tissues recovered by the elapse of time under the irradiation procedure employed in this study and also that bone formation was carried out in the physiological process. We think that bone transplantation after irradiation should be done after recovery from the radiation damage to the periosteal cells and the blood vessels.

Implanted octacalcium phosphate (OCP) stimulates osteogenesis by osteoblastic cells and/or committed osteoprogenitors in rat calvarial periosteum.

Our previous studies demonstrated that the octacalcium phosphate (OCP) causes new appositional bone formation on the OCP when implanted into the subperiosteal region of murine calvaria. The OCP may stimulate the cell population committed to the osteoblastic differentiation in the periosteum and have them express the phenotype. The present study was designed to investigate which periosteal cell population is involved in bone formation on the OCP with applying the OCP implants on top of and underneath the periosteum. The periosteum of the rat parietal bones was flapped and the OCP was implanted on top of or underneath the periosteum, in which the implantation sites were defined using the membrane filter. The histology was examined to see if new appositional bone formation occurs on the OCP implant under each condition. New bone was deposited on the OCP on the bone surface separated from the periosteum by the filter, whereas no bone was formed either under the periosteum separated from the bone surface by the filter or on the periosteum. The present study suggests that the OCP acts on osteoblasts, bone lining cells and/or their closely committed progenitors on the bone surface to express the phenotype and deposit new bone on the OCP implant.

Bone augmentation by recombinant human BMP-2 and collagen on adult rat parietal bone

A composite of recombinant human bone morphogenetic protein-2 (rhBMP-2) and collagen was implanted beneath the cranial periosteum of 10-month-old rats to observe bone development and absorbent change of carrier collagen. The rhBMP-2/collagen onlay implant resulted in active bone formation and the augmented bone was connected directly with the original bone, whereas the collagen alone resulted in neither bone nor cartilage. The ossification process in the rhBMP-2/collagen occurred directly through bone formation, similar to intramembranous ossification. The carrier collage fibers were found in the woven bone and were completely absorbed at 8 weeks in the presence of rhBMP-2, while the collagen alone implant remained encapsulated by a thin, fibrous connective tissue. Our results indicate that rhBMP-2/collagen is an effective material as a biological only implant, showing osteoinductive properties and being completely replaced by new bone. Carrier collagen not only plays a role in rhBMP-2 delivery, but also provides a cell anchorage for cell differentiation and remains as an artificial matrix in woven bone.

Bone augmentation by recombinant human BMP-2 and collagen on adult rat parietal bone

A composite of recombinant human bone morphogenetic protein-2 (rhBMP-2) and collagen was implanted beneath the cranial periosteum of 10-month-old rats to observe bone development and absorbent change of carrier collagen. The rhBMP-2/collagen onlay implant resulted in active bone formation and the augmented bone was connected directly with the original bone, whereas the collagen alone resulted in neither bone nor cartilage. The ossification process in the rhBMP-2/collagen occurred directly through bone formation, similar to intramembranous ossification. The carrier collagen fibers were found in the woven bone and were completely absorbed at 8 weeks in the presence of rhBMP-2, while the collagen alone implant remained encapsulated by a thin, fibrous connective tissue. Our results indicate that rhBMP-2/collagen is an effective material as a biological onlay implant, showing osteoinductive properties and being completely replaced by new bone. Carrier collagen not only plays a role in rhBMP-2 delivery, but also provides a cell anchorage for cell differentiation and remains as an artificial matrix in woven bone.