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Effects of long-term bisphosphonate (BP) administration on the metabolism of healthy bone and the concomitant changes in imaging are unclear. Hence, we aimed to retrospectively investigate the effects of long-term BP administration on the intact parietal bone using the standardised uptake value (SUV) derived from single photon emission computed tomography (SPECT). We enrolled 29 patients who had odontogenic infection, osteoporosis, bone metastasis cancer, or rheumatoid arthritis, and classified them into BP-naïve: A (14 patients) and BP-treated: B, < 4 years (7 patients) and C, ≥ 4 years (8 patients) groups. We measured the maximum bilateral SUV (SUVmax) of the parietal bone using quantitative bone SPECT software. There were significant differences in the duration of BP administration and SUVmax of the parietal bone among the diseases (P < 0.0001 and P = 0.0086, respectively). There was a positive correlation between the duration of BP administration and SUVmax of the parietal bone (r s  = 0.65, P = 0.0002). The SUVmax was significantly different between A and B (P = 0.02) and between A and C (P = 0.0024) groups. This is the first report on the correlation between long-term BP administration and the SUVmax of the parietal bone using the quantitative bone SPECT analysis.

Background: Ketamine is a widely used anesthetic in experimental medicine. We have also used ketamine for surgical interventions and imaging in rats and found significantly impaired ossification between identically performed experiments, which only differed in the number of anesthetic events. In order to investigate this phenomenon, we estimated the absorbed ionizing radiation and also studied whether ketamine administration has disadvantageous effect on bone cell viability., Methods: Spongious bone chips and parietal bone disks were harvested from rats. Explants were incubated in stem cell media containing 0.02, 0.2 and 2 mM ketamine. After 3 days of incubation, tetrazolium-based spectrophotometric assay was performed to measure cell viability. Size-specific dose estimation was used to calculate ionizing radiation of computed tomography imaging., Results: We found that ketamine supplementation with 0.2 mM slightly decreased cell viability, while 2 mM caused significant reduction both in the spongious and cortical explants. The cumulative ionizing radiation was found to be negligible compared to irradiation dosages used to impair ossification., Conclusions: We conclude that multiple ketamine administration was responsible for the diminished regenerative potential of bone tissue in the present experimental setup. For this reason, we suggest that ketamine anesthesia should be avoided in studies investigating bone regeneration.

In experiments on Wistar rats, a simulated defect in the flat bones of the skull was filled with a collagen sponge of animal origin impregnated with BMP-2 or pure sponge; in control rats, the defect was left open. During follow-up, X-ray density of the collagen sponge in the experimental groups differed significantly. The results attest to the absence of spontaneous remodeling of the bone tissue under conditions modeled focal defect. Moreover, stimulation of reparative processes by the collagen matrix did not lead to positive dynamics. Saturation of the collagen sponge with BMP-2 in a concentration of 0.05 mg/ml allowed increasing Xray density of the bone starting from week 4.

Background: Polyetheretherketone (PEEK) exhibits stable chemical properties, excellent biocompatibility, and rational mechanical properties that are similar to those of human cortical bone, but the lack of bioactivity impedes its clinical application., Methods: In this study, hydroxyapatite (HA) was incorporated into PEEK to fabricate HA/PEEK biocomposite using a compounding and injection-molding technique. The tensile properties of the prepared HA/PEEK composites (HA content from 0 to 40 wt%) were tested to choose an optimal HA content. To evaluate the bioactivity of the composite, the cell attachment, proliferation, spreading and alkaline phosphatase (ALP) activity of MC3T3-E1 cells, and apatite formation after immersion in simulated body fluid (SBF), and osseointegration in a rabbit cranial defect model were investigated. The results were compared to those from ultra-high molecular weight polyethylene (UHMWPE) and pure PEEK., Results: By evaluating the tensile properties and elastic moduli of PEEK composite samples/PEEK composites with different HA contents, the 30 wt% HA/PEEK composite was chosen for use in the subsequent tests. The results of the cell tests demonstrated that PEEK composite samples/PEEK composite exhibited better cell attachment, proliferation, spreading, and higher ALP activity than those of UHMWPE and pure PEEK. Apatite islands formed on the HA/PEEK composite after immersion in SBF for 7 days and grew continuously with longer time periods. Animal tests indicated that bone contact and new bone formation around the HA/PEEK composite were more obvious than those around UHMWPE and pure PEEK., Conclusions: The HA/PEEK biocomposite created by a compounding and injection-molding technique exhibited enhanced osteogenesis and could be used as a candidate of orthopedic implants.

Background: Osteoinduction and subsequent bone formation rely on efficient mesenchymal stem cell (MSC) recruitment. It is also known that migration is induced by gradients of growth factors and cytokines. Degradation of Ca 2+ -containing biomaterials mimics the bone remodeling compartment producing a localized calcium-rich osteoinductive microenvironment. The aim of our study was to determine the effect of calcium sulfate (CaSO 4 ) on MSC migration. In addition, to evaluate the influence of CaSO 4 on MSC differentiation and the potential molecular mechanisms involved., Methods: A circular calvarial bone defect (5 mm diameter) was created in the parietal bone of 35 Balb-C mice. We prepared and implanted a cell-free agarose/gelatin scaffold alone or in combination with different CaSO 4 concentrations into the bone defects. After 7 weeks, we determined the new bone regenerated by micro-CT and histological analysis. In vitro, we evaluated the CaSO 4 effects on MSC migration by both wound healing and agarose spot assays. Osteoblastic gene expression after BMP-2 and CaSO 4 treatment was also evaluated by qPCR., Results: CaSO 4 increased MSC migration and bone formation in a concentration-dependent manner. Micro-CT analysis showed that the addition of CaSO 4 significantly enhanced bone regeneration compared to the scaffold alone. The histological evaluation confirmed an increased number of endogenous cells recruited into the cell-free CaSO 4 -containing scaffolds. Furthermore, MSC migration in vitro and active AKT levels were attenuated when CaSO 4 and BMP-2 were in combination. Addition of LY294002 and Wortmannin abrogated the CaSO 4 effects on MSC migration., Conclusions: Specific CaSO 4 concentrations induce bone regeneration of calvarial defects in part by acting on the host's undifferentiated MSCs and promoting their migration. Progenitor cell recruitment is followed by a gradual increment in osteoblast gene expression. Moreover, CaSO 4 regulates BMP-2-induced MSC migration by differentially activating the PI3K/AKT pathway. Altogether, these results suggest that CaSO 4 scaffolds could have potential applications for bone regeneration.

Hyaluronic acid (HA) filler injection is widely used for soft-tissue augmentation. Complications associated with HA filling are not uncommon; however, HA-induced alopecia is a rarely reported complication that could result in severe secondary psychological trauma. The etiology, clinical traits, treatment strategies, outcomes, and possible reversibility of HA-induced alopecia have not been characterized. Here, we report a case in which bilateral temple injections of 6.5 mL of HA led to persistent pain over the left scalp for several days. Although the pain was relieved at day 9 after 600 U of hyaluronidase were injected in the left temple, the patient developed localized alopecia at the left temporoparietal region with central skin necrosis at day 15. After topical applications of recombinant bovine basic fibroblast growth factor gel and 2% minoxidil spay, the necrotic skin wound was healed at day 42. Hair regrowth and normal hair density were restored at day 74. Analyses of Doppler ultrasound examinations and histopathology of the skin biopsy suggested that mild ischemia of the left temporoparietal region led to reversible alopecia, while the permanent hair loss in the left parietal area was associated with severe skin ischemia. Therefore, the key to treatment would be to focus on the effective correction of severe ischemia-induced skin necrosis to prevent permanent hair loss. Level of Evidence V This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

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., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Objective: The aim of this study is to analyze histologically the effect of CAPE on bone healing of Critical Size Defect (CSD) in rat calvaria., Study Design: Thirty-two 3-month-old male rats were used. The animals were randomly divided into four groups. Group A received isotonic saline solution, Group B received CAPE (50 mmol/kg) locally, Group C received CAPE (100 mmol/kg) locally and Group D received CAPE (10 mmol/kg/day i.p. for 28 days) systematically. A 5-mm diameter calvarial defect was created in the right side of the parietal bone without damaging the underlying dura mater. Twenty-eight days after the surgery, all the animals were sacrificed. The original defect area was removed from the animal's calvarium bone en bloc. Beginning at the center of the surgical defect, serial sections of 6 µm thick were cut longitudinally. The sections were stained with hematoxylin and eosin for analysis under a light microscope. The sections were analyzed for the presence of inflammatory infiltrate, connective tissue formation and new bone formation. Computer-assisted histomorphometic measurements were carried out with an automated image analysis system., Results: The total new bone areas were significantly greater in group D than in all groups and group C was statistically insignificant from the other groups (p < 0.05). Group B had a greater, but not statistically significant (p > 0.05), amount of total regenerated bone area than the control group., Conclusion: The results indicate that 100 mmol/kg topical and 10 mmol/kg/day systemic application of CAPE increases bone healing, especially with systemic application.

Background: Application of recombinant human bone morphogenetic protein-2 (rhBMP-2) has been associated with significant adverse events in craniofacial settings, including swelling and seroma formation. Recent work has demonstrated an inverse relationship between bone formation/maturation and rhBMP-2 dose, frequency/severity of adverse events increasing with rising dose., Objective: The objective of this study was to determine the most effective dose for rhBMP-2 soak-loaded onto an absorbable collagen sponge (ACS) carrier for bone formation/maturation using an established defect model., Methods: One hundred sixty-eight outbred male Sprague-Dawley rats, age 11-13 weeks, weight 325-375 g randomized into seven groups of 24 subdivided into groups of eight, were used to provide radiographic and light microscopy observations of bone formation/maturation and aberrant healing events at 2, 4 and 8 weeks following application of rhBMP-2/ACS into critical-size, ø8-mm, through-through, calvarial osteotomy defects for a dose of 1.25, 2.5, 5.0, 10.0 and 20.0 μg rhBMP-2/defect, or serve as ACS or sham-surgery controls., Results: rhBMP-2 dosages ≥ 2.5 μg/defect showed histological defect closure >90% within 2 weeks, and complete resolution within 4 weeks. Adverse healing events including swelling, excessive bone formation or seroma formation could not be determined with certainty in this defect model. Notably ACS control sites showed complete defect closure at the 8-week healing interval., Conclusions: rhBMP-2/ACS accelerates local bone formation in the rat critical-size through-through calvarial defect model once reaching an osteoinductive dose threshold. This threshold may already be reached at a 1.25-/2.5-μg dose in this model. No further enhancement to bone formation/maturation may be observed adding rhBMP-2 above the 2.5-μg dose. The 1.25-20.0 μg dose range did not invoke appreciable aberrant healing events., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Autologous bone grafts are usually well consolidated after 4 to 5 months but can be incompletely interlocked with the native bone. This study investigated the effect of acid demineralization of the graft-bed interface on graft consolidation., Methods: Onlay bone grafts were performed on the calvaria of 36 guinea pigs. Half of the animals had the graft-bed contacting surfaces demineralized with 50% citric acid (pH 1.0) for 3 minutes (test group). The other half received no demineralization (control group). The bone grafts were immobilized by a resorbable membrane glued to the recipient bed with cyanoacrylate. After 7, 30, and 90 days, specimens (n = 6) were obtained for light microscopy. Data from qualitative analysis and computerized histomorphometry were statistically processed at a significance level of 5%., Results: Osteogenesis was not seen at the interface after 7 days. After 30 days, the test group showed 34.39% ± 13.4% of the interface area filled with mineralized tissue, compared to 17.14% ± 8.6% in the control group (P = 0.026). After 90 days, the mean percentages of mineralized tissue at the interface in the test and control specimens were 54.00% ± 11.23% and 38.65% ± 7.76% (P = 0.041), respectively. Within groups, a higher percentage of the area filled with mineralized tissue was seen at 90 days compared to 30 days (P = 0.004 for control and 0.041 for test)., Conclusions: Demineralization of the contacting surfaces between autologous bone graft and bone bed improved new bone formation and bone consolidation. These data need to be confirmed in humans.

Background: Although enamel matrix derivative (EMD) has demonstrated the ability to promote angiogenesis and osteogenesis both in vitro and in vivo, the specific elements within the EMD compound responsible for these effects remain unknown., Methods: Nine different protein pools from a commercially produced EMD were collected based on molecular weight. Six of these pools, along with the complete EMD unfractionated compound and positive and negative controls, were tested for their ability to induce bone formation in a calvarial induction assay. Immunocytochemistry of phosphorylated SMAD1/5/8 (phospho-SMAD), osterix, and vascular endothelial growth factor A (VEGF-A) was carried out at selected time points. Finally, proteomic analysis was completed to determine the specific protein-peptide content of the various osteoinductive pools., Results: One of the lower-molecular-weight pools tested, pool 7, showed bone induction responses significantly greater than those of the other pools and the complete EMD compound and was concentration dependent. Dynamic bone formation rate analysis demonstrated that pool 7 was optimally active at the 5- to 10-μg concentration. It was demonstrated that EMD and pool 7 induced phospho-SMAD, osterix, and VEGF-A, which is indicative of increased bone morphogenetic protein (BMP) signaling. Proteomic composition analysis demonstrated that pool 7 had the highest concentration of the biologically active amelogenin-leucine-rich amelogenin peptide and ameloblastin 17-kDa peptides., Conclusions: These studies demonstrate that the low-molecular-weight protein pools (7 to 17 kDa) within EMD have greater osteoinductive potential than the commercially available complete EMD compound and that the mechanism of action, in part, is through increased BMP signaling and increased osterix and VEGF-A. With this information, selected components of EMD can now be formulated for optimal osteo- and angio-genesis.

Particulate bone augmentation is an established clinical alternative to regenerate bone. However, in regions of poor bone quality or previously infected sites, the clinical outcomes are more inconsistent. For that purpose, peptides have been added to particulate materials in an attempt to render them with antibacterial properties or to improve their osseoconductivity. For instance, competence-stimulating peptide (CSP) has been studied to decrease the division rate of Streptococcus mutans. Also, the addition of a specific short amino acid sequence peptide derived from type I collagen (P-15) to the bone substitutes has been introduced in an attempt to increase its osseoconductivity. The present study hypothesized that xenogeneic graft materials with and without CSP would present improved host-to-biomaterial response when used in combination with P-15. Particulate graft materials with and without P-15, OsteoGraf with CSP and OsteoGraf, were implanted in an 8-mm rabbit calvarial defect for 4 weeks, and thereafter, histological and histomorphometrical evaluation was performed. The results showed that both OsteoGraf and CSP groups with the addition of P-15 induced bone growth towards the center of the defect. Furthermore, the addition of CSP to Osteograf showed a tendency to increase its osteoconductivity when combined with P-15. The results of the current study suggested that P-15 had some impact on osteogenesis; however, the effect differed between different bone substitute materials. Further investigation is necessary to clarify its effectiveness when used in combination with bone substitutes.

Object: Several materials, such as polyethylene glycol (PEG) hydrogel and fibrin glue, have been used to seal dural incisions after brain and spinal surgeries. Although the use of PEG sealant is gaining popularity, it can be associated with postoperative cerebrospinal fluid leakage and infection. However, the reasons for this association are currently unknown. The present study aimed to investigate the effects of PEG sealant and fibrin glue on wound healing and brain damage in vivo., Methods: Oval-shaped bone defects and dural defects were created bilaterally over the parietal lobes of 22 Japanese white rabbits. The dural defects were covered with 0.5 ml of fibrin glue on one side and 0.5 ml of PEG sealant on the other side. Dural regeneration and brain damage were investigated in each harvested brain and dura mater using light microscopy., Results: Dural regeneration was more effective in the presence of fibrin glue than it was with PEG sealant (p = 0.014). Of the 22 rabbits, 11 showed thick (Grades ++ and +++) dural regeneration by 28 days postsurgery in the hemisphere where fibrin glue was used, whereas Grade +++ dural regeneration was not observed in the PEG hydrogel hemisphere, and only 4 rabbits showed Grade ++ regeneration. Abscess and granulation formation also tended to be more severe when PEG hydrogel sealant was used. No Grade ++ granulation/abscess formation was observed with fibrin glue, and Grade + was only observed in 13 of 22 rabbits. Conversely, with PEG hydrogel sealant, only 2 rabbits did not show granulation/abscess formation, and Grade +, ++, and +++ granulation/abscess formation was observed in 8, 7, and 5 rabbits, respectively. The extent of cortical damage was significantly greater in rabbits with abscesses and granulations, compared with rabbits without these lesions (p = 0.007)., Conclusions: Dural regeneration tended to occur more rapidly with fibrin glue, whereas granulation was more likely with PEG hydrogel sealant, which led to postoperative complications. Histological analysis indicated that PEG hydrogel sealant inhibited the normal tissue healing process and that outcomes were improved by the use of fibrin glue.

This study aims to analyze the effect of the low-level laser therapy (LLLT) and ozone therapy on the bone healing of critical size defect (CSD) in rat calvaria. A total of 30 Wistar male rats were used. A 5-mm-diameter trephine bur was used to create CSD on the right side of the parietal bone of each rat calvarium. Once the bone was excised, a synthetic biphasic calcium phosphate graft material was implanted to all the bone defect sites. The animals were randomly divided into 3 groups as follows: the control group (n = 10), which received no LLLT or ozone therapy; the LLLT group (n = 10), which received only LLLT (120 seconds, 3 times a week for 2 weeks); and the ozone therapy group (n = 10) (120 seconds, 3 times a week for 2 weeks). After 1 month, all the rats were killed, and the sections were examined to evaluate the presence of inflammatory infiltrate, connective tissue, and new bone formation areas. Histomorphometric analyses showed that in the LLLT and ozone groups, the new bone areas were significantly higher than in the control group (P < 0.05). In the LLLT group, higher new bone areas were found than in the ozone group (P < 0.05). This study demonstrated that both ozone and laser therapies had a positive effect on bone formation in rat calvarial defect, compared with the control group; however, ozone therapy was more effective than LLLT (808 nm; 0.1 W; 4 J/cm(2); 0.028 cm(2), continuous wave mode).

Background: Isotretinoin is a vitamin A derivative, indicated for the treatment of patients with severe acne, which shows several side effects on bone metabolism., Objective: This study analyzed the process of bone repair in rats receiving 7.5 mg/kg/day of oral isotretinoin., Methods: Thirty-three male albino Wistar rats, at approximately 60 days of age, were randomly assigned to control (n = 15) and experimental (n = 18) groups. Only the experimental group underwent oral isotretinoin therapy. In both groups, a 2-mm cavity was established in the calvarium of each animal. The animals were euthanize 21, 28 and 90 days postoperatively. The parietal bone was removed and the surgical specimens underwent histological examination. Computed histomorphometry allowed the measurement of the total area of bone defects and the proportion of newly formed bone at the different observation time points., Results: In the experimental group, the results, expressed as mean percentage of newly formed bone, were: 25.37% (±9.14) at day 21; 41.78% (±7.00) at day 28; and 57.51% (±11.62) at day 90. In the control group, the results were: 17.10% (±9.23) at day 21; 34.42% (±7.70) at day 28; and 48.49% (±16.40) at day 90., Conclusion: These results enabled us to conclude that isotretinoin promoted acceleration in the process of new bone formation in rat calvaria, although this increase was not statistically significant., (Copyright © 2012 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

The objective of this study was to valuate 2 substances as potential carriers of fibroblast growth factor 1 (FGF-1) in a rat craniectomy model: gelatin sponge (Spongostan; Ferrosan A/S, Søborg, Denmark) and natural bone mineral (Bio-Oss; Geistlich Biomaterials, Wolhusen, Switzerland).Forty-eight adult male Sprague-Dawley rats were used. A 5-mm-diameter circular craniectomy was performed in the left parietal bone. Animals were divided into 6 experimental groups of 8 rats, each group receiving a different treatment: control (no substance added), Spongostan, Bio-Oss, FGF, FGF + Spongostan, and FGF + Bio-Oss. Animals were killed 12 weeks after surgery.Descriptive histology and stereology were used, the latter to measure the volumes of regenerated bone and Bio-Oss remaining in the defect. Analysis of variance was used to determine differences in bone regeneration between groups, and Mann-Whitney U test was used to compare the volume of remaining Bio-Oss particles.Histologically, the control defects behaved like critical size defects, showing incomplete bone regeneration. Only the FGF + Spongostan group achieved nearly complete bone regeneration. Bio-Oss particles seemed to reduce centripetal bone regeneration. Spongostan by itself did not interfere with spontaneous bone healing.Stereologic measurements of the volume of new bone growth, measured in cubic millimeter, were as follows: control group, 3.86 ± 1.03; Bio-Oss, 2.26 ± 1.06; Spongostan, 3.00 ± 0.81; FGF, 3.99 ± 1.85; FGF + Bio-Oss, 3.02 ± 1.88; and FGF + Spongostan, 8.93 ± 1.28. Analysis of variance showed a statistically significant difference between the FGF + Spongostan group and the other groups (P < 0.001). Comparison among the other groups did not show significant differences.Fibroblast growth factor 1 with a Spongostan carrier has shown great efficacy for bone regeneration in cranial critical size defects in rats. Bio-Oss did not produce a regenerative effect, either alone or with FGF-1.

Skeletal bone losses are mainly filled with autologous graft or artificial materials. Osteoblasts are essential to maintain bone homeostasis and bone repair through a matrix synthesis. We have previously demonstrated that adherence and regenerative matrix composition are fundamental to bone healing, even in critical situations. In this work the critical size defect technique was used to evaluate the systemic activity on bone regeneration of a novel mixture of extracellular polysaccharides. A 5mm diameter hole was made in each parietal bone of male Wistar rats. The right parietal bone hole was filled with a mixture of hyaluronic acid, chondroitin 6 sulphate, and dermatan sulphate mixed with 2.5% NaCl solution, while the left hole was left free of material and untreated and considered as control. Twenty-one days after surgery, the holes and surrounding tissues were examined visually, using X-rays, and by histological staining. Using the matrix substitute, bone healing was almost complete after 21 days in the treated hole and always complete in the control side due to some systemic effect. Neovascularization was also observed along with organized trabecular bone on both sides. No abnormal bone growth or connective tissue abnormalities were noted. At the end of the experiment, 95.1% (± 3.2) bone healing (n=20) was observed on the treated side; conversely, healing bone and histological structure were better on the control side., (Copyright © 2012 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Craniofacial sutures are bone growth fronts that respond and adapt to biomechanical environments. Little is known of the role sutures play in regulating the skull biomechanical environment during patency and fusion conditions, especially how delayed or premature suture fusion will impact skull biomechanics. Tgf-β3 has been shown to prevent or delay suture fusion over the short term in rat skulls, yet the long-term patency or its consequences in treated sutures is not known. It was therefore hypothesized that Tgf-β3 had a long-term impact to prevent suture fusion and thus alter the skull biomechanics. In this study, collagen gels containing 3 ng Tgf-β3 were surgically placed superficial to the posterior interfrontal suture (IFS) and deep to the periosteum in postnatal day 9 (P9) rats. At P9, P24, and P70, biting forces and strains over left parietal bone, posterior IFS, and sagittal suture were measured with masticatory muscles bilaterally stimulated, after which the rats were sacrificed and suture patency analyzed histologically. Results demonstrated that Tgf-β3 treated sutures showed less fusion over time than control groups, and strain patterns in the skulls of the Tgf-β3-treated group were different from that of the control group. Although bite force increased with age, no alterations in bite force were attributable to Tgf-β3 treatment. These findings suggest that the continued presence of patent sutures can affect strain patterns, perhaps when higher bite forces are present as in adult animals., (Copyright © 2012 Wiley Periodicals, Inc.)

Background and Objectives: The aim of this study was to compare culture-expanded, bone marrow-derived mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) loaded to biphasic calcium phosphate (BCP) bone ceramic in the repair of rat calvarial bone., Materials and Methods: Critical-size (7 mm dia.) calvarial defects were prepared in the frontal-parietal bones of 90 adult female Sprague-Dawley rats. Rats were randomly divided into 5 groups, according to defect filling, as follows: Group I (n = 21), BCP; Group II (n = 21), BCP+PRP; Group III (n = 21), BCP+MSC; Group IV (n = 21), BCP+PRP+MSC; Group V (n = 6) (control), no treatment. Animals were sacrificed at 2, 8 and 12 weeks postsurgery and bone regeneration was evaluated both histologically and immunohistochemically., Results: Statistically significant differences were observed in bone osteoblastic activity in calvarial defects among the groups (p < 0.05). PRP and MSC used in combination with BCP as a defect filling resulted in greater osteoblastic bone formation activity when compared to the use of BCP alone., Conclusions: The combination of mesenchymal stem cells, platelet rich plasma and synthetic bone substitute was found to be more effective in inducing new bone formation (osteogenesis) than the use of platelet rich plasma combined with synthetic bone substitute and the use of synthetic bone substitute alone.

This investigation examined the effects of pharmacologically induced precocious puberty on cranial growth in Wistar rats. Forty-eight female newborn Wistar rats were divided into two groups: a control group (C) and an experimental group (E), with four subgroups of six animals each. The time interval from birth until sacrifice differed between the subgroups, and was set at 30, 60, 90, and 120 days. An intramuscular single dose (300 μg) of steroid hormone danazol was administered on day 5 after birth, as a means of inducing precocious puberty. Alizarin (2 mg/100 g) was administered to three animals in each subgroup three days prior to sacrifice. Body mass and dates corresponding to the beginning of the oestrous cycle were recorded. Craniometric measurements were undertaken. Histological analysis using light and fluorescence microscopy was then carried out to qualitatively and quantitatively evaluate the spheno-occipital synchondrosis and to visualize bone deposition patterns. The results were analysed with a Student's t-test and analysis of variance. Precocious puberty was effectively induced and differences between groups denoted an earlier maturation in the experimental rats. In qualitative analysis, a significant increase of total synchondrosis width was noted only in group E60, in comparison with C60, and an increase in the E90 subgroup cortical bone width compared with the C90 subgroup. Histomorphometrically, a statistical difference between total width values of subgroups E60 (434.3 μm) and C60 (323.5 μm) was detected. However, body mass and macroscopic measurements did not show statistically significant differences. An appropriate model for studying bone growth associated with precocious puberty in Wistar female rats was not achieved using steroid hormone danazol, when evaluated at 30 day intervals.

The aim of this study was to investigate the effects of systemically applied zoledronic acid (ZA) on osteoblastic bone formation and relapse in the rat sagittal suture after expansion. Eighteen 12-week-old male Wistar rats were divided into three groups. In groups 1 and 2, a saline solution was given subcutaneously after expansion and the retention period lasted for 14 and 7 days, respectively. In group 3, 0.1 mg of ZA was diluted with saline and given subcutaneously after expansion: the retention period lasted for 7 days. Computed tomography (CT) measurements were obtained at the start of the study (T1), after expansion (T2), after the retention period (T3), and after the follow-up period (T4). The amount of expansion and relapse and the density of the newly formed bone in the expansion area were measured. The mean bone density values in hounsfield unit (HU) of the newly formed bone were recorded using MX View Workstation. Data were analysed using the Kruskal-Wallis, Friedman, Wilcoxon, and Mann-Whitney U-tests. The results showed that there were significant differences between the groups in the density of newly formed bone after the retention period (P < 0.05). Statistically significant differences were observed when the relapse percentages were compared between the groups (P < 0.05). ZA stimulated bone formation and decreased the relapse ratio after expansion in the rat sagittal suture.

This study analyzed the newly formed bone tissue after application of recombinant human BMP-2 (rhBMP-2) and P-1 (extracted from Hevea brasiliensis) proteins, 2 weeks after the creation of a critical bone defect in male Wistar rats treated or not with a low-intensity laser (GaAlAs 780 nm, 60 mW of power, and energy density dose of 30 J/cm(2)). The animals were divided into two major groups: (1) bone defect plus low-intensity laser treatment and (2) bone defect without laser irradiation. The following subgroups were also analyzed: (a) 5 μg of pure rhBMP-2; (b) 5 μg of pure P-1 fraction; (c) 5 μg of rhBMP-2/monoolein gel; (d) 5 μg of P-1 fraction/monoolein gel; (e) pure monoolein gel. Comparisons of the groups receiving laser treatment with those that did not receive laser irradiation show differences in the areas of new bone tissue. The group treated with 5 μg of rhBMP-2 and laser irradiation was not significantly different (P >0.05) than the nonirradiated group that received the same treatment. The irradiated, rhBMP-2/monoolein gel treatment group showed a lower area of bone formation than the nonirradiated, rhBMP-2/gel monoolein treatment group (P < 0.001). The area of new bone tissue in the other nonirradiated and irradiated groups was not significantly different (P > 0.05). Furthermore, the group that received the 5 μg of rhBMP-2 application showed the greatest bone formation. We conclude that the laser treatment did not interfere with the area of new bone tissue growth and that the greatest stimulus for bone formation involved application of the rhBMP-2 protein., (Copyright © 2011 Wiley Periodicals, Inc.)

We investigated bone repair in sensory-denervated rats, compared with controls, to elucidate the involvement of sensory neurons. Nine-week-old male Wistar rats received subcutaneous injections of capsaicin to denervate sensory neurons. Rats treated with the same amount of vehicle served as controls. A standardized bone defect was created on the parietal bone. We measured the amount of repaired bone with quantitative radiographic analysis and the mRNA expressions of osteocalcin and cathepsin K with real-time polymerase chain reaction (PCR). Quantitative radiographic analysis showed that the standard deviations and coefficients of variation for the amount of repaired bone were much higher in the capsaicin-treated group than in the control group at any time point, which means that larger individual differences in the amount of repaired bone were found in capsaicin-treated rats than controls. Furthermore, radiographs showed radiolucency in pre-existing bone surrounding the standardized defect only in the capsaicin-treated group, and histological observation demonstrated some multinuclear cells corresponding to the radiolucent area. Real-time PCR indicated that there was no significant difference in the mRNA expression levels of osteocalcin and cathepsin K between the control group and the capsaicin-treated group. These results suggest that capsaicin-induced sensory denervation affects the bone defect repair.

Background: Recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered on an absorbable collagen sponge is a U.S. Food and Drug Administration-approved therapy effective at generating bone formation. In pediatric patients for whom other therapeutic options have been exhausted, rhBMP-2 is used off-label to address problematic bony defects. In the skeletally immature patient, the safety of rhBMP-2 therapy remains uncertain. Experiments are needed that investigate the effect of rhBMP-2 on growth and development in clinically relevant models., Methods: Ten juvenile rabbits underwent creation of a parietal skull defect that was treated with either 0.2 mg/cc rhBMP-2/absorbable collagen sponge or a neutral buffer solution/absorbable collagen sponge. Amalgam markers were placed at suture confluences to track suture separation and skull growth. Cranial growth was assessed radiographically at 10, 25, 42, and 84 days of age. Means and standard deviations for the various craniofacial growth variables were calculated and compared. Mean differences were considered significant for values of p < 0.05. At 84 days, sutures were analyzed by means of micro-computed tomographic scanning and histologic staining., Results: Treatment with rhBMP-2 resulted in fusion of the coronal sutures bilaterally, with variable fusion of the sagittal suture by cephalometric, radiographic, and histologic analysis. There were statistically significant changes to coronal suture growth, sagittal suture growth, skull height, craniofacial length, and intracranial volume (p < 0.05)., Conclusions: The use of rhBMP-2 in this juvenile animal model resulted in skeletal changes that may be undesirable in a clinical setting. The appearance of these fused sutures suggested a direct effect of rhBMP-2. Further work is required to limit the effect of rhBMP-2 to the target defect when used in the immature skeleton.

Objective: This study compared the healing of 2 laser ablation units, erbium YAG and femtosecond lasers versus conventional mechanical cutting with carbide and diamond drills to explore future applications for bone surgery. The effects of laser or mechanical ablation combined with rhBMP-7 were also investigated., Methods and Materials: Following defect standardization, a full-thickness circular defect was created on the parietal bones of 160 mice divided into 4 groups: carbide drill, diamond drill, erbium YAG laser, and femtosecond laser. Each of the 4 ablation groups was treated with and without BMP 7. Hard tissue healing was assessed using microcomputerized tomography at 3 and 12 weeks postsurgical time points., Results: The femtosecond laser created wounds that showed slightly delayed bone healing during the observation period when compared with mechanical drilling, although the difference was not statistically significant. The Er:YAG laser showed a healing rate similar to that of the mechanically ablated groups. When BMP 7 was added to the surgical sites, bone wound closure occurred at a similar rate in all test groups., Conclusions: The femtosecond and Er:YAG lasers are 2 laser modalities suitable for bone ablation that are comparable to mechanical instrumentation in terms of bone healing. This study suggested that BMP-7 may be used to enhance bone healing with success regardless of the ablative modality used, whether laser or mechanical drilling., (Copyright © 2010 Mosby, Inc. All rights reserved.)

The objective of the study is to compare the amount of new bone produced by Buguzhi (Psoralea corylifolia fruit) extract in collagen matrix to that produced and collagen matrix in vivo. Eighteen bone defects, 5 mm by 10 mm, were created in the parietal bone of 9 New Zealand white rabbits. Six defects were grafted with Buguzhi extract mixed with collagen matrix. Six defects were grafted with collagen matrix alone (positive control) and 6 were left empty (negative control). Animals were sacrificed on day 14 and the defects were dissected and prepared for histological assessment. Quantitative analysis of new bone formation and bone cells was made on 100 sections (50 sections for each group) using image analysis. A total of 275% more new bone was present in defects grafted with Buguzhi extract in collagen matrix than those grafted with collagen matrix. No bone was formed in the negative control group. The amount of bone cells was also significantly greater in the Buguzhi group than in the positive control group. To conclude, Buguzhi extract in collagen matrix has the effect of increasing new bone formation locally in vivo. Buguzhi extract in collagen matrix can be used as a bone graft material.

Suture expansion osteogenesis, which induces active bone formation within the distracted area by mechanical stress, provides an alternative form of treatment of some bone deficiency problems in craniofacial field, such as craniosynostosis, palate cleft, or narrow maxilla. However, how to stimulate new bone formation within the distracted area remains a great challenge. In this study, the effect of recombinant human bone morphogenetic protein (rhBMP-4) on bone formation induced by mechanical stimuli was investigated. The expanded midsagittal sutures of the rat calvaria were maintained in an organ culture system in the absence (control group) or presence (experimental group) of rhBMP-4 (25 or 50 ng/mL). Proliferating cell nuclear antigen, alkaline phosphatase (ALP) activity, and messenger RNA levels of core-binding factor alpha 1 (Cbfa1) and ALP were detected to determine the cell proliferation and differentiation. Bone formation within the suture was evaluated by histologic and fluorescent examination. No difference in the number of proliferating cell nuclear antigen-positive cells was found between the control and the experimental groups, whereas ALPase activity and messenger RNA levels of Cbfa1 and ALP in the experimental group were higher than that in the control group. Bone formation was accelerated in the rhBMP-4-treated group when compared with the control group. In addition, the amounts of bone formation and cell differentiation in response to 25 ng/mL of rhBMP-4 were almost equal to those induced by 50 ng/mL of rhBMP-4. Our results suggest that application of exogenous rhBMP-4 could enhance new bone formation within the rapid expanded sutures by stimulating osteoblast differentiation.

The feature of osteoconductivity, and expression of inductive BMP and transcription factors (Runx2 and Osterix) for osteoblast differentiation, which was related to conductive bone formation, were observed in experimentally created defects in rat femoral and parietal bones filled with beta-tricalcium phosphate (beta-TCP) or carbonate apatite (CAP). Femoral cortical bone defects were repaired by conductive bone formed by osteoblasts differentiated around beta-TCP and CAP, and immunohistochemical observation revealed that the osteoblasts expressed BMPs, Runx2, and Osterix. However, the repair in parietal bone defects was incomplete despite the beta-TCP and CAP filling. Only cells, which differentiated around beta-TCP or CAP, and formed conductive bone expressed BMPs, Runx2, and Osterix. These findings revealed that the osteoconductivity of calcium phosphate materials required the expression of BMPs as the prerequisite for Runx2 and Osterix expression. Therefore, it is suggested that when calcium phosphate ceramics are used as bone substitute materials, BMPs are essential for osteoconductivity.

Objective: Fluoxetine is one of the most common medications used for the treatment of depression. Recent studies have demonstrated the possible effect of this drug on bone. The purpose of this study was to evaluate the effect of flouxetine on bone in Sprague-Dawley rats randomly divided into 3 groups (n = 7)., Study Design: Two calvarial defects, 5 mm diameter, were prepared in parietal bone. The left defects were filled with natural bovine bone mineral (NBBM) and the right defects were left untreated. The first group did not receive fluoxetine. The second group and the third group received 7.5 mg and 15 mg flouxetine, respectively, daily by gastric gavage. The animals were killed 8 weeks after surgery, and the amount of bone regeneration was evaluated using histometric analysis., Results: The defects showed dose-dependent increase in bone formation. The bone fill length had statistical significant differences between NBBM/flouxetine (15 mg) group and other groups (P < .05). The presence of the NBBM had positive effect on the bone formation in all groups in so far as the maximum amount of the increasing effect was seen in those rats filled with NBBM that received 15 mg flouxetine (P < .05). The minimum bone length in fluoxetine-treated defects was seen in 7.5 mg defects (0.36 mm), Conclusion: Fluoxetine may improve the amount of bone regeneration in the rat calvarial defects.

Despite the insufficient number of experimental studies, platelet-rich plasma (PRP) including high amounts of growth factors is introduced to clinical use rapidly. The aim of this study was to compare the effects of PRP and platelet-poor plasma (PPP) on healing of critical-size bone defects.Bilateral full-thickness, critical-size bone defects were created in the parietal bones of 32 rabbits, which had been studied in 4 groups. Saline, thrombin solution, PPP, and PRP were applied to the created defects before closure. Radiologic defect area measurement results at 0, 4, and 16 weeks were compared between the groups. In addition, densities of the newly formed bones at 16th week were studied. Histologic parameters (primary and secondary bone trabecula, neovascularization, and bone marrow and connective tissue formation) were compared between 4- and 16-week groups.More rapid decrease in defect size was observed in groups 3 and 4 than in groups 1 and 2, both in the 4th and 16th weeks. Newly formed bone densities were also found to be higher in these 2 groups. New bone formation was detected to be more rapid considering histologic parameters, in groups 3 and 4 at 4th and 16th weeks.Study demonstrates that PRP and PPP might have favorable effects on bone healing. Although we cannot reveal any statistical difference between these 2 substances considering osteoinductive potential, PRP group has demonstrated superior results compared with fibrin glue group. Higher platelet concentrations may expose beneficial effects of PRP.

Introduction: The purpose of the study was to compare the efficacy on rat skull defects of two bone growth factors derived from the GDF-5 family., Material and Methods: The study was conducted on 17 adult Wistar rats. On each animal, two symmetrical 6-mm wide, full-thickness, skull defects were carried out in the parietal regions. In 15 out of 17 animals, both experimental defects were filled by the implants. In the group I (n=2), both defects were left empty for control. The 15 other rats were divided into 3 groups: In group II (n=5), a collagen sponge was implanted. In group III (n=5), a collagen sponge impregnated with rhGDF-5 (the genuine dimeric form) was implanted. In group IV (n=5), a collagen sponge impregnated with rhGDF-5C465A (a monomeric form of GDF-5) was implanted. All animals were sacrificed at 8 weeks. The harvested specimens were processed for contact radiography and standard histological examination. The quantitative results were assessed with a semi-quantitative histological scoring system., Results: One animal in the group II was excluded because it died of unknown reasons. In group I, no bone healing was observed in the defects. In group II, no bone healing was observed in 4 out of 10 defects, and partial bone healing was observed in 5 out of 10 defects. In group III, partial bone healing was also observed in 3 out of 8 defects and complete bone healing in 4 out of 8 defects. In group IV, partial bone healing was observed in 8 out of 10 defects and complete bone healing in 2 out of 10 defects., Conclusion: Bone healing was improved in all treated groups. Further studies are necessary to determine the optimal formulation of these composite implants.

Premature fusion of calvarial sutures is the result of a long and complex reaction, and several growth factors including transforming growth factor beta and basic fibroblast growth factor have important role in this event. Several prostaglandins have important functions in local bone modeling and remodeling by autocrine and paracrine mechanisms. Although effects of prostaglandins on long bones were studied both experimentally and clinically, there are limited data about cranial bones and sutures. In this study, we investigated the effect of iloprost-a stable prostacyclin analogue, which is widely used for the treatment of pulmonary arterial hypertension even in early pregnancy, to rat calvarial sutures. In 2 study groups, iloprost was injected intraperitoneally 10 and 15 microg kg d, respectively. In the third group, dexamethasone 2 mg kg d + iloprost 15 microg kg d was injected intraperitoneally to antagonize the effects of iloprost. In every group, 4 rats were killed at the postoperative 15, 30, and 45 days, and specimens including the sagittal and frontal sutures were excised immediately. Routine histological and immunohistological staining were performed on the specimens. Morphological measurements were performed on the skulls, also. In histological evaluation, bone formation in the both frontal and sagittal suture area was increased and accelerated in iloprost groups. Dexamethasone inhibited the effects of iloprost on the third group. Expressions of transforming growth factor beta and basic fibroblast growth factor were also increased in immunohistological staining. In morphological measurements, statistically significant differences were found between control and study groups. Iloprost did not fused the rat calvarial sutures prematurely, but it narrowed the sagittal and frontal sutures especially after the second week of the study. This situation might effect the sutures of the babies of the pregnant patients with pulmonary arterial hypertension treated with iloprost. Cranial sutures, calvarial bones, and cranial shape of the babies of the pregnant patients who were treated with iloprost should be monitored to clarify the topic.

Background: The use of platelet-rich plasma (PRP) in bone augmentation procedures is well documented; however, the exact benefit of this material is not yet established., Purpose: This study aimed to evaluate the benefits of using PRP, when only used, and compare it to Bio-Oss (Geistlich Biomaterials, Wolhusen, Switzerland) in vertical bone augmentation capacity., Materials and Methods: The study was performed in calvaria of eight adult female New Zealand rabbits using titanium bone conduction cylinder. Two titanium cylinders were fixed into perforated slits made on the parietal bone of each rabbit. On each rabbit, one chamber was grafted with Bio-Oss, and the contralateral was filled with PRP. Animals were sacrificed 4 weeks after intervention and biopsies were taken. Densitometric, histological, and histomorphometric analyses were performed to evaluate bone mineral density, vertical bone augmentation, and remaining graft volume, respectively. Statistical analyses were performed with Mann-Whitney test, using a significance level of p < .05., Results: Densitometric and histomorphometric data analysis revealed that mean bone mineral densities and bone augmentation were significantly lower in the cylinders treated with PRP (p < .0001) 4 weeks after implantation., Conclusion: This study showed no beneficial effect of using PRP on osseous regeneration. In addition, it was emphasized that Bio-Oss presents good osteoconductive properties by achieving suitable bone volume values.

Objective: Puerarin is one of the major phytoestrogens isolated from Pueraria lobata, a Chinese medicine known as Gegen. Our laboratory compared the amount of new bone produced by puerarin in collagen matrix (carrier) to that produced by the collagen matrix alone., Method: Eighteen bone defects, 5mm by 10mm were created in the parietal bone of nine New Zealand White rabbits. In the experimental group, six defects were grafted with puerarin solution mixed with collagen matrix. In the control groups, six defects were grafted with collagen matrix alone (active control) and six were left empty (passive control). Animals were killed on day 14 and the defects were dissected and prepared for histological assessment. Serial sections were cut across each defect. No new bone was formed in the passive control group. Quantitative analysis of new bone formation was made on 100 sections (10 sections in each defect, in five defects randomly selected in each of the experimental group and active control group) using image analysis., Results: A total of 554% more new bone was present in defects grafted with puerarin in collagen matrix than those grafted with the collagen matrix alone., Conclusion: Puerarin in collagen matrix has the effect of increasing new bone formation locally and can be used for bone grafting or for bone induction often required in surgery.

Purpose: This study was conducted to histomorphologically determine the quantity of bone formation induced by TAK-778, a 3-benzothiepin derivative, (Takeda Pharmaceutical Co.Ltd.), in various amounts, using b-tricalcium phosphate granules (beta-TCP; OLYMPUS Corp.) as a carrier for the osteogenetic agent., Methods: Ten-week-old female SD rats were used. An incision was made over the parietal region of the head. The cranial periosteum was ablated and a titanium tube was fixed with an adhesive resin cement to the central part of the head, through which a mixture of TAK-778 and beta-TCP was implanted under various conditions. Tissue specimens were prepared at 4, 8, and 16 weeks after the implantation for histomorphological examination, and the proportion of new bone formation was compared at fixed time points using the NIH imaging software. The amount of new bone formation was examined every week after the implantation of TAK-778 at various doses, and the mean values were compared using Fisher's PSLD test (P< 0.05)., Results: The histomorphological observations revealed new bone formation in all the groups, irrespective of the amount and the duration of implantation of TAK-778. A comparative study revealed that the amount of new bone formation was the largest at 16 weeks following the implantation of a mixture of beta-TCP and 100 mg of TAK-778., Conclusions: 1. The present study confirmed the acceleration of new bone formation soon after TAK-778 implantation. 2. The results suggested that the action of TAK-778 could be maintained over time if the agent was used in combination with beta-TCP. 3. The time-course of bone formation differed depending on the proportion of TAK-778 and beta-TCP in the mixture used. 4. TAK-778 at the dose of 10 mg or 50 mg was more effective than that at the dose of 100 mg for the early formation of new bone. These results indicate that TAK-778 accelerates the formation of new bone and that beta-TCP is a useful carrier for TAK-778.

Background: The condition of alveolar bone influences the success and subsequent esthetics of implant treatment. This study investigated the early effects of bioactive glass (BG) on bone augmentation within a hemispherical titanium cap in rabbit parietal bone., Methods: Twelve adult male Japanese white rabbits were used. One titanium cap (test site) was packed with BG in a collagen gel, and the other (control site) was packed with the collagen gel alone. After 1 and 3 months, animals were euthanized, and the experimental area was examined using fluorescence and light microscopy., Results: Newly generated bone was observed at 1 and 3 months of healing. Although bone was also generated without BG, newly mineralized bone was generated sooner with BG present for guided bone augmentation than without BG. At 1 and 3 months, the BG was not bioabsorbed completely, and some particles remained., Conclusion: New bone is generated at an early stage of bone formation using BG for bone augmentation.

Purpose: We compared the amount of new bone produced by Gusuibu in collagen grafts to that produced by bone grafts and collagen grafts., Materials and Methods: Twenty bone defects were created in the parietal bone of 14 New Zealand White rabbits. In the experimental group, 5 defects were grafted with Gusuibu extract mixed with absorbable collagen sponge, and 5 defects were grafted with autogenous endochondral bone. In the control groups, 5 defects were grafted with absorbable collagen sponge alone (active control) and 5 were left empty (passive control). Animals were killed on day 14 and the defects were dissected and prepared for histologic assessment. Serial sections were cut across each defect. Quantitative analysis of new bone formation was made on 150 sections using image analysis., Results: A total of 24% and 90% more new bone were present in defects grafted with Gusuibu in collagen grafts than those grafted with bone and collagen, respectively. No bone was formed in the passive control group., Conclusions: Gusuibu in collagen grafts have the effect of increased new bone formation locally and can be used as a bone graft material.

In this study a digital measurement technique has been proposed to quantify bone formation on histological images. Two standard parietal defects were created in 30 adult rabbits. The animals were divided into six groups. Four animals of each group were randomly chosen as experimental group in which osteopontin-coated hydroxyapatite (OPN-HA) and hydroxyapatite (HA) were inserted alternatively in created defects. To observe the spontaneous healing process of defects, one animal of each group was used as control animal and these created defects did not receive any implants. The animals were sacrificed after 1, 2, 6, 12, 18 and 30 weeks. The histological sections were magnified (x100) and scanned digitally. The newly formed bone surfaces within the healing area were indicated and quantified by means of Adobe Photoshop 7 software. This measuring technique was found to be reliable and reproducible. The results of this study show no significant differences in bone formation between the OPN-HA and non-coated HA defects, although a significant difference in bone formation was measured at the margins of the defects treated with OPN-HA., (Copyright (c) 2007 S. Karger AG, Basel.)

Purpose: The purpose of this work was to study the bone tissue reaction after porous polyethylene (Polipore) implantation into surgical defects in the parietal bones of rats with streptozotocin-induced diabetes, treated with salmon calcitonin., Materials and Methods: Porous polyethylene implants were placed in bone defects created in 36 adult female rats. The rats were divided into 3 equal groups: diabetic treated with calcitonin (DCa), diabetic (D), and control (C). The animals of the DCa group received applications of salmon calcitonin on alternating days immediately after the surgery until sacrifice. The rats were sacrificed after 15, 30, 60, and 90 days, and the defects were examined histologically and statistically through histomorphometric analysis., Results: Histomorphometric analysis showed that there was no statistically significant difference in the mean quantity of inflammatory cells among all study groups after 15 and 90 days. At 30 days, a statistically significant difference was observed between the D and C groups and the D and DCa groups. At 60 days, there was no statistically significant difference between the D and DCa groups., Discussion: Porous polyethylene can be considered an option for implant material when there are investigations that prove its biocompatibility and stability in the host tissues. Salmon calcitonin positively aided the bone repair and attenuated the inflammatory response until 30 days after the surgery., Conclusion: Porous polyethylene was tolerated by the host tissues in all groups, and moderate chronic inflammatory reaction was observed up to the 90-day period. Salmon calcitonin attenuated the inflammatory response up until 30 days.

This study investigates the effects of intermittent parathyroid hormone (PTH(1-34)) treatment on bone regeneration and mechanical strength of critically sized rat calvarial bone defects covered with expanded membranes. A full-thickness bone defect (diameter 5 mm) was trephined in the central part of the parietal bones in 20-month-old female Wistar rats. The bone defects were covered with an exocranial and an endocranial expanded polytetrafluoroethylene membrane. The animals were killed 35 days after operation. 60 microg PTH(1-34)/kg was administered daily during the healing period, and control animals with calvarial bone defects were given vehicle. Mechanical testing was performed by a punch out testing procedure by placing a steel punch (diameter 3.5 mm) in the center of the healed defect. After mechanical testing, the newly formed tissue inside the defect was removed and the dry weight and ash weight were measured. PTH(1-34) increased dry weight by 48%, ash weight by 51%, and ash concentration by 26%. PTH(1-34) also augmented the mechanical strength of the new bone formed inside the defect by increasing ultimate stiffness by 87%. No differences in body weight were found between the vehicle-injected and the PTH-treated animals during the experiment. The experiment demonstrates that intermittent PTH(1-34) treatment increases bone deposition and enhances mechanical strength of healing rat calvarial defects covered with expanded polytetrafluoroethylene membranes.

Critical Size Defect (CSD) technique was used to evaluate the systemic activities on bone regeneration capacity of a newly discovered hyaluronic acid-like exopolysaccharide synthesized by a bacteria originating from a deep sea hydrothermal vent. Some systemic effects were previously detected on earlier experiments. A 5 mm diameter hole was made on each parietal bone of male rats. The right hole was filled with 0.5 mg of a new bacterial exopolysaccharide referenced HE 800, while the left hole remained free of any treatment. After 21 days, the holes and surrounding tissues were examined by direct examination, X-rays, and histological staining. Using HE 800, bone healing was almost complete after only 21 days in the treated hole and always complete in the control side by some systemic effect. Neovascularization was also observed along with an organized trabecular bone on both sides. No abnormal bone growth or conjunctival abnormalities were noticed. At the end of the experiment, 90.1% ( +/- 5.2) bone healing (n = 20) was observed on the treated side; conversely, the control side animals demonstrate an amazing healing 100% (+/- 0.5) by a systemic effect.

Background: In vitro studies demonstrated that proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) modulates bone metabolism., Objective: The aim of our study was to confirm the ability of TNF-alpha to induce osteoclastogenesis and bone resorption in an in vivo experiment, with the use of calvarial model in mice., Materials and Methods: Twenty C57-Black mice were divided into four groups with five animals in each. The first group was infused subcutaneously on their back with recombinant mouse (rm) TNF-alpha via osmotic minipumps for 3 d, the second group was similarly infused with phosphate-buffered saline (PBS), the third group was infused with rmTNF-alpha to the region above the parietal bone and the fourth group with PBS in the same manner. Number of osteoclasts on parietal bone was determined morphometrically. Serum calcium and phosphates were monitored colorimetrically., Results: Serum calcium level and number of osteoclasts on parietal bone were significantly greater after infusion of rmTNF-alpha above the parietal bone, whereas after subcutaneous delivery these parameters were similar to the control group., Conclusion: We are concluding that TNF-alpha has the ability to change the bone metabolism in a paracrine manner only.

Critical size defect (CSD) technique was used to evaluate the bone regeneration capacity of a newly discovered hyaluronic acid-like exopolysaccharide synthesized by a bacteria originating from a deep sea hydrothermal vent. A 5 mm-diameter hole was made on each parietal bone of male rats. The right hole was filled with either a new bacterial exopolysaccharide referenced HE 800 or with collagen used as negative control, while the left hole remained free of any treatment. After 15 days, the holes and surrounding tissues were examined by direct examination, X-ray films, and histological staining. Using HE 800, bone healing was almost complete after only 15 days, with osteoblasts onto lying external bone surfaces and enhancing osteocyte inclusion. Neovascularization was also observed along with an organized trabecular bone. No abnormal bone growth or conjunctival abnormalities were noticed. At the end of the experiment, 95.9% (+/-6.2) bone healing (n = 20) was observed. Conversely, the collagen-treated animals did not demonstrate significant healing-17.8% (+/-18.1).

During mammalian evolution, expansion of the cerebral hemispheres was accompanied by expansion of the frontal and parietal bones of the skull vault and deployment of the coronal (fronto-parietal) and sagittal (parietal-parietal) sutures as major growth centres. Using a transgenic mouse with a permanent neural crest cell lineage marker, Wnt1-Cre/R26R, we show that both sutures are formed at a neural crest-mesoderm interface: the frontal bones are neural crest-derived and the parietal bones mesodermal, with a tongue of neural crest between the two parietal bones. By detailed analysis of neural crest migration pathways using X-gal staining, and mesodermal tracing by DiI labelling, we show that the neural crest-mesodermal tissue juxtaposition that later forms the coronal suture is established at E9.5 as the caudal boundary of the frontonasal mesenchyme. As the cerebral hemispheres expand, they extend caudally, passing beneath the neural crest-mesodermal interface within the dermis, carrying with them a layer of neural crest cells that forms their meningeal covering. Exposure of embryos to retinoic acid at E10.0 reduces this meningeal neural crest and inhibits parietal ossification, suggesting that intramembranous ossification of this mesodermal bone requires interaction with neural crest-derived meninges, whereas ossification of the neural crest-derived frontal bone is autonomous. These observations provide new perspectives on skull evolution and on human genetic abnormalities of skull growth and ossification.

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.

Objective: Our previous radiographic examinations have indicated that the synthetic octacalcium phosphate (OCP) may provide the core for nucleating multiple osteogenic sites in the experimentally created cranial defect., Design: The present study was designed to confirm the possibility that the implanted OCP causes the osteoinduction as well as the osteoconduction in the rat cranial defect., Materials and Methods: Standardized defects were created in male Wistar rat calvaria, and the OCP granules were implanted into the defect. The sham operated rats were processed in the same way except that nothing was implanted. The rats were fixed at 4 weeks after implantation of OCP or the sham operation. We examined bone formed on the implanted OCP, analyzing serial sections histologically combined with immunohistochemistry for the bone specific protein, osteocalcin., Results: In the defects treated with OCP, the radiopacity was scattered throughout the defect besides being observed along the defect margin of the parietal bone. Examination of the serial sections showed that some of new bones on the implanted OCP were formed away from the defect margin of the parietal bone with regard to both histological identification and specific molecular marker., Conclusions: The present study suggested that the implanted OCP can serve as a core for initiating bone formation and cause the osteoinduction as well as the osteoconduction in the defect.

Aseptic loosening is the most common cause of orthopaedic implant failure. This process is thought to be due to osteolysis induced by implant-derived wear particles. Teitelbaum and colleagues have recently developed a promising murine calvarial model of wear particle-induced osteolysis. However, prior to this study, this model had only been assessed qualitatively. We now report a reproducible, quantitative version of the calvarial model of wear particle-induced osteolysis, in which the extent of osteolysis (and repair) of entire parietal bones is assessed by histomorphometry of contact microradiographs. Using this model, we found that the osteolytic response is transient and rapidly repaired in one month old mice. The extent of osteolysis peaks 7 days after particle implantation and returns to baseline levels by 13 days. A similar amount of osteolysis and even more extensive repair is observed when particles are implanted repeatedly. In contrast, aged mice develop progressive osteolysis with no detectable repair. As a result, 26 month old mice have approximately 17-fold more osteolysis than one month old mice 21 days after particle implantation. Skeletally mature, adult mice (4-16 months old) show an intermediate pattern of response. Osteolysis in these mice peaks at 7 days after particle implantation but it is repaired more slowly than in the one month old mice. Taken together, these results underscore the role of an imbalance between bone resorption and bone formation in the development of aseptic loosening and suggest that agents that stimulate bone formation maybe useful in prevention or treatment of aseptic loosening.

Butyl-2-cyanoacrylate is an easily applied, biocompatible, bioresorbable polymer glue that provides an alternative to conventional rigid fixation techniques. Our aim was to determine if cyanoacrylate fixation of the bone flap in a rabbit craniotomy model provides the healing and strength afforded by plate and screw fixation. We also investigated the inflammatory responses of adjacent tissues including the scalp, cranium, and brain. A unilateral parietal bone flap was elevated in 33 adult New Zealand rabbits. The bone was fixed in position with cyanoacrylate (n = 13), fixed with a microplate and screws (n = 14), or was replaced without fixation (sham-control, n = 6). Normal scar formation and no residual polymer were found in scalp specimens. Neuropathologic analysis identified the presence of residual polymer on the surface of 2 of the 13 rabbit brains. Histopathologic analysis of the bone flap-to-skull interface revealed no difference in the degree but rather in the quality of inflammation and healing between the plate and screw and polymer fixation groups. Microdensitometric analysis of the bone gap revealed nearly equivalent bone density in the cyanoacrylate and plated groups, tending to less density in the sham group (p = 0.11 and 0.09, respectively). An additional study focusing on neurotoxicity was performed in 20 adult rabbits with 3-week and 11-week recovery periods and similarly found the absence of a marked inflammatory response to the polymer. In conclusion, bone healing and soft-tissue inflammation were comparable between cyanoacrylate and plate and screw fixation groups. Although butyl-2-cyanoacrylate glue fixation may provide a reasonable alternative to hardware fixation, further investigations are necessary to identify its ideal utilization.

Insulin-like growth factor-II (IGF-II) plays an important role in skeletal remodeling, however, little is known about its effect on bone formation in vivo. In our study of the stimulation of bone formation in vivo by IGF II we injected recombinant human IGF-II into the parietal bones of neonatal rats once a day for 12 days. The bone mineral density measured by dual energy X-ray absorptiometry and the thickness of IGF-II-injected parietal bones increased in a dose-dependent manner. The layers of osteoblasts were observed along the IGF-II-injected side.

Transforming growth factor-beta (TGF-beta) stimulates new bone formation when administered locally in vivo. The extracellular matrix protein tenascin-C, which is secreted by osteoblasts but absent from mineralized bone matrix, supports differentiation of cultured osteoblast-like cells. The current study was undertaken to determine whether expression patterns of tenascin-C in TGF-beta-treated bone cells are in agreement with a role for this protein as a mediator of TGF-beta-stimulated new bone formation. Expression of tenascin-C was investigated by immunohistochemistry in calvarial bones of mice to which TGF-beta had been locally administered. Three days after initiation of daily TGF-beta treatment, strong staining for tenascin-C was seen in regions where periosteal osteoprogenitor cells were undergoing proliferation in response to TGF-beta; by comparison, staining of periosteal surfaces of control bones was weak and discontinuous. Seven days after initiation of a course of five daily injections of TGF-beta, tenascin-C staining was still enhanced in treated bones. The ability of TGF-beta to regulate expression of tenascin-C in osteoblast-like cells in vitro was investigated using an osteosarcoma-derived cell line (ROS 17/2.8). TGF-beta caused a small but significant increase in secretion of tenascin-C into the medium, as determined by a solid phase dot immunoassay quantitated by densitometry. Western and northern blot analysis indicated that TGF-beta did not influence the pattern of expression of tenascin-C splice variants. These results indicate that TGF-beta stimulates osteoblastic tenascin-C expression and suggest that tenascin-C may act as a mediator of TGF-beta-induced new bone formation.