Non-critical Sized Bone Defects Research Articles

Osseointegration of photodynamic active biomaterials for bone regeneration in an animal bone model over a period of 12 months.

Previous efforts led to the development of two different polymeric biomaterials for periodontal regeneration with antibacterial photodynamic surface activity. The present study aimed to investigate osseointegration and bone formation of both materials in an ovine model. Both biomaterials: 1) urethane dimethacrylate-based Biomaterial 1 (BioM1) and 2) tri-armed oligoester-urethane methacrylate-based Biomaterial 2 (BioM2) are enriched with beta-tri-calcium phosphate and the photosensitizer meso-tetra(hydroxyphenyl)chlorin (mTHPC). These materials were implanted in non-critical size bone defects in the sheep femur (n=16) and tibia (n=8). Empty defects served as controls (n=16). Polyfluorochrome sequential bone labeling was carried out at baseline and after 3, 6, and 12 months. Animals were sacrificed after 12 months. Bone specimens (n=40) were fixed and subjected to microtomographic analysis (µCT) for the evaluation of the bone-volume-fraction (BV/TV), trabecular number and trabecular thickness. Subsequently, histological sections were arranged and polyfluorochrome sequential bone labeling was analyzed by confocal laser scanning microscopy (cLSM). cLSM analysis revealed that highest remodeling and bone formation activity occurred during the second half of the study period (6-12 months). Bone formation in the tibia was significantly lower for the control (2.71±1.26%) as compared to BioM1 (6.01±2.99%) and BioM2 (6.45±2.12%); (p=0.006, p=0004). Micro-computed tomography revealed a BV/TV volume fraction of 44.72±9.01% in femur defects filled with BioM1 which was significantly higher compared to the control (32.27±7.02%; p=0.01). Bone architecture (trabecular number, trabecular thickness) did not significantly differ from the self-healed defects. Both biomaterials, especially BioM1 showed good osseointegration and bone formation characteristics and can be recommended for further examination in periodontal regeneration studies.

Histological and Histomorphometric Evaluation of Implanted Photodynamic Active Biomaterials for Periodontal Bone Regeneration in an Animal Study

Recently, our group developed two different polymeric biomaterials with photodynamic antimicrobial surface activity for periodontal bone regeneration. The aim of the present study was to analyze the biocompatibility and osseointegration of these materials in vivo. Two biomaterials based on urethane dimethacrylate (BioM1) and tri-armed oligoester-urethane methacrylate (BioM2) that additionally contained ß-tricalcium phosphate and the photosensitizer mTHPC (meso-tetra(hydroxyphenyl)chlorin) were implanted in non-critical size bone defects in the femur (n = 16) and tibia (n = 8) of eight female domestic sheep. Bone specimens were harvested and histomorphometrically analyzed after 12 months. BioM1 degraded to a lower extent which resulted in a mean remnant square size of 17.4 mm², while 12.2 mm² was estimated for BioM2 (p = 0.007). For BioM1, a total percentage of new formed bone by 30.3% was found which was significant higher compared to BioM2 (8.4%, p < 0.001). Furthermore, BioM1 was afflicted by significant lower soft tissue formation (3.3%) as compared to BioM2 (29.5%). Additionally, a bone-to-biomaterial ratio of 81.9% was detected for BioM1, while 8.5% was recorded for BioM2. Implantation of BioM2 caused accumulation of inflammatory cells and led to fibrous encapsulation. BioM1 (photosensitizer-armed urethane dimethacrylate) showed favorable regenerative characteristics and can be recommended for further studies.

OSTEOGENIC POTENTIAL OF NANOSTRUCTURED CARBONATED HYDROXIAPATITE MICROSPHERES ASSOCIATED WITH MESENCHYMAL STEM CELL IN VITRO AND IN VIVO

<h3>Background</h3> Bone tissue is capable of self-healing in the event of injuries. However, situations such as the patient‘s age or size of the damage can compromise natural healing, bringing to the fore the need for interventions to treat bone injuries. Tissue engineering presents treatment alternatives involving the use of cells and associated biomaterials. In this context, studies using biomaterials claim that carbonated hydroxyapatite (CHA) is more resorbable than hydroxyapatite (HA) and has biological similarity to bone hydroxyapatite. <h3>Methods</h3> Parameters of indirect contact (cytotoxicity, proliferation, and migration) of the extract obtained from spheres of carbonated hydroxyapatite associated with murine bone marrow stem cells (BMSCs) in vitro were evaluated. Then the osteogenic potential of CHA extract in murine BMSCs in vitro was assessed by quantification of calcium matrix, alkaline phosphatase levels and gene expression Col1A1 and Bglap. Non-critical size bone defects were performed in rat femur. After 14 days of healing, bone blocks were obtained for analysis from treated animals and compared to non-treated animals by histological and histomorphometric evaluation. <h3>Results</h3> The CHA extract showed osteoinductive properties in vitro without the need to add osteogenic inducing medium. The ability to induce osteogenic differentiation in undifferentiated cells was confirmed by increased activity of alkaline phosphatase, formation of mineralized nodules and increased expression of the Col1A1 and Bglap genes after 7 and 14 days of differentiation. All groups presented bone neoformation at the injury site 14 days after the operation. Histomorphometric analysis showed no statistical difference between groups in the parameters of bone formation, biomaterial and connective tissue. <h3>Conclusion</h3> The CHA spheres have in vitro osteoinductive properties and, when loaded with murine BMSCs, have the potential for bone repair and regeneration in vivo, presenting biocompatibility and osteoconductive, making it a promising alternative for bone tissue engineering applications.

Preventive Moderate Continuous Running-Exercise Conditioning Improves the Healing of Non-Critical Size Bone Defects in Male Wistar Rats: A Pilot Study Using µCT.

Although physical exercise has unquestionable benefits on bone health, its effects on bone healing have been poorly investigated. This study evaluated the effects of preemptive moderate continuous running on the healing of non-critical sized bone defects in rats by µCT. We hypothesized that a preemptive running exercise would quicken bone healing. Twenty 5-week-old, male, Wistar rats were randomly allocated to one of the following groups (n = 10): sedentary control (SED) or continuous running (EX, 45 min/d, 5 d/week at moderate speed, for 8 consecutive weeks). A 2 mm diameter bone defect was then performed in the right tibia and femur. No exercise was performed during a 4 week-convalescence. Healing-tissue trabecular microarchitectural parameters were assessed once a week for 4 weeks using µCT and plasma bone turnover markers measured at the end of the study protocol (time point T12). At T12, bone volume fraction (BV/TV; BV: bone volume, TV: tissue volume) of the healing tissue in tibiae and femurs from EX rats was higher compared to that in SED rats (p = 0.001). BV/TV in EX rats was also higher in tibiae than in femurs (p < 0.01). The bone mineral density of the healing tissue in femurs from EX rats was higher compared to that in femurs from SED rats (p < 0.03). N-terminal telopeptide of collagen type I in EX rats was decreased compared to SED rats (p < 0.05), while no differences were observed for alkaline phosphatase and parathyroid hormone. The study provides evidence that preemptive moderate continuous running improves the healing of non-critical sized bone defects in male Wistar rats.

Transmission Electron Microscopic Examination of the Healing Power Effect of Milled Teeth Versus Beta- Tri Calcium Phosphate Grafted in Calvarial Bone Defect in Rats.

Objectives: to evaluate the effect of milled teeth versus beta tricalcium phosphate bone graft material on new bone formation at the cell level using transmission electron microscope Materials and methods: Forty-five adult male albino rats, were divided into three main groups (n=15); control and two experimental groups. Under general anesthesia, 1.5 cm incision was done exposing the cranial bone. Then two noncritical size bone defects were created one in each parital sides of the rat calvarial bone. In Group I (control group): the bilateral calvarial bony defects were left to heal spontaneously. In Group II the bilateral calvarial bony defects completely filled with β-TCP. In Group III: the bilateral calvarial bony defects completely filled with tooth ash. Five rats from each group were sacrificed after 7, 15, and 30 days postoperatively. Results: tooth ash group: showed more new bone formation. Different types of cells appeared more active: multiple lymphocytes, fibroblast, osteoprogenitor cells, osteoblasts. Multinucleated osteoclasts with prominent vacuolated cytoplasm and ruffled border in Howship’s lacunae were clearly seen. At day 30, disappearing of the tooth ash and infiltration of different types of cells; osteoprogenitor cells, bone lining cells, and osteoblasts while β-TCP particles were still seen in the sections. Conclusion: Bone activity, formation and maturity were ahead in tooth ash filled calvarial bony defects. Tooth ash had a role in the stimulation of osteoblastic activity in matrix formation and its mineralization.

The Obliteration of Noncritical Size Bone Defects With Bone Dust or Bone Replacement Material (Bioactive Glass S53P4).

Bone dust (BD) harvested during operation may be suitable as an autologous obliteration material for noncritical size defects. Bioactive glass (BA) can be an alternative. To treat noncritical size defects, BD and BA are commonly used for obliteration techniques. However, the optimal harvesting method and parameters for BD have not been examined. In this study, we analyzed the osseoregenerative potential of both materials. Thirteen female merino sheep (7-yr old) underwent surgery on the frontal calvaria. Three defects were inserted. The first defect was considered a reference and remained unfilled, the second defect was filled with BD from the calvaria bone, and the third defect was filled with BA S53P4. The animals were sacrificed after 3 weeks. To evaluate bone regeneration, we used digital volume tomography, bone density measurement, fluorochrome sequence labeling, and histological analysis. All analyses showed quantitative and qualitative bone regeneration 3 weeks after operation. The control blank defect showed significantly less new bone growth than the BD-filled defect. Moreover, bone regeneration occurred from the surrounding bone and showed only a defect bridge in the BD-filled defect. The BA completely filled the defect and had the highest density although the same amount of new mineralized bone generated as in the reference. BD and BA seemed to be suitable bone replacement materials for obliteration techniques because they completely filled the defects. Thus, BD harvested under standardized conditions provided a higher level of osteoreparation potential for the generation of woven bone and establishment of defect bridges.

Histological Evaluation of Bone Repair with Hydroxyapatite: A Systematic Review.

The aim of this study was to evaluate the morphological bone response in animal experiments by applying hydroxyapatite grafts in critical and non-critical size bone defects. Current report followed the guidelines established by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. Animal experiments were selected by assessing repair of bone defects with hydroxyapatite as bone graft and with blood clot only as control. Eight articles were identified in specialized literature and included in the meta-analysis. Statistical analysis was carried out with a random-effect model (p=0.05). Subgroup analyses were further performed to investigate bone repair in critical and non-critical bone defects. Comprehensive analysis of bone repair outcome showed a statistically significant difference between hydroxyapatite and blood clot control (p<0.05). Subgroup analyses showed statistically significant difference for critical bone defects (p<0.05). No statistically significant difference was reported in non-critical bone defects (p>0.05). Although animal studies revealed a high risk of bias and results should be interpreted with caution, the literature suggests that non-critical bone defects may heal spontaneously and without the need of a bone graft. Conversely, when critical-size defects are present, the use of hydroxyapatite bone graft improves the bone repair process.

The effect of L-PRF membranes on bone healing in rabbit tibiae bone defects: micro-CT and biomarker results

More insight into the biological fundamentals of leukocyte platelet-rich fibrin (L-PRF) guided healing is necessary to recommend its application, in particular in deficient bone sites that need to support implants. This study investigated the short-term bone healing effect of L-PRF treatment in cylindrical non-critical sized bone defects with 3 mm diameter and 6 mm depth in tibiae of 18 adult male New Zealand White rabbits. After a randomization process, 96 bone defects were prepared and half of them were filled with a L-PRF membrane, while untreated defects in the opposite tibia served as control group. The rabbits were euthanized after 7, 14 or 28 days of healing. The bone healing of the cortical and medullary areas was investigated by micro-CT, while the expression of molecular markers (RUNX2, VEGFA, COL1A2 and BMP2) was assessed by qRT-PCR. Treatment with L-PRF did not affect the micro-structural bone characteristics of the repaired bone tissue, except for a decrease in the trabecular connectivity at the cortical level after 14 days of healing. At this time, RUNX2 and VEGFA mRNA levels were significantly lower in the treated defects. L-PRF membranes thus had a temporary negative influence on the bone microarchitecture (Tb.Pf) and on the RUNX2 and VEGFA expression during early bone healing. Overall, L-PRF treatment did not enhance bone regeneration in these non-critical size defects after 28 days.

Effects of low-level laser therapy on the expression of osteogenic genes during the initial stages of bone healing in rats: a microarray analysis.

This study evaluated the morphological changes produced by LLLT on the initial stages of bone healing and also studied the pathways that stimulate the expression of genes related to bone cell proliferation and differentiation. One hundred Wistar rats were divided into control and treated groups. Noncritical size bone defects were surgically created at the upper third of the tibia. Laser irradiation (Ga-Al-As laser 830nm, 30mW, 94s, 2.8J) was performed for 1, 2, 3, 5, and 7 sessions. Histopathology revealed that treated animals produced increased amount of newly formed bone at the site of the injury. Moreover, microarray analysis evidenced that LLLT produced a significant increase in the expression TGF-β, BMP, FGF, and RUNX-2 that could stimulate osteoblast proliferation and differentiation, which may be related to improving the deposition of newly formed bone at the site of the injury. Thus, it is possible to conclude that LLLT improves bone healing by producing a significant increase in the expression of osteogenic genes.

Effects of low-level laser therapy on the expression of osteogenic genes related in the initial stages of bone defects in rats

We evaluate the effects of low-level laser therapy (LLLT) on the histological modifications and temporal osteogenic genes expression during the initial phase of bone healing in a model of bone defect in rats. Sixty-four Wistar rats were divided into control and treated groups. Noncritical size bone defects were surgically created at the upper third of the tibia. Laser irradiation (Ga-Al-As laser 830 nm, 30 mW, 0.028 cm², 1.071 W/cm², 1 min and 34 s, 2.8 Joules, 100 J/cm²) was performed for 1, 2, 3, and 5 sessions. Histopathology revealed that treated animals presented higher inflammatory cells recruitment, especially 12 and 36 h postsurgery. Also, a better tissue organization at the site of the injury, with the presence of granulation tissue and new bone formation was observed on days three and five postsurgery in the treated animals. The quantitative real time polymerase chain reaction showed that LLLT produced a significantly increase in mRNA expression of Runx-2, 12 h and three days post-surgery, a significant upregulation of alkaline phosphatase mRNA expression after 36 h and three days post-surgery and a significant increase of osteocalcin mRNA expression after three and five days. We concluded that LLLT modulated the inflammatory process and accelerated bone repair, and this advanced repair pattern in the laser-treated groups may be related to the higher mRNA expression of genes presented by these animals.

Ultrasound therapy modulates osteocalcin expression during bone repair in rats

The aim of this study was to measure the temporal pattern of the protein expression of RUNX2, RANKL, OPG, and osteocalcin after ultrasound therapy during the process of bone healing by immunohistochemistry. The animals were randomly distributed into two groups: control or ultrasound-treated group. A non-critical size bone defects were surgically created at the upper third of the tibia. The treatments started 24 h post-surgery, and they are performed for 3, 6, and 12 sessions, with an interval of 48 h. A low-intensity pulsed ultrasound (1.5 MHz, 1:4 duty cycle, intensity SATA 30 mW/cm 2, 20 min/session, stationary mode application) was used. On days 7, 13, and 25 post-injury, rats were killed individually by carbon dioxide asphyxia. The tibias were removed for analysis. The histopathological analysis pointed out no remarkable differences between groups for all periods evaluated. However, immunohistochemical data revealed that ultrasound therapy produced an up-regulation of osteocalcin at day 7th and 13th post-surgery. Taken together, our results indicate that ultrasound therapy modulates osteocalcin expression during bone repair in rats as depicted by differential immunopression at the initial and intermediate stages of recovery.

Ameloblastin is not implicated in bone remodelling and repair

Ameloblastin (AMBN) is an enamel matrix protein produced by ameloblasts. It has been suggested that AMBN might also be implicated in craniofacial bone formation. Our objective was to determine whether AMBN has an effect on osteogenic mineralisation and influences bone remodelling and repair. MC3T3-E1 cells were screened for endogenous expression of enamel proteins using real time PCR. Various osteogenic cells were infected with lentivirus encoding for AMBN and protein expression was verified using immunochemistry. Cultures were stained with alizarin red and mineralisation was quantified. Healing bone was probed for expression of AMBN by DNA microarray analysis. Tooth extraction, experimental tooth movement (ETM), and creation of a non-critical size bone defect in the tibia (BDT) were carried out in wild type and AMBN(Δ5-6) mutant mice. Tissues were processed for immunolabelling of AMBN and Bril, an osteoblast specific protein associated with active bone formation. MC3T3-E1 cells and healing bone showed no significant expression of AMBN. Overexpression of AMBN in osteogenic cultures induced no noticeable changes in mineralisation. In wild type mice, AMBN was immunodetected in ameloblasts and enamel, but not in normal bone, and at sites where bone remodelling and repair were induced. Bone remodelling during ETM and BDT repair in AMBN(Δ5-6) mice were not significantly different from that in wild type animals. Our results suggest that AMBN does not influence osteogenic activity in vitro under the conditions used, and does not participate in craniofacial bone remodelling under mechanical stress and in repair of non-critical size bone defects.

Low-Level Laser Therapy Induces Differential Expression of Osteogenic Genes During Bone Repair in Rats

The aim of this study was to measure the temporal pattern of the expression of osteogenic genes after low-level laser therapy during the process of bone healing. We used quantitative real-time polymerase chain reaction (qPCR) along with histology to assess gene expression following laser irradiation on created bone defects in tibias of rats. The animals were randomly distributed into two groups: control or laser-irradiated group. Noncritical size bone defects were surgically created at the upper third of the tibia. Laser irradiation started 24 h post-surgery and was performed for 3, 6, and 12 sessions, with an interval of 48 h. A 830 nm laser, 50 J/cm(2), 30 mW, was used. On days 7, 13, and 25 post-injury, rats were sacrificed individually by carbon dioxide asphyxia. The tibias were removed for analysis. The histological results revealed intense new bone formation surrounded by highly vascularized connective tissue presenting slight osteogenic activity, with primary bone deposition in the group exposed to laser in the intermediary (13 days) and late stages of repair (25 days). The quantitative real-time PCR showed that laser irradiation produced an upregulation of BMP-4 at day 13 post-surgery and an upregulation of BMP4, ALP, and Runx 2 at day 25 after surgery. Our results indicate that laser therapy improves bone repair in rats as depicted by differential histopathological and osteogenic genes expression, mainly at the late stages of recovery.