Created Critical-size Defects Research Articles

Bone marrow coagulated and low-level laser therapy accelerate bone healing by enhancing angiogenesis, cell proliferation, osteoblast differentiation, and mineralization.

The present study evaluated bone marrow aspirate (BMA) and low-level laser therapy (LLLT) on bone healing. It was created critical-size defects (CSD) of 5 mm diameter in rat calvaria of 64 rats. Animals were randomly divided into four groups: Control (blood clot), BMA (coagulated BMA), LLLT (laser irradiation and blood clot), and BMA/LLLT (laser irradiation and coagulated BMA). Euthanasia was performed at 15 or 30 days postoperative. Immunohistochemical reactions were performed to identify vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA), runt-related transcription factor-2 (Runx2), bone morphogenetic protein-2 (BMP-2), osteocalcin (OCN), and osteopontin (OPN). The markers were quantified, and data were statistically analyzed. Groups BMA/LLLT and LLLT presented significantly higher VEGF expression than group control. Group BMA/LLLT presented a significantly higher expression of PCNA than all experimental groups. Groups BMA and BMA/LLLT presented significantly higher expression of BMP-2 than all experimental groups. Groups LLLT and BMA/LLLT presented significantly higher expression of OPN than groups control and BMA. Groups LLLT, BMA, and BMA/LLLT presented a significantly higher expression of OCN than group control. It can be concluded that the association of BMA and LLLT enhanced bone healing by improving expression of VEGF, PCNA, Runx2, BMP-2, OPN, and OCN.

Effects of low-level laser therapy on bone healing of critical-size defects treated with bovine bone graft

ObjectiveTo histomorphometrically analyze the effect of low-level laser therapy (LLLT) on bone formation process in surgically created critical-size defects (CSDs) treated with bovine bone graft (BBG) and its influence over particles' resorption of BBG. MethodsA 10-mm diameter CSD was surgically created in the calvaria of 64 male rats, which were distributed into 4 experimental groups: the C group (control), only blood clot; the LLLT group, LLLT (GaAlAs, 660nm) and blood clot; the BBG group, CSD filled with BBG; the BBG/LLLT group, LLLT and CSD filled with BBG. Animals were euthanized at either 30 or 60days post-operation. A histological analysis was performed. Additionally, the percentage of newly formed bone area (NFBA) and remaining particles areas (RPA) of BBG were histometrically evaluated and data statistically analyzed. ResultsThe LLLT (5.82±2.05; 7.34±1.01) group presented significantly greater NFBA when compared to the C group (1.61±0.30; 5.59±0.94) at 30 and 60days post-operation (p<0.05). The BBG/LLLT group (7.39±1.45; 9.44±2.36) presented significantly greater NFBA than the BBG group (3.85±1.56; 8.02±0.63) at 30 and 60days postoperation (p<0.05). There was no significant difference in the mean percentage of implanted material RPA between the BBG and the BBG/LLLT groups. ConclusionsLLLT can improve bone formation process in CSD filled or not with BBG in rat calvaria, but it is not able to accelerate particles resorption of this material in the interior of bone defect.

Effects of oestrogen deficiency and 17β-estradiol therapy on bone healing in calvarial critical size defects treated with bovine bone graft

ObjectiveTo histomorphometrically analyze the effect of ovariectomy-induced oestrogen deficiency and 17β-estradiol therapy on bone healing of surgically created critical-size defects (CSDs) treated with bovine bone graft (BBG). MethodsForty-eight female rats were randomly assigned to the following 3 experimental groups (n=16): sham-operated animals (SHAM), ovariectomized animals (OVX) and ovariectomized animals treated with oestrogen (OVX+E2). OVX+E2 animals received daily subcutaneous injections of 17β-estradiol (20μg/kg) from 8 days after ovariectomy until euthanasia. Thirty days after the surgery, an 8mm CSD was surgically created in each calvaria of all animals and filled with BBG. Animals were euthanized at either 30 or 60 days postoperative. A histological analysis, percentage of Newly Formed Bone Area (NFBA), osteoblast and osteoclast number was histomorphometrically performed (p≤0.05). ResultsAt 30 days, SHAM group (8.82%±2.93) had significantly greater NFBA than OVX (4.66%±1.35) and OVX+E2 groups (5.85%±4.08) (p≤0.05). At 60 days, SHAM group (11.51%±3.08) and OVX+E2 group (9.84%±1.87) had significantly greater NFBA than OVX animals (5.12%±0.68) (p≤0.05). Fewer osteoblasts were observed in the OVX group at 30 (763.40±121.60) and 60 (696.60±80.92) days than in the SHAM group at 30 days (1356.00±95.38). Fewer osteoclasts were observed in the OVX+E2 group (3.25±2.16) than in the SHAM (9.75±1.82) and OVX (12.75±1.47) groups at 30 days (p≤0.05). ConclusionsOestrogen deficiency compromises bone healing in calvarial CSDs treated with BBG in ovariectomized rats. After 60 days post-surgery, 17β-estradiol therapy improved bone healing in calvarial CSDs treated with BBG in ovariectomized rats.

Bone marrow aspirate combined with low-level laser therapy: A new therapeutic approach to enhance bone healing

This study evaluated the influence of bone marrow aspirate (BMA), low-level laser therapy (LLLT) and their combination on bone healing in surgically created critical-size defects (CSDs) in rat calvaria. 40 rats were divided into four groups: C (control), BMA, LLLT and BMA/LLLT. A 5mmdiameter CSD was created in the calvarium of each animal. In Group C, the defect was filled by blood clot only. In Group BMA, the defect was filled with BMA. In groups LLLT and BMA/LLLT, the defect received laser irradiation (InGaAlP laser), was filled with blood clot or BMA respectively, and irradiated again. Animals were euthanized 30days postoperatively. Histomorphometric and immunohistochemical analyses were performed. Newly formed bone area (NFBA) was calculated as percentage of the total area of the original defect. Proliferating cell nuclear antigen (PCNA), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) immunohistochemical staining were performed. PCNA-positive, Runx2-positive and OCN-positive cells were quantified. Data were statistically analyzed. Group BMA/LLLT had significantly greater NFBA than groups C, BMA or LLLT. Group BMA presented significantly greater NFBA than control, while group LLLT did not. Group BMA/LLLT presented a significantly higher number of PCNA-positive and OCN-positive cells than any of the other groups. Groups BMA/LLLT and BMA showed a significantly lower number of Runx2-positive cells than groups C or LLLT. The combination of BMA/LLLT yielded significantly greater bone formation in surgically created CSD in rat calvaria when compared to control, or either treatment alone.

Effects of LLLT in combination with bisphosphonate on bone healing in critical size defects: a histological and histometric study in rat calvaria

The purpose of this study was to analyze histologically the effect of low-level laser therapy (LLLT) in combination with bisphosphonate on bone healing in surgically created critical size defects (CSD) in rat calvaria. One hundred Wistar female rats sham operated (sham) and ovariectomized (Ovx) were maintained untreated for 1month to allow for the development of osteopenia in the Ovx animals. A CSD was made in the calvarium of each rat, and the animals were divided into five groups according to following treatments: (1) sham rats (control), (2) Ovx rats, (3) Ovx rats treated with LLLT, (4) Ovx rats treated with bisphosphonate, and (5) Ovx rats treated with bisphosphonate and LLLT. Groups 4 and 5 were irrigated with 1ml of bisphosphonate, and groups 3 and 5 were submitted to LLLT (GaAlAs), 660nm, 24J, and 0.4285W/cm(2) on the CSD. Ten animals of each treatment were killed at 30 and 60days. Histomorphometric assessments, using image analysis software, and histological analyses were performed. No defect was completely regenerated with the bone. Histometrically, it can be observed that groups 3 (37.49 ± 1.94%, 43.11 ± 2.39%) and 5 (35.05 ± 1.57%, 41.07 ± 1.89%) showed a significant bone neoformation when compared to groups 1 (16.81 ± 1.57%, 27.54 ± 1.49%), 2 (11.68 ± 0.98%, 22.51 ± 1.05%), and 4 (14.62 ± 1.70%, 25.67 ± 1.41%) in all experimental periods (P < 0.05). It was possible to conclude that the LLLT associated or not with bisphosphonate treatment was effective for stimulating bone formation in CSD in the calvaria of rats submitted to ovariectomy.

Bone healing in critical-size defects treated with new bioactive glass/calcium sulfate: a histologic and histometric study in rat calvaria.

This study analyzed histologically the influence of new spherical bioactive glass (NBG) particles with or without a calcium sulfate (CS) barrier on bone healing in surgically created critical-size defects (CSD) in rat calvaria. A CSD was made in each calvarium of 60 rats, which were divided into three groups: C (control): the defect was filled with blood clot only; NBG: the defect was filled with NBG only; and NBG/CS: the defect was filled with NBG covered by CS barrier. Subgroups were euthanized at 4 or 12 weeks. Amounts of new bone and remnants of implanted materials were calculated as percentages of total area of the original defect. Data were statistically analyzed. In contrast to Group C, thickness throughout defects in Groups NBG and NBG/CS was similar to the original calvarium. At 4 weeks, Group C had significantly more bone formation than Group NBG/CS. No significant differences were found between Group NBG and either Group C or Group NBG/CS. At 12 weeks, Group C had significantly more bone formation than Group NBG or NBG/CS. NBG particles, used with or without a CS barrier, maintained volume and contour of area grafted in CSD. Presence of remaining NBG particles might have accounted for smaller amount of new bone in Groups NBG and NBG/CS at 12 weeks post-operative.

Bone healing in critical-size defects treated with platelet-rich plasma: a histologic and histometric study in the calvaria of diabetic rat

The objective of this study was to histologically analyze the influence of the platelet-rich plasma (PRP) on bone healing in surgically created critical-size defects (CSD) in the calvaria of diabetic rats. A total of 20 diabetic rats were divided into 2 groups: C (control) and PRP. A 5-mm diameter CSD was created in the calvarium of each animal. In Group C, the defect was filled by blood clot only. In Group PRP, 0.35 mL of PRP was placed in the defects. All animals were humanely killed 30 days postoperatively. Histometric and histologic analyses were performed. Data were statistically analyzed (t test, P < .05). No defect completely regenerated with bone. Group PRP had a statistically greater amount of bone formation than Group C (37.22% +/- 6.00% and 21.68% +/- 11.35%, respectively). PRP placed in the defects significantly enhanced bone healing in CSD in the calvaria of diabetic rats both qualitatively and quantitatively.

Effect of platelet‐rich plasma on bone healing of autogenous bone grafts in critical‐size defects

This study histologically analysed the effect of autogenous platelet-rich plasma (PRP), prepared according to a new semiautomatic system, on healing of autogenous bone (AB) grafts placed in surgically created critical-size defects (CSD) in rabbit calvaria. Sixty rabbits were divided into three groups: C, AB and AB/PRP. A CSD was created in the calvarium of each animal. In Group C (control), the defect was filled by blood clot only. In Group AB (autogenous bone graft), the defect was filled with particulate autogenous bone. In Group AB/PRP (autogenous bone graft with platelet-rich plasma), it was filled with particulate autogenous bone combined with PRP. All groups were divided into subgroups (n=10) and euthanized at 4 or 12 weeks post-operatively. Histometric and histologic analyses were performed. Data were statistically analysed (anova, t-test, p<0.05). Group C presented significantly less bone formation compared with Group AB and AB/PRP in both periods of analysis (p<0.001). At 4 weeks, Group AB/PRP showed a statistically greater amount of bone formation than Group AB (64.44 +/- 15.0%versus 46.88 +/- 14.15%; p=0.0181). At 12 weeks, no statistically significant differences were observed between Groups AB and AB/PRP (75.0 +/- 8.11%versus 77.90 +/- 8.13%; p>0.05). It is notable that the amount of new bone formation in Group AB/PRP at 4 weeks was similar to that of Group AB at 12 weeks (p>0.05). Within its limitation, the present study has indicated that (i) AB and AB/PRP significantly improved bone formation and (ii) a beneficial effect of PRP was limited to an initial healing period of 4 weeks.

Influence of the proportion of particulate autogenous bone graft/platelet-rich plasma on bone healing in critical-size defects: An immunohistochemical analysis in rat calvaria

This immunohistochemical analysis evaluated the influence of the proportion of particulate autogenous bone (AB) graft/platelet-rich plasma (PRP) on bone healing in surgically created critical-size defects (CSD) in rat calvaria. 25 rats were divided into 5 groups: C (control), AB, AB/PRP-50, AB/PRP-100 and AB/PRP-150. A 5 mm diameter critical-size defect was created in the calvarium of each animal. In Group C, defect was filled by blood clot only. In Group AB, defect was filled with AB graft. In Groups AB/PRP-50, AB/PRP-100 and AB/PRP-150, defects were filled with AB graft combined with 50, 100 and 150 μl of PRP, respectively. Animals were euthanized at 30 days post-operative. Osteocalcin (OC) and osteopontin (OP) immunohistochemical staining were performed. OP-positive and OC-positive cells were quantified. The amount of vital bone (VBA) and remaining bone graft particles (RPA) were calculated as percentage of the total area of the original defect. Data were statistically analyzed (ANOVA, Tukey, p<0.05).Group AB/PRP-100 presented a significantly higher number of OP-positive cells than groups C, AB/PRP-50 and AB/PRP-150 (p<0.05) and also a significantly higher number of OC-positive cells than all of the other groups (p<0.05). The number of OP-positive cells in Group AB/PRP-150 was significantly lower than in Group AB (p<0.05). Statistically significant differences were not found between groups with regard to either VBA or RPA (p>0.05). However, clear trends were indicated by an increase in VBA in Group AB/PRP-100 when compared to groups C, AB and AB/PRP-150 and an increase in RPA in Group AB/PRP-150 when compared to groups AB, AB/PRP-50 and AB/PRP-100.The proportion AB graft/PRP seems to influence bone healing in CSD in rat calvaria. The highest expression of osteocalcin and osteopontin, and thus the most developed healing, was found in Group AB/PRP-100 indicating that there may be an optimal proportion beyond which increasing the concentration PRP is deleterious.

Influence of the ratio of particulate autogenous bone graft/platelet-rich plasma on bone healing in critical-size defects: A histologic and histometric study in rat calvaria

The purpose of this study was to analyze histomorphometrically the influence of the ratio of particulate autogenous bone (AB) graft/platelet-rich plasma (PRP) on bone healing in surgically created critical-size defects (CSD) in rat calvaria. Fifty rats were divided into five groups: Group C (control), Group AB, Group AB/PRP-50, Group AB/PRP-100, and Group AB/PRP-150. A 5-mm diameter critical-size defect was created in the calvarium of each animal. In Group C, the defect was filled by blood clot only. In Group AB, the defect was filled with 0.01 mL of AB graft. In Groups AB/PRP-50, AB/PRP-100, and AB/PRP-150, the defects were filled with 0.01 mL of AB graft combined with 50, 100, and 150 microL of PRP, respectively. All animals were euthanized at 30 days postoperative. Histomorphometry, using image analysis software, and histology analyses were performed. New Bone Area (NBA) and the remaining bone graft particles area (RPA) were calculated as a percentage of the total area of the original defect. Percentage data were transformed into arccosine for analysis. No defect completely regenerated with bone. Group AB/PRP-50 (41.78 +/- 13.48%) had a significantly greater NBA than Groups C (19.29 +/- 5.11%), AB (27.43 +/- 10.90%) or AB/PRP-150 (19.17 +/- 8.45%) (p < 0.05). No significant differences were observed between groups AB/PRP-50 and AB/PRP-100 or among groups AB, AB/PRP-100, and AB/PRP-150 with regard to NBA (p > 0.05). Group AB/PRP-150 (31.59 +/- 3.22%) had a significantly greater RPA than Groups AB (19.09 +/- 5.21%), AB/PRP-50 (17.33 +/- 4.43%), and AB/PRP-100 (19.72 +/- 3.62%) (p < 0.001). No significant differences were observed among groups AB, AB/PRP-50, and AB/PRP-100 with regard to RPA (p > 0.05). The ratio AB graft/PRP influences bone healing in surgically created CSD in rat calvaria.

Bone healing in critical‐size defects treated with platelet‐rich plasma activated by two different methods. A histologic and histometric study in rat calvaria

The purpose of this study was to analyze histologically the influence of platelet-rich plasma (PRP) coagulated with two different activators on bone healing in surgically created critical-size defects (CSD) in rat calvaria. Forty-eight rats were divided into three groups: C, PRP-C and PRP-T. An 8 mm diameter CSD was created in the calvarium of each animal. In group C, the defect was filled by a blood clot only. In groups PRP-C and PRP-T, the defect was filled with PRP activated with either calcium chloride or thromboplastin solution, respectively. Each group was divided into two subgroups (n = 8 per subgroup) and killed at either 4 or 12 weeks postoperatively. Histologic and histometric analyses were performed. The amount of new bone formed was calculated as a percentage of the total area of the original defect. Percentage data were transformed into arccosine for statistical analysis (analysis of variance, Tukey's post hoc test, p < 0.05). No defect completely regenerated with bone. Group PRP-C had a statistically greater amount of bone formation than groups C and PRP-T at both time points of analysis. No statistically significant differences were observed between groups C and PRP-T. It can be concluded that the type of activator used to initiate PRP clot formation influences its biological effect on bone healing in CSD in rat calvaria.

Bone healing in critical‐size defects treated with bioactive glass/calcium sulfate: a histologic and histometric study in rat calvaria

The purpose of this study was to analyze histologically the influence of bioactive glass (BG) with or without a calcium sulfate (CS) barrier on bone healing in surgically created critical-size defects (CSD) in rat calvaria. A CSD was made in each calvarium of 48 rats. They were divided into three groups: C (control): blood clot only; BG: defect filled with BG; and BG/CS: defect filled with BG covered by a CS barrier. Animals were euthanized at 4 or 12 weeks. Formation of new bone was evaluated histomorphometrically. No defect completely regenerated with bone. BG particles were observed in Groups BG and BG/CS at both periods of analysis. The thickness throughout the healing area in Groups BG and BG/CS was similar to the original calvarium, while Group C presented a thin connective tissue in the center of the defect in both periods of analysis. At 4 weeks, Groups C and BG/CS presented significantly more bone formation than Group BG. No significant differences were found between Groups C and BG/CS. At 12 weeks, no significant differences in the amount of bone formation were observed among the three groups. When comparing 4 and 12 weeks, there was a significant increase in new bone formation within groups BG and BG/CS, but not C. BG particles, used with or without a CS barrier, maintained the volume and contour of the area grafted in CSD. However, they did not lead to a significant difference in bone formation when compared with control at 12 weeks post-operative.