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.

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

AimThis 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.Material and MethodsSixty 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).ResultsGroup 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).ConclusionWithin 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.

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

Objective. 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.Study design. 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).Results. 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).Conclusion. PRP placed in the defects significantly enhanced bone healing in CSD in the calvaria of diabetic rats both qualitatively and quantitatively. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109: 72-78)

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

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Influence of sample type and collection method on Streptococcus mutans and Lactobacillus spp. counts in the oral cavity

Objectives: This study compared three methods of Streptococcus mutans and Lactobacillus spp. detection in the oral cavity: saliva swab (SS)-sample of stimulated saliva collected with swab; whole saliva (WS)-sample of 2 ml of stimulated saliva; and the dental plaque method (DP)-plaque sample of all dental surfaces.Methods: Thirty children were included in this study. In the first 15 children, the SS and WS methods were carried out before the dental plaque collection, and in the following 15, the sequence was inverted to evaluate possible interference of the methods sequence. The samples were diluted and inoculated in SB20 and Rogosa agar, respectively for S. mutans and Lactobacillus spp., at 37 degrees C for 48 h.Results: the results (cfu/mL) of S. mutans were analysed by the statistical Friedman's test. The levels of Lactobacillus spp. were analysed by descriptive statistics due to the high proportion of zero counts in the culture. In the first sequence of methods, the number of S. mutans counted for the SS method was inferior to DP and WS (P < 0.05), and the results for the WS and DP methods were similar. The detection of Lactobacillus spp. was observed just by the WS (100 %) and SS (14.3 %) methods. However, in the second experimental set the number of S. mutans detected by the DP method was similar to those of the SS and WS, however, the WS method showed higher values than SS (P < 0.05). A greater number of Lactobacillus spp. was detected by the WS method (100 %), followed by SS (55.5 %) and DP (33.3 %).Conclusions: the dental plaque collection and the sample of stimulated whole saliva presented similar results in the S. mutans count. The most suitable method to detect the Lactobacillus spp. level in the oral cavity is the stimulated whole saliva method. (c) 2004 Elsevier Ltd. All rights reserved.

Platelet-rich plasma stimulates cytokine expression and alkaline phosphatase activity in osteoblast-derived osteosarcoma cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bioglass Associated With Leukocyte-Poor Platelet-Rich Plasma in the Rabbit Maxillary Sinus: Histomorphometric, Densitometric, and Fractal Analysis

The objective of the present study was to evaluate the outcomes of autogenous bone graft (AB) and bioglass (BG) associated or not with leukocyte-poor platelet-rich plasma (LP-PRP) in the rabbit maxillary sinus (MS) by histomorphometric and radiographic analysis. Twenty rabbits divided into 2 groups (G1, G2) were submitted to sinus lift surgery. In G1, 10 MS were grafted with AB and 10 MS were grafted with BG. In G2, 10 MS were grafted with AB + LP-PRP and 10 MS were grafted with BG + LP-PRP. After 90 days, the animals were killed and specimens were obtained, x-rayed, and submitted to histomorphometric, radiographic bone density (RD) and fractal dimension analysis. Radiographic bone density mean values (SD), expressed as aluminum equivalent in mm, of AB, BG, AB + LP-PRP, and BG + LP-PRP groups were 1.79 (0.31), 2.04 (0.39), 1.61 (0.28), and 1.53 (0.30), respectively. Significant differences (P < 0.05) were observed between BG and AB, and BG + PRP and BG. Fractal dimension mean values were 1.48 (0.04), 1.35 (0.08), 1.44 (0.04), and 1.44 (0.06), respectively. Significant differences were observed between BG and AB, and AB + LP-PRP and BG. Mean values for the percentage of bone inside MS were 63.30 (8.60), 52.65 (10.41), 55.25 (7.01), and 51.07 (10.25), respectively. No differences were found. No correlations were observed among percentage of bone, RD and FD. Histological analysis showed that MS treated with AB presented mature and new bone formation. The other groups showed minor bone formation. Within the limitations of this study, the results indicated that at a 90-day time end point, AB yielded better results than AB + LP-PRP, BG, and BG + LP-PRP and should be considered the primary material for MS augmentation.

Effect of Platelet-Rich Plasma on Peri-Implant Bone Repair: A Histologic Study in Dogs

The present study evaluated the effect of platelet-rich plasma (PRP) on pen-implant bone healing. A total of 9 mongrel dogs received 36 dental implants with sandblasted acid-etched surface in lower jaws in a split-mouth design: in the PRP group (n = 18 implants) the implants were placed in association with PRP, and in the control group (n = 18 implants) the implants were placed without PRP. Biopsies were obtained and prepared for histologic and histometric analysis after 15, 30, and 55 days of healing. The biopsies retrieved at 15 days showed delicate bone trabeculae formed by immature bone with presence of numerous osteoblasts for both groups. At 30 days the trabeculae presented reversal lines and evident lamellar disposition, where some thread spaces were filled by bone and dense connective tissue. At 55 days, bone healing was not altered in the control group, and histologic aspects were variable for the group treated with PRP. There was no significant difference between the groups for bone-to-implant contact (P > .05). PRP did not enhance bone formation around sandblasted acid-etched implants.

Human Platelet Antigen Genotype Is Associated With Progression of Fibrosis in Chronic Hepatitis C

Although progression of fibrosis in the chronic hepatitis C depends on environmental, viral, and host factors, genetic polymorphisms have been associated recently with this progression, including the expression of integrins, adhesion proteins. Some integrins expressed on the platelet membrane show polymorphic antigenic determinants called human platelet antigens (HPA), where the major ones are HPA-1, -3, -5. The association between HCV infection and HPA-5b has been demonstrated. Similarly, the HPA profile could determine if HPA is related to progression of fibrosis. The goal of this study was to evaluate the association between the frequencies of HPA-1, -3, and -5 and degree of fibrosis in HCV-infected patients. Genomic DNA from 143 HCV-infected patients was used as the source for HPA genotyping by PCR-SSP or PCR-RFLP. Progression of fibrosis was evaluated using the METAVIR scoring system, and the patients were grouped according to degree of fibrosis into G1 (n = 81, with F1, portal fibrosis without septa or F2, few septa) and G2 (n = 62, with F3, numerous septa, or F4, cirrhosis). Statistical analysis was performed using the proportional odds model. The genotypic frequency of HPA-1a/1b was significantly higher in the patients in G2. To evaluate the influence of the time of infection to the development of fibrosis and its effect on the genetic factor HPA-1, 96 patients from 143 studied were evaluated considering the time of HCV infection, and these results suggest that the HPA-1a/1b genotype promotes the development of fibrosis in HCV infection with time. J. Med. Virol. 84: 56-60, 2012. (C) 2011 Wiley Periodicals, Inc.

Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops jararacussu snake venom

An acidic (pI similar to 4.5) phospholipase A(2) (BthA-I-PLA(2)) was isolated from Bothrops jararacussu snake venom by ion-exchange chromatography on a CM-Sepharose column followed by reverse phase chromatography on an RP-HPLC C-18 column. It is an similar to13.7 kDa single chain Asp49 PLA(2) with approximately 122 amino acid residues, 7 disulfide bridges, and the following N-terminal sequence: 'SLWQFGKMINYVMJGESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCC(51). Crystals of this acidic protein diffracted beyond 2.0 Angstrom resolution. These crystals are monoclinic and have unit cell dimensions of a = 33.9, b = 63.8, c = 49.1 Angstrom, and beta = 104.0degrees. Although not myotoxic, cytotoxic, or lethal, the protein was catalytically 3-4 tithes more active than BthTX-II, a basic D49 myotoxic PLA(2) from the same venom and other Bothrops venoms. Although it showed no toxic activity, it was able to induce time-independent edema, this activity being inhibited by EDTA. In addition, BthA-I-PLA(2) caused a hypotensive response in the rat and inhibited platelet aggregation, Catalytic, antiplatelet and other activities were abolished by chemical modification with 4-bromophenacyl bromide, which is known to covalently bind to His48 of the catalytic site. Antibodies raised against crude B. jararacussu venom recognized this acidic PLA(2), while anti-Asp49-BthTX-II recognized it weakly and anti-Lys49-BthTX-I showed the least cross-reaction. These data confirm that myotoxicity does not necessarily correlate with catalytic activity in native PLA(2) homologues and that either of these two activities may exist alone. BthA-I-PLA(2), in addition to representing a relevant molecular model of catalytic activity, is also a promising hypotensive agent and platelet aggregation inhibitor for further studies. (C) 2002 Elsevier B.V. All rights reserved.

Crystal structure of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) in the monomeric and dimeric states: insights into its oligomeric state

Phospholipases A(2) belong to the superfamily of proteins which hydrolyzes the sn-2 acyl groups of membrane phospholipids to release arachidonic acid and lysophospholipids. An acidic phospholipase A(2) isolated from Bothrops juraracussu snake venom presents a high catalytic, platelet aggregation inhibition and hypotensive activities. This protein was crystallized in two oligomeric states: monomeric and dimeric. The crystal structures were solved at 1.79 and 1.90 Angstrom resolution, respectively, for the two states. It was identified a Na+ ion at the center of Ca2+-binding site of the monomeric form. A novel dimeric conformation with the active sites exposed to the solvent was observed. Conformational states of the molecule may be due to the physicochemical conditions used in the crystallization experiments. We suggest dimeric state is one found in vivo. (C) 2004 Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray diffraction studies of BmooPLA(2)-I, a platelet-aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops moojeni venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Platelet hyperaggregability in high-fat fed rats: A role for intraplatelet reactive-oxygen species production

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crystallization and preliminary diffraction data of a platelet-aggregation inhibitor from the venom of Agkistrodon piscivorus piscivorus (North American water moccasin)

Applaggin (Agkistrodon piscivorus piscivorus platelet-aggregation inhibitor) is a potent inhibitor of blood platelet aggregation derived from the venom of the North American water moccasin, the protein consists of 71 amino acids, is rich in cysteines, contains the sequence-recognition site of adhesion proteins at positions 50-52 (Arg-Gly-Asp) and shares high sequence homology with other snake-venom disintegrins such as echistatin, kistrin and trigramin, Single crystals of applaggin have been grown and X-ray diffraction data have been collected to a resolution of 3.2 Angstrom. The crystals belong to space group P4(1)2(1)2 (or its enantiomorph), with unit-cell dimensions a = b = 63.35, c = 74.18 Angstrom and two molecules per asymmetric unit. Molecular replacement using models constructed from the NMR structures of echistatin and kistrin has not been successful in producing a trial structure for applaggin.