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L-PRP diminishes bone matrix formation around autogenous bone grafts associated with changes in osteocalcin and PPAR-γ immunoexpression
Int. J. Oral Maxillofac. Surg. 2014; 43: 261–268 http://dx.doi.org/10.1016/j.ijom.2013.07.739, available online at http://www.sciencedirect.com
Research Paper Bone Regeneration
L-PRP diminishes bone matrix formation around autogenous bone grafts associated with changes in osteocalcin and PPAR-g immunoexpression
G. S. Portela, D. X. Cerci, G. Pedrotti, M. R. Araujo, T. M. Deliberador, J. C. Zielak, T. A. Costa-Casagrande, C. C. Gonzaga, A. F. Giovanini School of Dentistry, Positivo University, Curitiba, Parana´, Brazil
G.S. Portela, D.X. Cerci, G. Pedrotti, M.R. Araujo, T.M. Deliberador, J.C. Zielak, T.A. Costa-Casagrande, C.C. Gonzaga, A.F. Giovanini: L-PRP diminishes bone matrix formation around autogenous bone grafts associated with changes in osteocalcin and PPAR-g immunoexpression. Int. J. Oral Maxillofac. Surg. 2014; 43: 261–268. # 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. Leucocyte- and platelet-rich plasma (L-PRP) is an autogenous platelet concentrate enriched with leukocytes that releases various growth factors responsible for the proliferation, regulation, and differentiation of mesenchymal cells during wound healing. Since the bone and medullary tissue are contiguous and share the same origin, this study evaluated the effect of L-PRP on the repair of calvaria bone using histomorphometric analysis of the newly formed bone, and compared the results in the presence of osteocalcin (OC) and peroxisome proliferator-activated receptor gamma (PPAR-g) detected by immunohistochemistry. Artificial circular bone defects (5 mm diameter) were produced in the calvaria of 42 rats. The defects were treated with autograft, autograft combined with L-PRP, or without grafting material (sham). The animals were euthanized at 15 or 40 days postsurgery (n = 7 in each group). Data obtained were analyzed by Student–Newman–Keuls test for histomorphometric and immunohistochemical interpretation. The development of bone matrix was significantly less in the defects treated with L-PRP, while the medullary area composed of fatty cells was larger. This coincided with the minor expression of OC and expressive presence of PPAR-g. These results suggest that L-PRP may impair osteoneogenesis and alter the ratio of differentiation between bone matrix and fatty cells, increasing the medullary tissue.
0901-5027/020261 + 08 $36.00/0
Keywords: Platelet-rich plasma; Bone regeneration; Osteocalcin; PPAR-g. Accepted for publication 9 July 2013 Available online 8 August 2013
# 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
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Introduction
Platelet-rich plasma (PRP) is a non-toxic and non-antigenic autologous blood product enriched with platelets that has been considered an essential source of growth factors, which are thought to improve wound healing in both soft and hard tissue.1,2 Since Marx et al.3 reported that the use of PRP promotes faster maturation of autologous bone grafts and a consequent increase in new bone formation in mandibular defects, the use of PRP combined with autografts has been used frequently in the reconstruction of craniofacial deformities. However, the positive effects of PRP in osteoneogenesis are the subject of debate and several studies have revealed a failure of bone maturation when PRP is used.4,5 A relevant speculation in the divergent results produced by PRP combined with autograft has been assigned in particular to the fabrication of PRP. While the classical definition of PRP, as proposed by Marx et al.,3 would suggest a pure mixture of plasma and platelets, the usual definition of PRP has expanded to include a variety of final products; thus the PRP may vary markedly not only in the final concentration of platelets, but also in the amounts of leukocytes that are commonly included in the final preparation.6,7 Leukocytes are considered to play a key role in the preliminary phases of inflammation and they may contribute to the variation in results found in the literature regarding the efficiency of bone wound healing. This hypothesis seems likely given that these inflammatory cells commonly increase the levels of some platelet growth factors, especially transforming growth factor beta 1 (TGF-b1), which is also present in bone loss.8 The presence of platelet growth factors is considered important to bone turnover and repair, since they contribute to the chemotaxis of osteoprogenitor cells at the site of injury. In contrast, some studies have revealed that TGF-b1 in particular may also induce negative effects on osteoblastogenesis, since this cytokine antagonizes bone morphogenetic protein (BMP) expression and impairs osteoblast maturation; this may contribute to changes in the ratios of cellular differentiation during bone repair.9 Osteoblasts and adipocytes are the principal cells that constitute the microarchitecture of bone tissue. Commonly the adipocytes of the bone marrow and the osteoblasts are present in anatomically contiguous tissues and also derive from the same mesenchymal cell lineage.10
Whether adipogenesis or osteoneogenesis occurs is commonly associated with the activation of certain proteins and/or transcription factors, with one process suppressing the other. This constitutes the essential pathway by which cells commit to a specific differentiation lineage. The presence of these proteins detected by immunohistochemistry may identify a particular type of cell in bone development while the adipose differentiation pathways are inhibited, and vice versa. Osteocalcin (OC) is a vitamin K-dependent osteoblast-specific protein and is commonly produced and expressed by mature osteoblasts.11,12 OC may also accumulate in the extracellular matrix produced by osteoblasts and has a high affinity for hydroxyapatite and mineral ions, properties that are considered determinants for the development and deposition of bone matrix.13 Peroxisome proliferator-activated receptor gamma (PPAR-g) is expressed in adipocytes; these receptors are responsible for the regulation of the expression of genes that control lipid metabolism,14 and at the same time inhibit the actions of proinflammatory genes.15 In addition, PPAR-g inhibits osteoblastic differentiation and the expression of several anabolic mediators involved in bone formation, such as BMPs, type I collagen, and OC.16 It was hypothesized that leucocyte- and platelet-rich plasma (L-PRP) could alter the histophenotype of bone repair, changing the proportions of osteoblasts and adipocytes. Thus the aim of this study was to evaluate the ratio of OC to PPAR-g detected by immunohistochemistry, and to compare these results with a histomorphometric analysis of the bone matrix deposited in the presence and absence of L-PRP. Materials and methods
Forty-two male rats (Rattus norvegicus albinus, Wistar), 5–6 months old, weighing 450–500 g and with no previous disease were used in this study; the study protocol was approved by the institutional animal care and use committee. The rats were kept in a room with a controlled temperature (approximately 22 8C) and maintained under a 12-h light–dark cycle. PRP fabrication and quantification
A 3.2-ml sample of autologous blood was collected from each animal through cardiac puncture, into a syringe containing 0.35 ml of 10% sodium citrate. The blood samples were centrifuged at 200 g for
20 min at room temperature to separate the plasma containing the platelets from the red cells (Beckman J-6 M Induction Drive Centrifuge; Beckman Instruments Inc., Palo Alto, CA, USA). The plasma fraction was collected from the top of the supernatant. The remaining fractions were centrifuged once more at room temperature for 10 min at 400 g to separate the platelets. The platelet-poor plasma was removed from the upper level of the supernatant, leaving the PRP and buffy coat. Both the buffy coat and PRP (0.35 ml) were resuspended and activated with a mixture of 10% calcium chloride (0.05 ml/ml of PRP). They were then added to the previously prepared L-PRP and mixed for approximately 1 min until they formed a gel. The platelets and leukocytes in the LPRP were measured after centrifugation using a Coulter STKS haematology counting machine (Beckman-Coulter, Chicago, IL, USA). The average whole blood platelet count was 617.66 69.81 103 platelets/ ml, while the average L-PRP platelet count was 2826.44 309.12 103 platelets/ml (P 0.001). The average concentration of leukocytes in the L-PRP was 8.14 0.23 103 leukocytes/ml, while the average initial blood value was 3.18 0.91 103 leukocytes/ml (P 0.001). Surgical procedure
The rats were anesthetized by intramuscular injection of xylazine (5 mg/kg) and ketamine (70 mg/kg). The surgical region was shaved and aseptically prepared, and sterile barriers were created to limit the surgical area. A 5-cm midline dermo-periosteal incision was made to expose the calvarial surface with complete removal of the periosteum. Circular artificial defects of 5 mm in diameter were created with a trephine (Biomedical Research Instruments Inc., Silver Spring, MD, USA) under abundant saline solution irrigation in each rat. Bone fragments removed from the calvarial defects were particulated and used as autograft. In a randomized manner, one group received treatment with 0.01 ml of autograft and another group had the defect filled with 0.01 ml of autograft associated with 150 ml of L-PRP. The control animals (sham) underwent the same surgical procedures without the use of any grafting material. Soft tissues were repositioned and sutured to achieve primary closure (4-0 silk, Ethicon, Sao Paulo, SP, Brazil). Each animal received a prophylactic intramuscular injection of 24,000 IU of penicillin G benzathine and a daily dose of
L-PRP diminishes bone matrix formation around autogenous bone grafts associated with changes in osteocalcin 200 mg/kg/day of liquid acetaminophen administrated orally.
Euthanasia procedure and tissue processing
At 15 or 40 days postsurgery, animals were euthanized by brief exposure in a CO2 chamber until they ceased to move. The skull caps of each animal were removed in blocks using an inverted cone bur. Calvaria specimens obtained were fixed in 10% buffered formalin for 48 h and decalcified in 20% formic acid and sodium citrate for 7 days. The specimens were washed with tap water, dehydrated, cleared with xylene, and embedded in paraffin. Serial sections of 3 mm made parallel to the mid-sagittal suture were cut from the centre of each defect using a microtome (RM2155, Leica Microsystems GmbH, Nussloch, Germany) and stained with haematoxylin and eosin to permit histomorphological and histomorphometric evaluation.
Immunohistochemistry processing
Three-micrometre thicknesses of each specimen were deparaffinized and subjected to antigen retrieval in 1% trypsin solution (pH 1.8) for 60 min at 37 8C for the anti-OC and anti-PPAR-g antibodies. Slides containing the histological fragments were immersed in 3% hydrogen peroxide for 30 min to eliminate endogenous peroxidase activity, followed by incubation with 1% phosphate-buffered saline (PBS; pH 7.4). The sections were incubated with the primary antibody anti-OC (200 mg/ml; Santa Cruz Biotechnology Inc., CA, USA) with a dilution factor of 1:200, and anti-PPAR-g (200 mg/ml; Santa Cruz Biotechnology Inc., CA, USA) with a dilution factor of 1:150. A labelled streptavidin biotin antibody-binding detection system (Universal HRP Immunostaining Kit; Diagnostic BioSystems, Pleasanton, CA, USA) was employed to detect the primary antibodies. The immune reaction was observed with diaminobenzidine tetrachloride chromogen solution (Universal HRP Immunostaining Kit), which produces a brown precipitate at the antigen site. The specimens were counterstained with Harris haematoxylin. A negative control was included for all samples, using rabbit polyclonal isotype IgG (2 mg/ml, Abcam, Cambridge, UK) for 20 min at room temperature as a primary antibody. For each specimen, three slides were utilized for incubation with each antibody.
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Image analysis
L-PRP mixed with autograft
The images of both the histological and immunohistochemistry sections were captured by digital camera (SDC-310; Samsung, Gyeonggi-do, South Korea) coupled to a light microscope (Zeiss, Oberkochen, Germany) with an original magnification of 200. Each digital image was gathered and preserved at 300 dpi resolution, producing a virtual frame of 113 cm 80 cm. As it was not possible to capture the entire defect in one image at the magnification level used, a composite digital image of the whole defect was then constructed by combining five smaller images based on histomorphological reference structures, in particular the deposited bone trabeculae and blood vessels. All histomorphometric and immunohistochemical measurements were completed using the software Image J (National Institute of Health, Bethesda, MD, USA). A 1-mm calibration slide was used to calibrate all measurements. The histomorphometric data were compiled manually and expressed as areas (mm2). The perimeters of the deposited histological bone matrix were carefully traced and the areas computed. At the same time, positive OC and PPAR-g cells/mm2 were manually counted and tagged.
On day 15 postsurgery, inserted bone fragments were detected surrounded by rich mesenchymal cellular tissue. From day 15 to day 40 postsurgery, the amount of deposited bone matrix increased slightly, and a substantial quantity of medullary tissue represented by fatty cells was found.
Statistical analysis
Each parameter was evaluated within the monitoring period. Analysis of variance (ANOVA) was used to determine if there were significant differences among groups, followed by the Student–Newman–Keuls non-parametric test. A Pvalue of less than 0.05 was considered to be statistically significant.
Results Light microscopy analysis
The quantitative histomorphometric analysis data are given in Fig. 1. A brief description of the microscopic characteristics found in the groups is provided below and shown in Fig. 2.
Autograft
On postoperative day 15, bone fragments and new bone formation from the autograft bone were present. At this time point, discrete to mild granulation tissues were observed. On postoperative day 40, newly formed compact Haversian bone was identified.
Control – sham group
On day 15 postsurgery, the histological analysis revealed areas of granulation tissue composed of collagen and mesenchymal cells in the larger part of the defect. Further, there was evidence of newly formed immature bone that mimicked intramembranous ossification restricted to the peripheral area of the defect. At day 40 after surgery, granulation tissue was detected around a presence of both mature and immature Haversian bone tissue. Immunohistochemical results
The quantitative immunohistochemistry data are given in Fig. 1 (P < 0.05). In summary, the results can be described as follows. Osteocalcin (OC)
OC positivity was present in all specimens analyzed (Fig. 3). On day 15 postsurgery, the quantities of cells that exhibited positivity for this protein were significantly lower in the group that received L-PRP when compared to the autograft and sham groups. On postoperative day 40, the presence of OC had decreased in the autograft and autograft + L-PRP groups, as soon as the bone matrix or medullary tissue had matured; however the quantities of OC in the group that received L-PRP remained lower. In contrast, in the sham group, the presence of OC remained higher, expressed both in cells and bone matrix. Peroxisome proliferator-activated receptor gamma (PPAR-g)
A significant PPAR-g expression was seen only in the groups that received autograft (Fig. 4). In the earlier stages of the repair (15 days), the group that received only autograft presented scarce PPAR-g-positive cells, found around the blood vessels in the reparative granulation tissue, while the majority of cells that surrounded the grafted bone demonstrated positivity for PPAR-g in the autograft + L-PRP group. The pattern on day 40 postsurgery was
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Fig. 1. Graphical comparison of the groups analyzed. Average and standard deviation values of (A) bone matrix, (B) OC, and (C) PPAR-g. Values followed by the same superscript letter (a, b, and c) are statistically similar, P > 0.05.
similar, however the PPAR-g-positive cells in the specimens treated with LPRP exhibited an adipocyte-like histophenotype.
Discussion
The use of PRP in bone repair is based on the considerable number of platelets that
produce active growth factors, which can have a substantial impact on chemotaxis, proliferation, regulation, and differentiation of mesenchymal cells into osteoblasts.17 Despite the fact that activation of some growth factors by platelets is fundamental to osteoneogenesis, results demonstrating that PRP increases local bone formation1,3,17 have not been corroborated by some groups.4,5,18 The lack of agreement regarding the positive effect of PRP on osteoneogenesis may reflect variations in the PRP preparations used, which could alter the platelet quality and/or quantity, resulting in differences in the regenerative potential of PRP.6,7 A matter that should be taken into consideration in the variations in PRP preparations is the possible presence, or variation, in the amount of leukocytes.6,7 In a recent study performed by our group Diff id="43">[43_TD$DIFF],19 it was demonstrated that L-PRP derived from peripheral blood altered the immunoexpression of TGF-b and suppressed the immunoexpression of BMP2 in rabbits; the resulting positivity for BMP receptor 1B (BMPR1B) would contribute to minor bone matrix deposition and an increase in fatty cell development. Similarly, we have also previously demonstrated the intense presence of medullary tissue composed of adipocytes at sites treated with PRP.20,21 We revealed that the platelets present in PRP might demonstrate thrombogenic-like behaviour and contribute to the synthesis of collagen III, which is the type of collagen present in the medullary area. It is known that the concentration of leukocytes in cardiac blood is much lower than that in peripheral blood. Thus in the present study we tested the bone repair induced by autogenous L-PRP derived from cardiac blood in rats and compared the histomorphometric results to the presence of OC (which is considered a biomarker for mature osteoblasts) and also PPAR-g (which is a protein specific to adipocytes and foam cells). The results presented herein show that rats treated with L-PRP demonstrated lower new bone deposition when compared to rats that received autograft or the sham group (no grafting material). Yet, the bone morphology produced in the L-PRP group revealed thinner trabeculae and an enhanced medullary area composed of fatty cells, similar to appendicular bone. These histological features coincided with the intense presence of cells positive for PPAR-g and scarce OC-positive cells.
L-PRP diminishes bone matrix formation around autogenous bone grafts associated with changes in osteocalcin
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Fig. 2. Histological illustration of calvaria treated with autograft (A, D), autograft + L-PRP (B, E), and the sham group (C, F). Micrographs A and B demonstrate the grafted bone tissue at 15 days postoperative and also reveal the presence of angiogenesis (bv) and granulation tissue (bg). Micrograph C shows scarce bone formation in the sham group (control), particularly in the deep area of the defect, and intense quantities of fibrous tissue in the body of the defect (ft). Micrographs D, E, and F reveal the evolution of the repair in the three study groups on day 40 postsurgery. Micrograph D (autograft) shows intense bone matrix deposition, while micrograph E (autograft + L-PRP) shows intense fatty cell differentiation in the medullary area surrounded by trabecular bone. Micrograph F (sham) shows bone neoformation in the intense granulation tissue. Haematoxylin and eosin staining at 40 magnification; bg = grafted bone; bv = blood vessel; ft = fibrous tissue; hbt = Haversian bone tissue; hc = Haversian channel; tbt = trabecular bone tissue; ama = adipose medullary area.
OC is a highly conserved non-collagenous matrix protein found in bone tissue.22 The function of OC in bone matrix formation is controversial and some studies have revealed both induction and inhibition of matrix bone formation when OC is present.23,24 According to Poser and Price,25 the positive effect of mineralization induced by OC may occur due to the high affinity between OC and bone
mineral, especially apatite. This affinity enables it to regulate the rate of apatite crystal growth in solution,26 suggesting that OC may promote both mineral formation and mineral crystal growth, but without changes in osteoblast or osteoclast number. This indicates that OC may be an important terminal marker of osteoblast differentiation and bone matrix formation.27
In contrast, perceptible impaired bone matrix development has been shown in OC-knockout animal models. Using this method, Ducy et al.24 showed that OCdeficient animals had an accelerated rate of bone matrix formation without evident changes in osteoblast and osteoclast numbers, suggesting a negative action of OC on the mineralization process. These negative results for OC are also corroborated
Fig. 3. The presence of OC (brownish colour) among the study groups on day 15 (A, B, and C) and day 40 (D, E, and F) postsurgery. Micrograph A (autograft group) demonstrates intense quantities of OC-positive cells (arrows) among the grafted bone, while in micrograph B (autograft + LPRP) the OC cells are scarce. Micrograph C demonstrates the basal immunoexpression of OC in the sham (control) group. Micrographs D (autograft) and E (autograft + L-PRP) reveal decreased OC in the cells (arrows) and osteoid matrix (arrowheads), with bone or medullary tissue formed, while in micrograph G (sham) the presence of OC-positive cells (arrows) and osteoid matrix (arrowheads) remains higher. Magnification 100; bg = grafted bone; ama = adipose medullary area.
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Fig. 4. The presence of PPAR-g immunopositivity (brownish colour) among the study groups on day 15 (A, B, and C) and day 40 (D, E, and F) postsurgery. Micrograph A (autograft group) demonstrates scarce quantities of PPAR-g-positive cells (arrows) among the grafted bone, while micrograph B (autograft + L-PRP) shows intense immunopositivity for PPAR-g in the body of the defect among the grafted bone, except in osteoblasts (arrowheads) peripheral to the bone graft. Micrograph C demonstrates that the basal immunoexpression of PPAR-g in the sham (control) group is absent. Micrographs D (autograft) and E (autograft + L-PRP) reveal scarce and prevalent PPAR-g in the autograft, respectively, and the intense presence of protein in cells that exhibited adipocytic morphology in the autograft + L-PRP group at 40 days postsurgery. Micrograph G (sham) shows that the presence of PPAR-g-positive cells remained rare in the sham group. Magnification 100; bg = grafted bone.
by other authors who have described the persistent expression of OC to block both endochondral and intramembranous ossification,23 or even to induce loss of bone associated with the capacity of OC to support both chemotaxis28 and differentiation of osteoclasts in vitro.29 However, in the present study a positive relationship between OC and histological bone matrix formation was found. Thus the scarce immunohistochemical presence of OC may be considered an important reason for the minor bone quantities of bone matrix in the defects treated with L-PRP. A possible explanation for the lesser bone matrix deposition and significantly lower quantities of OC in specimens that received L-PRP may be the higher levels of TGF-b1 released by platelets at the sites of injury. Despite the fact that TGF-b1 has been considered important to bone remodelling, several studies have revealed that this cytokine is also a potent inhibitor of osteoblastic maturation, prior to the point at which the process is committed to the final terminal differentiation of osteoblasts, and likely suppresses OC immunoexpression.9 Based on this hypothesis, Kwok et al.30 evaluated the effect of TGF-b1 on the expression of osteoblast differentiation marker genes in osteoblast cells derived from rat calvaria. The authors found that
the administration of TGF-b1 in osteoblast cells inhibited the endogenous osteoblastic differentiation genes, including alkaline phosphatase and OC. These results corroborate those of the in vitro study performed by de Oliveira et al.31 and may help to explain the results of the present in vivo study in which only minor bone matrix was produced in the group receiving L-PRP. Concomitant to the decreased bone matrix formation when compared to both the control and autograft groups, the defects treated with L-PRP demonstrated the robust presence of adipose-like tissue formation, enlarging the medullary area, associated with a significant presence of PPAR-g. In fact, for in vivo studies, the outcome with regard to osteoblast and adipocyte differentiation has been found to be complex at reparative sites. Several studies have stated that angiogenesis is a fundamental factor in the enhancement of osteoneogenesis in calvarial bone repair.32,33 Steinbrech et al.33 add that the absence, or even a decrease of angiogenesis induces a hypoxia process. The authors explain that a lack of oxygen results in the down-regulation of vascular endothelial growth factor (VEGF), which is critical in bone formation, and at the same time this favours adipogenesis at bone sites associated with the inhibition of the
platelet growth factors. However this hypothesis32,33 does not seem to explain the presence of adipocyte-like cells in the specimens treated with L-PRP, because platelets release both TGF-b1 and VEGF. These growth factors possess anti-adipogenic properties and also promote the chemotaxis of stem cells and endothelial cells that exhibit the CD34 immunophenotype.19,20,34 Thus, the presence of adipocytes and/or foam-like cells in the calvaria bone repair induced by L-PRP may mimic a particular athero-pathological condition, where the higher number of platelets and fatty (foam) cells are simultaneous and dependent events.35 This hypothesis is based on the studies performed by Daub et al.36 and May et al.,37 which showed, in vitro, that the presence of high numbers of platelets does not only lead to recruitment and binding to CD34+ stem cells, but also supports the differentiation of these stem cells. These authors described that the coincubation of progenitor cells with platelets induced morphological changes from stem cells to mature foam cells, and indicated that a key mechanism in this differentiation process occurred due the phagocytosis of platelets by the progenitor cells. This hypothesis is supported by the findings published by Kruth38 and Curtiss et al.,39 who revealed that activated platelets may enhance both the rate of
L-PRP diminishes bone matrix formation around autogenous bone grafts associated with changes in osteocalcin cholesteryl ester formation and accumulation, as well as promote the storage of lipid droplets in cultured peripheral blood mononuclear cells, processes that are commonly associated with the expression of PPAR-g. The extrapolation of the results found in the present study to clinical situations would suggest that the use of L-PRP may be unfavourable in craniofacial bone repair, since the L-PRP produced excess fatty cell-like tissue, which would probably not be able to support, for example, the load of osseointegrated dental implants. In addition, bone that contains excess fatty cells that are PPAR-g-positive, as observed in the present study, possesses similarities to osteoporotic bone, having a different aetiology but presenting a similar histophenotype, and consequently a comparable structure. This study has some limitations. The first analysis was performed at only 15 days postsurgery and no conclusions could be drawn for the immediate effects of LPRP. The immunohistochemistry staining identifies only proteins present in cells and extracellular matrix, regardless of the time when they were expressed.2 In addition, this study focused only on histological craniofacial bone repair and the present results may not be extended to appendicular or axial bone repair, since craniofacial bones are derived from distinct embryological sources.40 However the present study adds important information that may contribute to the discussion over the clinical use of L-PRP. Funding
Giovanna Schirmer Portela received a scholarship from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq-BRAZIL). Competing interests
The authors do not have any conflict of interest or financial interests, either direct or indirect, in the products listed in the study. Ethical approval
This study was approved by the Institutional Animal Care and Use Committee of Positivo University, with protocol number 019/2009. References 1. Marx RE. Platelet-rich plasma: evidence to support its use. J Oral Maxillofac Surg 2004;62:489–96.
2. Thorwarth M, Wehrhan F, Schultze-Mosgau S, Wiltfang J, Schlegel KA. PRP modulates expression of bone matrix proteins in vivo without long-term effects on bone formation. Bone 2006;38:30–40. 3. Marx RE, Carlson ER, Eichstaedt RM, Schimmele SR, Strauss JE, Georgeff KR. Platelet-rich plasma: growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85: 638–46. 4. Choi BH, Im CJ, Huh JY, Suh JJ, Lee SH. Effect of platelet-rich plasma on bone regeneration in autogenous bone graft. Int J Oral Maxillofac Surg 2004;33:56–9. 5. Klongnoi B, Rupprecht S, Kessler P, Zimmermann R, Thorwarth M, Pongsiri S, Neukam FW, Wiltfang J, Schlegel KA. Lack of beneficial effects of platelet-rich plasma on sinus augmentation using a fluorohydroxyapatite or autogenous bone: an explorative study. J Clin Periodontol 2006;33:500–9. 6. Arnoczky SP, Delos D, Rodeo SA. What is platelet-rich plasma? Oper Tech Sports Med 2011;19:142–8. 7. Dohan Ehrenfest DM, Rasmusson L, Albrektsson T. Classification of platelet concentrates: from pure platelet-rich plasma (PPRP) to leucocyte- and platelet-rich fibrin (L-PRF). Trends Biotechnol 2009;27: 158–67. 8. Ehnert S, Baur J, Schmitt A, Neumaier M, Lucke M, Dooley S, Vester H, Wildemann B, Stockle U, Nussler AK. TGF-beta1 as possible link between loss of bone mineral density and chronic inflammation. PLoS One 2010;5:e14073. 9. Spinella-Jaegle S, Roman-Roman S, Faucheu C, Dunn FW, Kawai S, Gallea S, Stiot V, Blanchet AM, Courtois B, Baron R, Rawadi G. Opposite effects of bone morphogenetic protein-2 and transforming growth factor-beta1 on osteoblast differentiation. Bone 2001;29:323–30. 10. Gimble JM, Zvonic S, Floyd ZE, Kassem M, Nuttall ME. Playing with bone and fat. J Cell Biochem 2006;98:251–66. 11. Lian JB, Stein GS, Stein JL, van Wijnen AJ. Osteocalcin gene promoter: unlocking the secrets for regulation of osteoblast growth and differentiation. J Cell Biochem Suppl 1998;30:62–72. 12. Qi H, Aguiar DJ, Williams SM, La Pean A, Pan W, Verfaillie CM. Identification of genes responsible for osteoblast differentiation from human mesodermal progenitor cells. Proc Natl Acad Sci USA 2003;100:3305–10. 13. Birmingham E, Niebur GL, McHugh PE, Shaw G, Barry FP, McNamara LM. Osteogenic differentiation of mesenchymal stem cells is regulated by osteocyte and osteoblast cells in a simplified bone niche. Eur Cell Mater 2012;23:13–27. 14. Musri MM, Parrizas M. Epigenetic regulation of adipogenesis. Curr Opin Clin Nutr Metab Care 2012;15:342–9.
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15. Swanson CR, Joers V, Bondarenko V, Brunner K, Simmons HA, Ziegler TE, Kemnitz JW, Johnson JA, Emborg ME. The PPARgamma agonist pioglitazone modulates inflammation and induces neuroprotection in parkinsonian monkeys. J Neuroinflammation 2011;8:91. 16. Lecka-Czernik B, Moerman EJ, Grant DF, Lehmann JM, Manolagas SC, Jilka RL. Divergent effects of selective peroxisome proliferator-activated receptor-gamma 2 ligands on adipocyte versus osteoblast differentiation. Endocrinology 2002;143: 2376–84. 17. Intini G. The use of platelet-rich plasma in bone reconstruction therapy. Biomaterials 2009;30:4956–66. 18. Oliveira Filho MA, Nassif PA, Malafaia O, Ribas Filho JM, Ribas CA, Camacho AC, Stieven Filho E, Giovanini AF. Effects of a highly concentrated platelet-rich plasma on the bone repair using non-critical defects in the calvaria of rabbits. Acta Cir Bras 2010;25:28–33. 19. Giovanini AF, Grossi JR, Gonzaga CC, Zielak JC, Gohringer I, Vieira JD, Kuczera J, de Oliveira Filho MA, Deliberador TM. Leukocyte-platelet-rich plasma (L-PRP) induces an abnormal histophenotype in craniofacial bone repair associated with changes in the immunopositivity of the hematopoietic clusters of differentiation, osteoproteins, and TGF-beta1. Clin Implant Dent Relat Res 2012. http://dx.doi.org/10.1111/j.17088208.2012.00478.x. 20. Giovanini AF, Deliberador TM, Gonzaga CC, de Oliveira Filho MA, Gohringer I, Kuczera J, Zielak JC, de Andrade Urban C. Platelet-rich plasma diminishes calvarial bone repair associated with alterations in collagen matrix composition and elevated CD34+ cell prevalence. Bone 2010;46: 1597–603. 21. Giovanini AF, Gonzaga CC, Zielak JC, Deliberador TM, Kuczera J, Goringher I, de Oliveira Filho MA, Baratto-Filho F, Urban CA. Platelet-rich plasma (PRP) impairs the craniofacial bone repair associated with its elevated TGF-beta levels and modulates the co-expression between collagen III and alpha-smooth muscle actin. J Orthop Res 2011;29:457–63. 22. Huq NL, Rambaud SM, Teh LC, Davies AD, McCulloch B, Trotter MM, Chapman GE. Immunochemical detection and characterisation of osteocalcin from moa bone. Biochem Biophys Res Commun 1985;129: 714–20. 23. Yagami K, Suh JY, Enomoto-Iwamoto M, Koyama E, Abrams WR, Shapiro IM, Pacifici M, Iwamoto M. Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb. J Cell Biol 1999;147: 1097–108.
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24. Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C, Smith E, Bonadio J, Goldstein S, Gundberg C, Bradley A, Karsenty G. Increased bone formation in osteocalcindeficient mice. Nature 1996;382:448–52. 25. Poser JW, Price PA. A method for decarboxylation of gamma-carboxyglutamic acid in proteins, properties of the decarboxylated gamma-carboxyglutamic acid protein from calf bone. J Biol Chem 1979;254:431–6. 26. Hunter GK, Hauschka PV, Poole AR, Rosenberg LC, Goldberg HA. Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins. Biochem J 1996;317(Pt 1):59–64. 27. Starup-Linde J. Diabetes biochemical markers of bone turnover, diabetes control, and bone. Front Endocrinol (Lausanne) 2013;4:21. http://dx.doi.org/10.3389/fendo.2013.00021. 28. Malone JD, Teitelbaum SL, Griffin GL, Senior RM, Kahn AJ. Recruitment of osteoclast precursors by purified bone matrix constituents. J Cell Biol 1982;92:227–30. 29. Liggett Jr WH, Lian JB, Greenberger JS, Glowacki J. Osteocalcin promotes differentiation of osteoclast progenitors from murine long-term bone marrow cultures. J Cell Biochem 1994;55:190–9. 30. Kwok S, Partridge NC, Srinivasan N, Nair SV, Selvamurugan N. Mitogen activated protein kinase-dependent inhibition of osteocalcin gene expression by transforming growth factor-beta1. J Cell Biochem 2009;106: 161–9.
31. de Oliveira PT, de Oliva MA, Maximiano WM, Sebastiao KE, Crippa GE, Ciancaglini P, Beloti MM, Nanci A, Rosa AL. Effects of a mixture of growth factors and proteins on the development of the osteogenic phenotype in human alveolar bone cell cultures. J Histochem Cytochem 2008;56:629–38. 32. Schantz JT, Woodruff MA, Lam CX, Lim TC, Machens HG, Teoh SH, Hutmacher DW. Differentiation potential of mesenchymal progenitor cells following transplantation into calvarial defects. J Mech Behav Biomed Mater 2012;11:132–42. 33. Steinbrech DS, Mehrara BJ, Saadeh PB, Chin G, Dudziak ME, Gerrets RP, Gittes GK, Longaker MT. Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro. Plast Reconstr Surg 1999;104:738–47. 34. Giovanini AF, Deliberador TM, Tannuri Nemeth JE, Crivellaro VR, Portela GS, de Oliveira Filho MA, de Araujo MR, Zielak JC, Gonzaga CC. Leukocyte-platelet-rich plasma (L-PRP) impairs the osteoconductive capacity of the autograft associated to changes in the immunolocalization of TGF-beta1 and its co-expression with Wnt10b and CD34 cells. J Craniomaxillofac Surg 2013. http://dx.doi.org/10.1016/ j.jcms.2013.01.003. 35. Linden MD, Jackson DE. Platelets: pleiotropic roles in atherogenesis and atherothrombosis. Int J Biochem Cell Biol 2010;42:1762–6.
36. Daub K, Langer H, Seizer P, Stellos K, May AE, Goyal P, Bigalke B, Schonberger T, Geisler T, Siegel-Axel D, Oostendorp RA, Lindemann S, Gawaz M. Platelets induce differentiation of human CD34+ progenitor cells into foam cells and endothelial cells. FASEB J 2006;20:2559–61. 37. May AE, Seizer P, Gawaz M. Platelets: inflammatory firebugs of vascular walls. Arterioscler Thromb Vasc Biol 2008;28:s5–10. 38. Kruth HS. Platelet-mediated cholesterol accumulation in cultured aortic smooth muscle cells. Science 1985;227:1243–5. 39. Curtiss LK, Black AS, Takagi Y, Plow EF. New mechanism for foam cell generation in atherosclerotic lesions. J Clin Invest 1987;80:367–73. 40. van den Bos T, Speijer D, Bank RA, Bromme D, Everts V. Differences in matrix composition between calvaria and long bone in mice suggest differences in biomechanical properties and resorption: special emphasis on collagen. Bone 2008;43:459–68.
Corresponding author at: Positivo University Rua Pedro Viriato Parigot de Souza 5300 Curitiba Parana´ 81280-330 Brazil Tel.: +55 2141 33173000 fax: +55 2141 33173000 E-mail: [email protected]
Research Paper Bone Regeneration
L-PRP diminishes bone matrix formation around autogenous bone grafts associated with changes in osteocalcin and PPAR-g immunoexpression
G. S. Portela, D. X. Cerci, G. Pedrotti, M. R. Araujo, T. M. Deliberador, J. C. Zielak, T. A. Costa-Casagrande, C. C. Gonzaga, A. F. Giovanini School of Dentistry, Positivo University, Curitiba, Parana´, Brazil
G.S. Portela, D.X. Cerci, G. Pedrotti, M.R. Araujo, T.M. Deliberador, J.C. Zielak, T.A. Costa-Casagrande, C.C. Gonzaga, A.F. Giovanini: L-PRP diminishes bone matrix formation around autogenous bone grafts associated with changes in osteocalcin and PPAR-g immunoexpression. Int. J. Oral Maxillofac. Surg. 2014; 43: 261–268. # 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. Leucocyte- and platelet-rich plasma (L-PRP) is an autogenous platelet concentrate enriched with leukocytes that releases various growth factors responsible for the proliferation, regulation, and differentiation of mesenchymal cells during wound healing. Since the bone and medullary tissue are contiguous and share the same origin, this study evaluated the effect of L-PRP on the repair of calvaria bone using histomorphometric analysis of the newly formed bone, and compared the results in the presence of osteocalcin (OC) and peroxisome proliferator-activated receptor gamma (PPAR-g) detected by immunohistochemistry. Artificial circular bone defects (5 mm diameter) were produced in the calvaria of 42 rats. The defects were treated with autograft, autograft combined with L-PRP, or without grafting material (sham). The animals were euthanized at 15 or 40 days postsurgery (n = 7 in each group). Data obtained were analyzed by Student–Newman–Keuls test for histomorphometric and immunohistochemical interpretation. The development of bone matrix was significantly less in the defects treated with L-PRP, while the medullary area composed of fatty cells was larger. This coincided with the minor expression of OC and expressive presence of PPAR-g. These results suggest that L-PRP may impair osteoneogenesis and alter the ratio of differentiation between bone matrix and fatty cells, increasing the medullary tissue.
0901-5027/020261 + 08 $36.00/0
Keywords: Platelet-rich plasma; Bone regeneration; Osteocalcin; PPAR-g. Accepted for publication 9 July 2013 Available online 8 August 2013
# 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
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Introduction
Platelet-rich plasma (PRP) is a non-toxic and non-antigenic autologous blood product enriched with platelets that has been considered an essential source of growth factors, which are thought to improve wound healing in both soft and hard tissue.1,2 Since Marx et al.3 reported that the use of PRP promotes faster maturation of autologous bone grafts and a consequent increase in new bone formation in mandibular defects, the use of PRP combined with autografts has been used frequently in the reconstruction of craniofacial deformities. However, the positive effects of PRP in osteoneogenesis are the subject of debate and several studies have revealed a failure of bone maturation when PRP is used.4,5 A relevant speculation in the divergent results produced by PRP combined with autograft has been assigned in particular to the fabrication of PRP. While the classical definition of PRP, as proposed by Marx et al.,3 would suggest a pure mixture of plasma and platelets, the usual definition of PRP has expanded to include a variety of final products; thus the PRP may vary markedly not only in the final concentration of platelets, but also in the amounts of leukocytes that are commonly included in the final preparation.6,7 Leukocytes are considered to play a key role in the preliminary phases of inflammation and they may contribute to the variation in results found in the literature regarding the efficiency of bone wound healing. This hypothesis seems likely given that these inflammatory cells commonly increase the levels of some platelet growth factors, especially transforming growth factor beta 1 (TGF-b1), which is also present in bone loss.8 The presence of platelet growth factors is considered important to bone turnover and repair, since they contribute to the chemotaxis of osteoprogenitor cells at the site of injury. In contrast, some studies have revealed that TGF-b1 in particular may also induce negative effects on osteoblastogenesis, since this cytokine antagonizes bone morphogenetic protein (BMP) expression and impairs osteoblast maturation; this may contribute to changes in the ratios of cellular differentiation during bone repair.9 Osteoblasts and adipocytes are the principal cells that constitute the microarchitecture of bone tissue. Commonly the adipocytes of the bone marrow and the osteoblasts are present in anatomically contiguous tissues and also derive from the same mesenchymal cell lineage.10
Whether adipogenesis or osteoneogenesis occurs is commonly associated with the activation of certain proteins and/or transcription factors, with one process suppressing the other. This constitutes the essential pathway by which cells commit to a specific differentiation lineage. The presence of these proteins detected by immunohistochemistry may identify a particular type of cell in bone development while the adipose differentiation pathways are inhibited, and vice versa. Osteocalcin (OC) is a vitamin K-dependent osteoblast-specific protein and is commonly produced and expressed by mature osteoblasts.11,12 OC may also accumulate in the extracellular matrix produced by osteoblasts and has a high affinity for hydroxyapatite and mineral ions, properties that are considered determinants for the development and deposition of bone matrix.13 Peroxisome proliferator-activated receptor gamma (PPAR-g) is expressed in adipocytes; these receptors are responsible for the regulation of the expression of genes that control lipid metabolism,14 and at the same time inhibit the actions of proinflammatory genes.15 In addition, PPAR-g inhibits osteoblastic differentiation and the expression of several anabolic mediators involved in bone formation, such as BMPs, type I collagen, and OC.16 It was hypothesized that leucocyte- and platelet-rich plasma (L-PRP) could alter the histophenotype of bone repair, changing the proportions of osteoblasts and adipocytes. Thus the aim of this study was to evaluate the ratio of OC to PPAR-g detected by immunohistochemistry, and to compare these results with a histomorphometric analysis of the bone matrix deposited in the presence and absence of L-PRP. Materials and methods
Forty-two male rats (Rattus norvegicus albinus, Wistar), 5–6 months old, weighing 450–500 g and with no previous disease were used in this study; the study protocol was approved by the institutional animal care and use committee. The rats were kept in a room with a controlled temperature (approximately 22 8C) and maintained under a 12-h light–dark cycle. PRP fabrication and quantification
A 3.2-ml sample of autologous blood was collected from each animal through cardiac puncture, into a syringe containing 0.35 ml of 10% sodium citrate. The blood samples were centrifuged at 200 g for
20 min at room temperature to separate the plasma containing the platelets from the red cells (Beckman J-6 M Induction Drive Centrifuge; Beckman Instruments Inc., Palo Alto, CA, USA). The plasma fraction was collected from the top of the supernatant. The remaining fractions were centrifuged once more at room temperature for 10 min at 400 g to separate the platelets. The platelet-poor plasma was removed from the upper level of the supernatant, leaving the PRP and buffy coat. Both the buffy coat and PRP (0.35 ml) were resuspended and activated with a mixture of 10% calcium chloride (0.05 ml/ml of PRP). They were then added to the previously prepared L-PRP and mixed for approximately 1 min until they formed a gel. The platelets and leukocytes in the LPRP were measured after centrifugation using a Coulter STKS haematology counting machine (Beckman-Coulter, Chicago, IL, USA). The average whole blood platelet count was 617.66 69.81 103 platelets/ ml, while the average L-PRP platelet count was 2826.44 309.12 103 platelets/ml (P 0.001). The average concentration of leukocytes in the L-PRP was 8.14 0.23 103 leukocytes/ml, while the average initial blood value was 3.18 0.91 103 leukocytes/ml (P 0.001). Surgical procedure
The rats were anesthetized by intramuscular injection of xylazine (5 mg/kg) and ketamine (70 mg/kg). The surgical region was shaved and aseptically prepared, and sterile barriers were created to limit the surgical area. A 5-cm midline dermo-periosteal incision was made to expose the calvarial surface with complete removal of the periosteum. Circular artificial defects of 5 mm in diameter were created with a trephine (Biomedical Research Instruments Inc., Silver Spring, MD, USA) under abundant saline solution irrigation in each rat. Bone fragments removed from the calvarial defects were particulated and used as autograft. In a randomized manner, one group received treatment with 0.01 ml of autograft and another group had the defect filled with 0.01 ml of autograft associated with 150 ml of L-PRP. The control animals (sham) underwent the same surgical procedures without the use of any grafting material. Soft tissues were repositioned and sutured to achieve primary closure (4-0 silk, Ethicon, Sao Paulo, SP, Brazil). Each animal received a prophylactic intramuscular injection of 24,000 IU of penicillin G benzathine and a daily dose of
L-PRP diminishes bone matrix formation around autogenous bone grafts associated with changes in osteocalcin 200 mg/kg/day of liquid acetaminophen administrated orally.
Euthanasia procedure and tissue processing
At 15 or 40 days postsurgery, animals were euthanized by brief exposure in a CO2 chamber until they ceased to move. The skull caps of each animal were removed in blocks using an inverted cone bur. Calvaria specimens obtained were fixed in 10% buffered formalin for 48 h and decalcified in 20% formic acid and sodium citrate for 7 days. The specimens were washed with tap water, dehydrated, cleared with xylene, and embedded in paraffin. Serial sections of 3 mm made parallel to the mid-sagittal suture were cut from the centre of each defect using a microtome (RM2155, Leica Microsystems GmbH, Nussloch, Germany) and stained with haematoxylin and eosin to permit histomorphological and histomorphometric evaluation.
Immunohistochemistry processing
Three-micrometre thicknesses of each specimen were deparaffinized and subjected to antigen retrieval in 1% trypsin solution (pH 1.8) for 60 min at 37 8C for the anti-OC and anti-PPAR-g antibodies. Slides containing the histological fragments were immersed in 3% hydrogen peroxide for 30 min to eliminate endogenous peroxidase activity, followed by incubation with 1% phosphate-buffered saline (PBS; pH 7.4). The sections were incubated with the primary antibody anti-OC (200 mg/ml; Santa Cruz Biotechnology Inc., CA, USA) with a dilution factor of 1:200, and anti-PPAR-g (200 mg/ml; Santa Cruz Biotechnology Inc., CA, USA) with a dilution factor of 1:150. A labelled streptavidin biotin antibody-binding detection system (Universal HRP Immunostaining Kit; Diagnostic BioSystems, Pleasanton, CA, USA) was employed to detect the primary antibodies. The immune reaction was observed with diaminobenzidine tetrachloride chromogen solution (Universal HRP Immunostaining Kit), which produces a brown precipitate at the antigen site. The specimens were counterstained with Harris haematoxylin. A negative control was included for all samples, using rabbit polyclonal isotype IgG (2 mg/ml, Abcam, Cambridge, UK) for 20 min at room temperature as a primary antibody. For each specimen, three slides were utilized for incubation with each antibody.
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Image analysis
L-PRP mixed with autograft
The images of both the histological and immunohistochemistry sections were captured by digital camera (SDC-310; Samsung, Gyeonggi-do, South Korea) coupled to a light microscope (Zeiss, Oberkochen, Germany) with an original magnification of 200. Each digital image was gathered and preserved at 300 dpi resolution, producing a virtual frame of 113 cm 80 cm. As it was not possible to capture the entire defect in one image at the magnification level used, a composite digital image of the whole defect was then constructed by combining five smaller images based on histomorphological reference structures, in particular the deposited bone trabeculae and blood vessels. All histomorphometric and immunohistochemical measurements were completed using the software Image J (National Institute of Health, Bethesda, MD, USA). A 1-mm calibration slide was used to calibrate all measurements. The histomorphometric data were compiled manually and expressed as areas (mm2). The perimeters of the deposited histological bone matrix were carefully traced and the areas computed. At the same time, positive OC and PPAR-g cells/mm2 were manually counted and tagged.
On day 15 postsurgery, inserted bone fragments were detected surrounded by rich mesenchymal cellular tissue. From day 15 to day 40 postsurgery, the amount of deposited bone matrix increased slightly, and a substantial quantity of medullary tissue represented by fatty cells was found.
Statistical analysis
Each parameter was evaluated within the monitoring period. Analysis of variance (ANOVA) was used to determine if there were significant differences among groups, followed by the Student–Newman–Keuls non-parametric test. A Pvalue of less than 0.05 was considered to be statistically significant.
Results Light microscopy analysis
The quantitative histomorphometric analysis data are given in Fig. 1. A brief description of the microscopic characteristics found in the groups is provided below and shown in Fig. 2.
Autograft
On postoperative day 15, bone fragments and new bone formation from the autograft bone were present. At this time point, discrete to mild granulation tissues were observed. On postoperative day 40, newly formed compact Haversian bone was identified.
Control – sham group
On day 15 postsurgery, the histological analysis revealed areas of granulation tissue composed of collagen and mesenchymal cells in the larger part of the defect. Further, there was evidence of newly formed immature bone that mimicked intramembranous ossification restricted to the peripheral area of the defect. At day 40 after surgery, granulation tissue was detected around a presence of both mature and immature Haversian bone tissue. Immunohistochemical results
The quantitative immunohistochemistry data are given in Fig. 1 (P < 0.05). In summary, the results can be described as follows. Osteocalcin (OC)
OC positivity was present in all specimens analyzed (Fig. 3). On day 15 postsurgery, the quantities of cells that exhibited positivity for this protein were significantly lower in the group that received L-PRP when compared to the autograft and sham groups. On postoperative day 40, the presence of OC had decreased in the autograft and autograft + L-PRP groups, as soon as the bone matrix or medullary tissue had matured; however the quantities of OC in the group that received L-PRP remained lower. In contrast, in the sham group, the presence of OC remained higher, expressed both in cells and bone matrix. Peroxisome proliferator-activated receptor gamma (PPAR-g)
A significant PPAR-g expression was seen only in the groups that received autograft (Fig. 4). In the earlier stages of the repair (15 days), the group that received only autograft presented scarce PPAR-g-positive cells, found around the blood vessels in the reparative granulation tissue, while the majority of cells that surrounded the grafted bone demonstrated positivity for PPAR-g in the autograft + L-PRP group. The pattern on day 40 postsurgery was
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Fig. 1. Graphical comparison of the groups analyzed. Average and standard deviation values of (A) bone matrix, (B) OC, and (C) PPAR-g. Values followed by the same superscript letter (a, b, and c) are statistically similar, P > 0.05.
similar, however the PPAR-g-positive cells in the specimens treated with LPRP exhibited an adipocyte-like histophenotype.
Discussion
The use of PRP in bone repair is based on the considerable number of platelets that
produce active growth factors, which can have a substantial impact on chemotaxis, proliferation, regulation, and differentiation of mesenchymal cells into osteoblasts.17 Despite the fact that activation of some growth factors by platelets is fundamental to osteoneogenesis, results demonstrating that PRP increases local bone formation1,3,17 have not been corroborated by some groups.4,5,18 The lack of agreement regarding the positive effect of PRP on osteoneogenesis may reflect variations in the PRP preparations used, which could alter the platelet quality and/or quantity, resulting in differences in the regenerative potential of PRP.6,7 A matter that should be taken into consideration in the variations in PRP preparations is the possible presence, or variation, in the amount of leukocytes.6,7 In a recent study performed by our group Diff id="43">[43_TD$DIFF],19 it was demonstrated that L-PRP derived from peripheral blood altered the immunoexpression of TGF-b and suppressed the immunoexpression of BMP2 in rabbits; the resulting positivity for BMP receptor 1B (BMPR1B) would contribute to minor bone matrix deposition and an increase in fatty cell development. Similarly, we have also previously demonstrated the intense presence of medullary tissue composed of adipocytes at sites treated with PRP.20,21 We revealed that the platelets present in PRP might demonstrate thrombogenic-like behaviour and contribute to the synthesis of collagen III, which is the type of collagen present in the medullary area. It is known that the concentration of leukocytes in cardiac blood is much lower than that in peripheral blood. Thus in the present study we tested the bone repair induced by autogenous L-PRP derived from cardiac blood in rats and compared the histomorphometric results to the presence of OC (which is considered a biomarker for mature osteoblasts) and also PPAR-g (which is a protein specific to adipocytes and foam cells). The results presented herein show that rats treated with L-PRP demonstrated lower new bone deposition when compared to rats that received autograft or the sham group (no grafting material). Yet, the bone morphology produced in the L-PRP group revealed thinner trabeculae and an enhanced medullary area composed of fatty cells, similar to appendicular bone. These histological features coincided with the intense presence of cells positive for PPAR-g and scarce OC-positive cells.
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Fig. 2. Histological illustration of calvaria treated with autograft (A, D), autograft + L-PRP (B, E), and the sham group (C, F). Micrographs A and B demonstrate the grafted bone tissue at 15 days postoperative and also reveal the presence of angiogenesis (bv) and granulation tissue (bg). Micrograph C shows scarce bone formation in the sham group (control), particularly in the deep area of the defect, and intense quantities of fibrous tissue in the body of the defect (ft). Micrographs D, E, and F reveal the evolution of the repair in the three study groups on day 40 postsurgery. Micrograph D (autograft) shows intense bone matrix deposition, while micrograph E (autograft + L-PRP) shows intense fatty cell differentiation in the medullary area surrounded by trabecular bone. Micrograph F (sham) shows bone neoformation in the intense granulation tissue. Haematoxylin and eosin staining at 40 magnification; bg = grafted bone; bv = blood vessel; ft = fibrous tissue; hbt = Haversian bone tissue; hc = Haversian channel; tbt = trabecular bone tissue; ama = adipose medullary area.
OC is a highly conserved non-collagenous matrix protein found in bone tissue.22 The function of OC in bone matrix formation is controversial and some studies have revealed both induction and inhibition of matrix bone formation when OC is present.23,24 According to Poser and Price,25 the positive effect of mineralization induced by OC may occur due to the high affinity between OC and bone
mineral, especially apatite. This affinity enables it to regulate the rate of apatite crystal growth in solution,26 suggesting that OC may promote both mineral formation and mineral crystal growth, but without changes in osteoblast or osteoclast number. This indicates that OC may be an important terminal marker of osteoblast differentiation and bone matrix formation.27
In contrast, perceptible impaired bone matrix development has been shown in OC-knockout animal models. Using this method, Ducy et al.24 showed that OCdeficient animals had an accelerated rate of bone matrix formation without evident changes in osteoblast and osteoclast numbers, suggesting a negative action of OC on the mineralization process. These negative results for OC are also corroborated
Fig. 3. The presence of OC (brownish colour) among the study groups on day 15 (A, B, and C) and day 40 (D, E, and F) postsurgery. Micrograph A (autograft group) demonstrates intense quantities of OC-positive cells (arrows) among the grafted bone, while in micrograph B (autograft + LPRP) the OC cells are scarce. Micrograph C demonstrates the basal immunoexpression of OC in the sham (control) group. Micrographs D (autograft) and E (autograft + L-PRP) reveal decreased OC in the cells (arrows) and osteoid matrix (arrowheads), with bone or medullary tissue formed, while in micrograph G (sham) the presence of OC-positive cells (arrows) and osteoid matrix (arrowheads) remains higher. Magnification 100; bg = grafted bone; ama = adipose medullary area.
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Fig. 4. The presence of PPAR-g immunopositivity (brownish colour) among the study groups on day 15 (A, B, and C) and day 40 (D, E, and F) postsurgery. Micrograph A (autograft group) demonstrates scarce quantities of PPAR-g-positive cells (arrows) among the grafted bone, while micrograph B (autograft + L-PRP) shows intense immunopositivity for PPAR-g in the body of the defect among the grafted bone, except in osteoblasts (arrowheads) peripheral to the bone graft. Micrograph C demonstrates that the basal immunoexpression of PPAR-g in the sham (control) group is absent. Micrographs D (autograft) and E (autograft + L-PRP) reveal scarce and prevalent PPAR-g in the autograft, respectively, and the intense presence of protein in cells that exhibited adipocytic morphology in the autograft + L-PRP group at 40 days postsurgery. Micrograph G (sham) shows that the presence of PPAR-g-positive cells remained rare in the sham group. Magnification 100; bg = grafted bone.
by other authors who have described the persistent expression of OC to block both endochondral and intramembranous ossification,23 or even to induce loss of bone associated with the capacity of OC to support both chemotaxis28 and differentiation of osteoclasts in vitro.29 However, in the present study a positive relationship between OC and histological bone matrix formation was found. Thus the scarce immunohistochemical presence of OC may be considered an important reason for the minor bone quantities of bone matrix in the defects treated with L-PRP. A possible explanation for the lesser bone matrix deposition and significantly lower quantities of OC in specimens that received L-PRP may be the higher levels of TGF-b1 released by platelets at the sites of injury. Despite the fact that TGF-b1 has been considered important to bone remodelling, several studies have revealed that this cytokine is also a potent inhibitor of osteoblastic maturation, prior to the point at which the process is committed to the final terminal differentiation of osteoblasts, and likely suppresses OC immunoexpression.9 Based on this hypothesis, Kwok et al.30 evaluated the effect of TGF-b1 on the expression of osteoblast differentiation marker genes in osteoblast cells derived from rat calvaria. The authors found that
the administration of TGF-b1 in osteoblast cells inhibited the endogenous osteoblastic differentiation genes, including alkaline phosphatase and OC. These results corroborate those of the in vitro study performed by de Oliveira et al.31 and may help to explain the results of the present in vivo study in which only minor bone matrix was produced in the group receiving L-PRP. Concomitant to the decreased bone matrix formation when compared to both the control and autograft groups, the defects treated with L-PRP demonstrated the robust presence of adipose-like tissue formation, enlarging the medullary area, associated with a significant presence of PPAR-g. In fact, for in vivo studies, the outcome with regard to osteoblast and adipocyte differentiation has been found to be complex at reparative sites. Several studies have stated that angiogenesis is a fundamental factor in the enhancement of osteoneogenesis in calvarial bone repair.32,33 Steinbrech et al.33 add that the absence, or even a decrease of angiogenesis induces a hypoxia process. The authors explain that a lack of oxygen results in the down-regulation of vascular endothelial growth factor (VEGF), which is critical in bone formation, and at the same time this favours adipogenesis at bone sites associated with the inhibition of the
platelet growth factors. However this hypothesis32,33 does not seem to explain the presence of adipocyte-like cells in the specimens treated with L-PRP, because platelets release both TGF-b1 and VEGF. These growth factors possess anti-adipogenic properties and also promote the chemotaxis of stem cells and endothelial cells that exhibit the CD34 immunophenotype.19,20,34 Thus, the presence of adipocytes and/or foam-like cells in the calvaria bone repair induced by L-PRP may mimic a particular athero-pathological condition, where the higher number of platelets and fatty (foam) cells are simultaneous and dependent events.35 This hypothesis is based on the studies performed by Daub et al.36 and May et al.,37 which showed, in vitro, that the presence of high numbers of platelets does not only lead to recruitment and binding to CD34+ stem cells, but also supports the differentiation of these stem cells. These authors described that the coincubation of progenitor cells with platelets induced morphological changes from stem cells to mature foam cells, and indicated that a key mechanism in this differentiation process occurred due the phagocytosis of platelets by the progenitor cells. This hypothesis is supported by the findings published by Kruth38 and Curtiss et al.,39 who revealed that activated platelets may enhance both the rate of
L-PRP diminishes bone matrix formation around autogenous bone grafts associated with changes in osteocalcin cholesteryl ester formation and accumulation, as well as promote the storage of lipid droplets in cultured peripheral blood mononuclear cells, processes that are commonly associated with the expression of PPAR-g. The extrapolation of the results found in the present study to clinical situations would suggest that the use of L-PRP may be unfavourable in craniofacial bone repair, since the L-PRP produced excess fatty cell-like tissue, which would probably not be able to support, for example, the load of osseointegrated dental implants. In addition, bone that contains excess fatty cells that are PPAR-g-positive, as observed in the present study, possesses similarities to osteoporotic bone, having a different aetiology but presenting a similar histophenotype, and consequently a comparable structure. This study has some limitations. The first analysis was performed at only 15 days postsurgery and no conclusions could be drawn for the immediate effects of LPRP. The immunohistochemistry staining identifies only proteins present in cells and extracellular matrix, regardless of the time when they were expressed.2 In addition, this study focused only on histological craniofacial bone repair and the present results may not be extended to appendicular or axial bone repair, since craniofacial bones are derived from distinct embryological sources.40 However the present study adds important information that may contribute to the discussion over the clinical use of L-PRP. Funding
Giovanna Schirmer Portela received a scholarship from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq-BRAZIL). Competing interests
The authors do not have any conflict of interest or financial interests, either direct or indirect, in the products listed in the study. Ethical approval
This study was approved by the Institutional Animal Care and Use Committee of Positivo University, with protocol number 019/2009. References 1. Marx RE. Platelet-rich plasma: evidence to support its use. J Oral Maxillofac Surg 2004;62:489–96.
2. Thorwarth M, Wehrhan F, Schultze-Mosgau S, Wiltfang J, Schlegel KA. PRP modulates expression of bone matrix proteins in vivo without long-term effects on bone formation. Bone 2006;38:30–40. 3. Marx RE, Carlson ER, Eichstaedt RM, Schimmele SR, Strauss JE, Georgeff KR. Platelet-rich plasma: growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85: 638–46. 4. Choi BH, Im CJ, Huh JY, Suh JJ, Lee SH. Effect of platelet-rich plasma on bone regeneration in autogenous bone graft. Int J Oral Maxillofac Surg 2004;33:56–9. 5. Klongnoi B, Rupprecht S, Kessler P, Zimmermann R, Thorwarth M, Pongsiri S, Neukam FW, Wiltfang J, Schlegel KA. Lack of beneficial effects of platelet-rich plasma on sinus augmentation using a fluorohydroxyapatite or autogenous bone: an explorative study. J Clin Periodontol 2006;33:500–9. 6. Arnoczky SP, Delos D, Rodeo SA. What is platelet-rich plasma? Oper Tech Sports Med 2011;19:142–8. 7. Dohan Ehrenfest DM, Rasmusson L, Albrektsson T. Classification of platelet concentrates: from pure platelet-rich plasma (PPRP) to leucocyte- and platelet-rich fibrin (L-PRF). Trends Biotechnol 2009;27: 158–67. 8. Ehnert S, Baur J, Schmitt A, Neumaier M, Lucke M, Dooley S, Vester H, Wildemann B, Stockle U, Nussler AK. TGF-beta1 as possible link between loss of bone mineral density and chronic inflammation. PLoS One 2010;5:e14073. 9. Spinella-Jaegle S, Roman-Roman S, Faucheu C, Dunn FW, Kawai S, Gallea S, Stiot V, Blanchet AM, Courtois B, Baron R, Rawadi G. Opposite effects of bone morphogenetic protein-2 and transforming growth factor-beta1 on osteoblast differentiation. Bone 2001;29:323–30. 10. Gimble JM, Zvonic S, Floyd ZE, Kassem M, Nuttall ME. Playing with bone and fat. J Cell Biochem 2006;98:251–66. 11. Lian JB, Stein GS, Stein JL, van Wijnen AJ. Osteocalcin gene promoter: unlocking the secrets for regulation of osteoblast growth and differentiation. J Cell Biochem Suppl 1998;30:62–72. 12. Qi H, Aguiar DJ, Williams SM, La Pean A, Pan W, Verfaillie CM. Identification of genes responsible for osteoblast differentiation from human mesodermal progenitor cells. Proc Natl Acad Sci USA 2003;100:3305–10. 13. Birmingham E, Niebur GL, McHugh PE, Shaw G, Barry FP, McNamara LM. Osteogenic differentiation of mesenchymal stem cells is regulated by osteocyte and osteoblast cells in a simplified bone niche. Eur Cell Mater 2012;23:13–27. 14. Musri MM, Parrizas M. Epigenetic regulation of adipogenesis. Curr Opin Clin Nutr Metab Care 2012;15:342–9.
267
15. Swanson CR, Joers V, Bondarenko V, Brunner K, Simmons HA, Ziegler TE, Kemnitz JW, Johnson JA, Emborg ME. The PPARgamma agonist pioglitazone modulates inflammation and induces neuroprotection in parkinsonian monkeys. J Neuroinflammation 2011;8:91. 16. Lecka-Czernik B, Moerman EJ, Grant DF, Lehmann JM, Manolagas SC, Jilka RL. Divergent effects of selective peroxisome proliferator-activated receptor-gamma 2 ligands on adipocyte versus osteoblast differentiation. Endocrinology 2002;143: 2376–84. 17. Intini G. The use of platelet-rich plasma in bone reconstruction therapy. Biomaterials 2009;30:4956–66. 18. Oliveira Filho MA, Nassif PA, Malafaia O, Ribas Filho JM, Ribas CA, Camacho AC, Stieven Filho E, Giovanini AF. Effects of a highly concentrated platelet-rich plasma on the bone repair using non-critical defects in the calvaria of rabbits. Acta Cir Bras 2010;25:28–33. 19. Giovanini AF, Grossi JR, Gonzaga CC, Zielak JC, Gohringer I, Vieira JD, Kuczera J, de Oliveira Filho MA, Deliberador TM. Leukocyte-platelet-rich plasma (L-PRP) induces an abnormal histophenotype in craniofacial bone repair associated with changes in the immunopositivity of the hematopoietic clusters of differentiation, osteoproteins, and TGF-beta1. Clin Implant Dent Relat Res 2012. http://dx.doi.org/10.1111/j.17088208.2012.00478.x. 20. Giovanini AF, Deliberador TM, Gonzaga CC, de Oliveira Filho MA, Gohringer I, Kuczera J, Zielak JC, de Andrade Urban C. Platelet-rich plasma diminishes calvarial bone repair associated with alterations in collagen matrix composition and elevated CD34+ cell prevalence. Bone 2010;46: 1597–603. 21. Giovanini AF, Gonzaga CC, Zielak JC, Deliberador TM, Kuczera J, Goringher I, de Oliveira Filho MA, Baratto-Filho F, Urban CA. Platelet-rich plasma (PRP) impairs the craniofacial bone repair associated with its elevated TGF-beta levels and modulates the co-expression between collagen III and alpha-smooth muscle actin. J Orthop Res 2011;29:457–63. 22. Huq NL, Rambaud SM, Teh LC, Davies AD, McCulloch B, Trotter MM, Chapman GE. Immunochemical detection and characterisation of osteocalcin from moa bone. Biochem Biophys Res Commun 1985;129: 714–20. 23. Yagami K, Suh JY, Enomoto-Iwamoto M, Koyama E, Abrams WR, Shapiro IM, Pacifici M, Iwamoto M. Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb. J Cell Biol 1999;147: 1097–108.
268
Portela et al.
24. Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C, Smith E, Bonadio J, Goldstein S, Gundberg C, Bradley A, Karsenty G. Increased bone formation in osteocalcindeficient mice. Nature 1996;382:448–52. 25. Poser JW, Price PA. A method for decarboxylation of gamma-carboxyglutamic acid in proteins, properties of the decarboxylated gamma-carboxyglutamic acid protein from calf bone. J Biol Chem 1979;254:431–6. 26. Hunter GK, Hauschka PV, Poole AR, Rosenberg LC, Goldberg HA. Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins. Biochem J 1996;317(Pt 1):59–64. 27. Starup-Linde J. Diabetes biochemical markers of bone turnover, diabetes control, and bone. Front Endocrinol (Lausanne) 2013;4:21. http://dx.doi.org/10.3389/fendo.2013.00021. 28. Malone JD, Teitelbaum SL, Griffin GL, Senior RM, Kahn AJ. Recruitment of osteoclast precursors by purified bone matrix constituents. J Cell Biol 1982;92:227–30. 29. Liggett Jr WH, Lian JB, Greenberger JS, Glowacki J. Osteocalcin promotes differentiation of osteoclast progenitors from murine long-term bone marrow cultures. J Cell Biochem 1994;55:190–9. 30. Kwok S, Partridge NC, Srinivasan N, Nair SV, Selvamurugan N. Mitogen activated protein kinase-dependent inhibition of osteocalcin gene expression by transforming growth factor-beta1. J Cell Biochem 2009;106: 161–9.
31. de Oliveira PT, de Oliva MA, Maximiano WM, Sebastiao KE, Crippa GE, Ciancaglini P, Beloti MM, Nanci A, Rosa AL. Effects of a mixture of growth factors and proteins on the development of the osteogenic phenotype in human alveolar bone cell cultures. J Histochem Cytochem 2008;56:629–38. 32. Schantz JT, Woodruff MA, Lam CX, Lim TC, Machens HG, Teoh SH, Hutmacher DW. Differentiation potential of mesenchymal progenitor cells following transplantation into calvarial defects. J Mech Behav Biomed Mater 2012;11:132–42. 33. Steinbrech DS, Mehrara BJ, Saadeh PB, Chin G, Dudziak ME, Gerrets RP, Gittes GK, Longaker MT. Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro. Plast Reconstr Surg 1999;104:738–47. 34. Giovanini AF, Deliberador TM, Tannuri Nemeth JE, Crivellaro VR, Portela GS, de Oliveira Filho MA, de Araujo MR, Zielak JC, Gonzaga CC. Leukocyte-platelet-rich plasma (L-PRP) impairs the osteoconductive capacity of the autograft associated to changes in the immunolocalization of TGF-beta1 and its co-expression with Wnt10b and CD34 cells. J Craniomaxillofac Surg 2013. http://dx.doi.org/10.1016/ j.jcms.2013.01.003. 35. Linden MD, Jackson DE. Platelets: pleiotropic roles in atherogenesis and atherothrombosis. Int J Biochem Cell Biol 2010;42:1762–6.
36. Daub K, Langer H, Seizer P, Stellos K, May AE, Goyal P, Bigalke B, Schonberger T, Geisler T, Siegel-Axel D, Oostendorp RA, Lindemann S, Gawaz M. Platelets induce differentiation of human CD34+ progenitor cells into foam cells and endothelial cells. FASEB J 2006;20:2559–61. 37. May AE, Seizer P, Gawaz M. Platelets: inflammatory firebugs of vascular walls. Arterioscler Thromb Vasc Biol 2008;28:s5–10. 38. Kruth HS. Platelet-mediated cholesterol accumulation in cultured aortic smooth muscle cells. Science 1985;227:1243–5. 39. Curtiss LK, Black AS, Takagi Y, Plow EF. New mechanism for foam cell generation in atherosclerotic lesions. J Clin Invest 1987;80:367–73. 40. van den Bos T, Speijer D, Bank RA, Bromme D, Everts V. Differences in matrix composition between calvaria and long bone in mice suggest differences in biomechanical properties and resorption: special emphasis on collagen. Bone 2008;43:459–68.
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