PRP modulates expression of bone matrix proteins in vivo without long-term effects on bone formation

Bone 38 (2006) 30 – 40 www.elsevier.com/locate/bone

PRP modulates expression of bone matrix proteins in vivo without long-term effects on bone formation Michael Thorwarth a,b,⁎, Falk Wehrhan a,b , Stefan Schultze-Mosgau a,b , Jörg Wiltfang c , Karl Andreas Schlegel b a

Department of Oral and Maxillofacial Surgery/Plastic Surgery, University of Jena, Erlanger Allee 101, D-07747 Jena, Germany Department of Oral and Maxillofacial Surgery, University of Erlangen-Nuremberg, Glueckstrasse 11, D-91054 Erlangen, Germany Department of Oral and Maxillofacial Surgery, University of Schleswig-Holstein/campus Kiel, Arnold-Heller-Strasse 16, D-24105 Kiel, Germany b

c

Received 5 December 2004; revised 19 June 2005; accepted 22 June 2005 Available online 28 October 2005

Abstract This experimental study (domestic pig) examined the bone formation after filling defined defects of the frontal skull with autogenous bone or a deproteinized bovine bone matrix (DBBM) in combination with platelet-rich plasma (PRP). Six groups, both materials with and without PRP in two different concentrations (4.1× and 6.5× referring to untreated whole blood) were evaluated at 2, 4, 12, and 26 weeks by means of immunohistochemical staining for different bone matrix proteins, microradiography, light microscopy and polychromatic fluorescence labeling. The sequential expression of bone matrix proteins reflected the specific roles these proteins fulfil in the mineralization of hard tissue. Collagen I expression at 2 weeks was enhanced in all autogenous bone groups. No specific modification of the collagen I expression was found after use of DBBM with or without PRP. Osteopontin and especially osteonectin showed a remarkable enhancement at 4 weeks in nearly all autogenous bone and DBBM groups. These increased levels closely resembled the mineralization content evaluated by microradiography at that time. For the three autogenous bone groups, an expression peak for osteocalcin was demonstrated at 12 weeks, further reflecting the way of de novo bone formation. The microradiographic evaluation demonstrated a statistically significant enhancement in bone regeneration by PRP only after use of autogenous bone plus PRP at 2 weeks (P = 0.002). After 4 weeks, mineralization values after use of autogenous bone were significantly lower if PRP was added to the autogenous bone (P = 0.002). No long-term effects of the PRP administration were found in the mineralization process. In all DBBM groups, bone formation remained unchanged, confirming the lack of any osteoinductive capacity of PRP. PRP modulated the expression of bone matrix proteins in this experimental setting. However, an enhancement of bone formation was demonstrated only at 2 weeks after application of the higher PRP concentration in combination with autogenous bone. In conjunction with an anorganic bovine bone no effects of PRP on defect mineralization were discovered, demonstrating the lack of osteoinductive capacity in PRP as well as in DBBM. © 2005 Elsevier Inc. All rights reserved. Keywords: Bone regeneration; Platelet-rich plasma; Bone matrix proteins; Deproteinized bovine bone matrix; Prospective study

Introduction Due to its osteogenic potential, autogenous bone is the most effective bone graft material [1]. To avoid donor morbidity, bone substitutes may be used instead. A large number of materials are available. Despite the increase in the number of products, up to ⁎ Corresponding author. Department of Oral and Maxillofacial Surgery/Plastic Surgery, University of Jena, Erlanger Allee 101, D-07747 Jena, Germany. Fax: +49 3641 9 323 602. E-mail address: [email protected] (M. Thorwarth). 8756-3282/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2005.06.020

date there has not been a single ideal material [2]. In 1998, Marx et al. proposed the local application of platelet-rich plasma (PRP) to enhance the maturation of autologous bone grafts [3]. PRP is a concentrate of platelets who release the fundamental protein growth factors involved in wound healing—the rationale to use it in bone surgery. These are mainly the isoforms of platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and epithelial growth factor (EGF). PRP further contains proteins known to act as cell adhesion molecules for osteoconduction and as a matrix for bone, connective tissue, and

M. Thorwarth et al. / Bone 38 (2006) 30–40

epithelial migration. These cell adhesion molecules are fibrin itself, fibronectin, and vitronectin [4]. In 2002 Fennis et al. reported an experimental study on mandibular reconstruction where the use of PRP appeared to enhance bone healing considerably in combination with autogenous scaffolds [5]. However, the influence of PRP on bone transplants or bone substitutes remains to be determined [6]. At present, there is a lack of data on PRP to demonstrate new bone formation in either autologous bone grafts or anorganic bovine bone in the presence of PRP [7]. This investigation was designed to compare the regenerative potential of a routinely utilized bone graft material alone and after addition of PRP in two different concentrations. Application of the material was performed in the forehead area of female pigs, which closely resembles the conditions of the maxillofacial region because the bone is of desmal origin and does not depend on central blood supply [8]. The evaluated material is a deproteinized bovine bone matrix (DBBM), its osteoconductive properties were shown previously [9,10]. In long-term assessment it exhibited no signs of resorption [11]. The investigation was performed by means of microradiography and immunohistochemistry. Microradiograpic evaluation detects bone boundaries with accuracy and allows to obtain images from an entire defect, making it possible to measure areas of bone growth [12]. Although this allows to evaluate the final results of the achieved de novo bone formation, it does not explore the underlying biologic processes. Matrix proteins define the specific properties of the mineralized tissues and are expressed in a tightly controlled fashion. Their sequential expression depends on the location and differentiation stage of the producing cells [13]. Thus, this investigation was further based on evaluating the expression of certain bone matrix proteins during the osseous regeneration process. Material and methods Bone graft materials The deproteinized bovine bone matrix (Bio-Oss®, Geistlich Pharma AG, Wolhusen, Switzerland) is the inorganic component of bovine bone (i.e., the mineral). All organic material is removed by a stepwise annealing process (up to 300°C), followed by a chemical treatment (NaOH) that leaves a porous hydroxyapatite block material [14].

Selection of the study animal The adult domestic pig was the animal of choice, as it is especially suitable for the evaluation of bone healing and bone remodeling [15]. The pig’s morphological and anatomical characteristics allow the results obtained to be compared to humans. In comparison with a dog, bone regeneration rates in an adult pig have greater correlation with those in a human (pigs 1.2–1.5 mm per day; dogs 1.5–2.0 mm per day; humans 1.0–1.5 mm per day) [15]. Twenty-four adult female pigs were included in this study.1

Fabrication of platelet-rich plasma Two different techniques were used to produce autologous PRP. Both methods allowed to render approximately 0.5 ml of PRP for each defect. 1 The study was approved by the local animal committee of the government of Midfrankonia, Ansbach, Germany (approvalnr. 621-2532.31-5/00).

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Thrombocyte and leukocyte concentration as well as the levels of TGF-β1, PDGF-AB, and IGF-I were measured for both techniques prior to in vivo application as part of this study. The tube technique, further described as PRP1 (Curasan AG, Kleinostheim, Germany), provided a thrombocyte concentration 4.1 times higher to a total of 483.8 with a standard deviation (SD) of ±97.2 (thrombocytes 1000/μl) referring to untreated whole blood (118.0 ± 12.0 thrombocytes 1000/μl). Leukocytes were increased to 24.8 ± 8.9 referring to the untreated whole blood (4.3 ± 1.9 leucocytes 1000/μl) [16]. The Platelet Concentration Collection System (PCCS), a Food and Drug Administration (FDA)-cleared device which is further described as PRP2 (3i, Hanau, Germany), provided a platelet concentration 6.5 times higher to a total of 767 with an SD of ±108.7 (thrombocytes 1000/μl) [17]. The leukocyte count in this group was raised to 14.7 ± 6.8. Thus, using the PRP2 technique, higher thrombocyte values in comparison with the PRP1 method were obtained. This is in accordance with studies of Weibrich and Kleis and Marx et al. who evaluated these two preparation methods [4,18]. In our investigation, the growth factor content differed significantly for TGF-β1 (PRP1: 79.7 ng/ml vs. PRP2: 467.1 ng/ml) and PDGF-AB (PRP1: 314.1 ng/ml vs. PRP2: 251.8 ng/ml). This was less significant for IGF-I (PRP1: 69.5 ng/ml vs. PRP2: 91.0 ng/ml).

Test groups Six test groups were formed and examined at 4 different times (at 2, 4, 12, and 26 weeks). The following material combinations were randomly selected per bony defect. Group A Group B Group C Group D Group E Group F

particulated autogenous bone particulated autogenous bone and PRP1 (0.5 ml per defect) particulated autogenous bone and PRP2 (0.5 ml per defect) DBBM DBBM and PRP1 (0.5 ml per defect) DBBM and PRP2 (0.5 ml per defect)

Surgical procedure All surgical procedures were performed using intubation anesthesia. A perioperative antibiosis was administered 1 h preoperatively and for 2 days postoperatively (Streptomycin, 0.5 g/day, Gruenenthal, Stolberg, Germany). An incision was first made to the skin and the periosteum of the skull which created access to the neurocranium. Using a trephine drill (1 × 1 cm, Roland Schmid, Fuerth, Germany), identical bony defects were created. The size of the defects (10 mm diameter, 10 mm depth) met the requirements in dimension for a critical size defect in porcine species [19,20]. Without the use of a suitable augmentation material, such defects are not completely regenerated with bone by the organism but are partially filled with connective tissue [21]. The defects were positioned at least 10 mm apart to avoid biological interactions. The forehead area closely resembles the conditions of the maxillofacial region because this bone is of desmal origin and does not depend on central blood supply. The internal plate of the neurocranium remained completely intact during the procedure. The bone harvested with the trephine drill was used for filling the defects in groups A, B, and C. To produce particles of a defined size, it was crushed in a bone mill (Quetin Dental Products, Leimen, Germany) [22]. The periosteum and skin over the defects was finally sutured in two layers (Vicryl® 3.0; Vicryl® 1.0; Ethicon GmbH and Co KG, Norderstedt, Germany).

Polychromatic fluorescence labeling Starting with the 14th post-operative day, a polychromatic fluorescence labeling was performed. Sequential administration of fluorescent dyes allows to follow the direction and the topographic localization of new bone formation. During the mineralization process, the fluorescent dyes are incorporated in the matrix of the front of mineralization by chelation [23]. Rolytetracycline (12 mg/ kg body weight, Sigma-Aldrich GmbH; Taufkirchen, Germany), calcein blue (30 mg/kg body weight, Sigma-Aldrich); alizarine complex (30 mg/kg body weight, Sigma-Aldrich); and xylenol orange (20 mg/kg body weight, SigmaAldrich) were used. The animals received intramuscular injections of the fluorescent dyes in a 2% NaHCO3 solution.

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Necropsy

Immunohistochemistry

Following the protocol, the animals were sacrificed to allow recovery of the material. The pigs were sedated by an intramuscular injection of azaperone (1 mg/kg) and midazolam (1 mg/kg) in the neck. They were then euthanized by an intravascular injection of 20% pentobarbital solution into an ear vein until cardiac arrest occurred.

Immunohistological staining was performed in accordance to an established method [26]. After the samples had been cut to 5–10 μm using a microtome (Leica microsystems, Heidelberg, Germany), they were pretreated in citrate buffer (collagen I staining) and 0.1% trypsin (BMP-2 staining), respectively. Then endogenous peroxidase was blocked by incubation in 3% H2O2 solution for 15 min. Subsequently, unspecific binding of the secondary antibody was blocked using serum-free protein-block solution (Protein Block Serum Free, DAKO Diagnostics GmbH, Hamburg, Germany). Then the primary antibodies against BMP-2, collagen I, osteocalcin, osteonectin, and osteopontin were added (Table 1). For amplification of staining, a secondary biotinylated antibodies (DAKO) were used. Finally, the addition of StreptAB/HRP (DAKO) made it possible to bind the actual dye AEC+ (DAKO) to the labeled epitopes. The procedure was completed by nuclear counterstaining using hematoxylin. All samples were accompanied by a negative control. From every animal, one additional bone sample was recovered from an untreated area of the frontal skull and the activity of the original bone was taken as standard expression (0).

Removal and preparation of the specimens The skull caps of the sacrificed animals were removed and immediately frozen at minus 80°C. Before preparing the specimens, an X-ray was taken (40– 45 kV, 0.25 mA and 5 min) in a Faxitron® cabinet X-ray unit (Faxitron Cabinet X-ray Systems, Illinois, USA) in order to detect the defects. The individual bone defects were separated using a standard cutting system (Exakt Apparatebau GmbH, Norderstedt, Germany). Immersion fixation was carried out using 1.4% paraformaldehyde at 4°C to render the organic matrix insoluble. The specimens were dehydrated in an ascending alcohol series at room temperature in a dehydration unit (Shandon Citadel 1000, Shandon GmbH, Frankfurt, Germany). Xylol was used as an intermediary fixation. Technovit 9100® (Heraeus Kulzer, Kulzer Division, Werheim, Germany), a special resin which allows quantitative evidence of bone matrix proteins and is also suitable for the cutting and grinding technique according to Donath, was used for embedding [24]. This method combines the morphological superiority of plastic embedded bone tissue with the advantages of specific enzyme histochemical and immunochemical markers. It permits good preservation of morphological details, the survival of antigenic determinants and the retention of enzyme activity [25].

Analyzing the images Microradiography produces images of mineralized structures. Bone and the bone augmentation material were both radio-opaque, but could always be clearly distinguished from one another due to the structure of tested material. Newly formed bone was discriminated from local bone by the lower degree of mineralization, the different architecture of the spongiosa, and the lack of alignment of the trabecular trajectory. Osiris image analysis software (Digital Imaging Unit, University Hospital of Geneva, Switzerland) was used for analyzing the images. With this software, it is possible to determine the percentage of individual grey values or color scaling in a grey scale image. For evaluation of the microradiographic images, these percentages were classified into groups of local bone, bone augmentation material, and voids. The recorded percentages were then compared with one another. A quantitative analysis of the bone matrix proteins was performed using a protocol described previously [26,27]. The stained sections were examined under a light microscope (Axioskop, Zeiss, Jena, Germany) and subsequently fed into a computer with the attached video camera using the KS 300 software (Zeiss, Jena, Germany). Osiris image analysis software was used to analyze at least 40 microscopic fields in two sections. This number has been shown previously to be sufficient to obtain representative data with a small confidence interval [28]. The threshold of positive staining (red) was determined interactively and the determined threshold was used to automatically analyze the images of the section. The ratio of the immunostained and unstained bone matrix area was calculated. Two separate measurements were performed in all sections. The recorded percentages were then compared with one another and correlated to the expression in untreated bone. All values were finally applied to an open scale, the expression of untreated bone acting as neutral axis (0), respectively.

Microradiography Microradiography detects bone boundaries with accuracy and allows to obtain images from an entire defect, making it possible to measure areas of bone growth [12]. To produce microradiographs, the undecalcified resin embedded sections were reduced to 150–80 μm using a grinding unit (Exakt Apparatebau GmbH, Norderstedt). Subsequently, the samples were irradiated in the Faxitron® cabinet X-ray unit using 11 kV tube voltage and 0.25 mA for 6 min, with virtually no gap between the specimen and the film in order to attain an exposure as high as possible. The developed X-rays (Kodak, Stuttgart, Germany) were scanned with an AGFA scanner at 1200 dpi and 12-bit grayscale and stored in Tiff-format.

Histological evaluation The sectioned specimens were further reduced to 30 μm. Subsequently, they were polished and then stirred continuously for 5 min in 10% H2O2, rinsed under cold running water, dried, and stained for 15 min in Toluidine blue O solution. Excess stain was removed by rinsing the specimens under running water. After allowing them to dry briefly, the sections were coated with Technovit® 7200 (Heraeus Kulzer, Kulzer Division, Werheim, Germany) and polymerized under a blue light for 8 min. The stained sections were then examined under a light microscope (Axioskop, Zeiss, Jena, Germany) and were subsequently fed into a computer with the attached video camera. The KS 300 software (Zeiss, Jena, Germany) was used.

Statistics The images were each analyzed by two test persons and the values were aggregated for each specimen. For a description of continuous variables without assumption of normal distribution, median values and for a graphical description

Table 1 Specifications of the antibodies used for immunohistochemical staining Isotype

Manufacturer

Concentration

Source

Specificity

Collagen I

IgG

Novacastra Laboratories Ltd., England

1:10

Osteocalcin

IgG

Takara Biomedicals Europe, France

1:1500

Osteonectin

IgG

Takara Biomedicals Europe, France

1:500

Osteopontin

IgG

ChemIcon Inc., USA

1:100

Polyclonal goat anti human Monoclonal mouse anti human Monoclonal mouse anti human Polyclonal rabbit anti human

Carboxy-terminus of mature collagen α1 type I Epitope including γ-carboxylated residue at position 17 Bone- and platelet-derived osteonectin Amino acids 75–90 of osteopontin

M. Thorwarth et al. / Bone 38 (2006) 30–40 box plots are given. The Wilcoxon test was used for comparison of dependent samples of continuous data where normal distribution cannot be assumed. Twotailed P values of equal to or smaller than 0.05 were considered significant. All calculations and the graphic imaging were carried out using the AXUM-program (Math-Software, Cambridge/MA, USA). Due to the small number of individual probes (n = 5 per group per time), only absolute numbers representing the mean findings and standard deviations are shown for immunohistochemical results.

Results All the test animals survived the operation and each of the post-operative examinations. During the course of healing, there were no differences between the tested DBBM and autogenous bone with regard to local tissue complications. All animals could finally be evaluated. The histological assessment demonstrated that a connective tissue separation layer between the bone replacement material and the local bone did not form during the study. Thus, the osseointegration of autogenous bone and DBBM with or without PRP could be evaluated positively.

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4 weeks Mineralization content. Four weeks after surgery, autogenous bone alone reached a rate of 69.8% (±2.49) at this time. Using PRP1 the mineralization was significantly lower 58.2% (±3.74) (P = 0.002), similar to the values achieved after use of PRP2 53.8% (±5.27) (P = 0.002). In reference to the evaluated crosssectional area, microradiographic analysis demonstrated a bony consolidation in approximately 2/3 of the former defect area. The hydroxylapatite alone at 4 weeks achieved a mineralization of 38.4% (±5.89). Used with PRP1 the mineralization was 42.8% (±6.84) and PRP2 showed a mineralization of 42.8% (±6.55) (Fig. 1). The bony in growth took place from the peripheral areas. Microradiographically visible bone tissue was only found in 30– 40% of the cross-sectional area in the DBBM groups (Fig. 2). Material degradation. Four weeks after surgery, the DBBM was present in 23.4% (±3.53) of the cross-sectional area. Used with PRP1, a level of 15.9% (±2.38) was found. PRP2 showed a value of 21.6% (±6.30) (Fig. 1).

Microradiography 12 weeks 2 weeks Mineralization content. A mineralization rate of 38.0% (±9.90) could be seen when autogenous bone alone was placed in the defect. Adding PRP1 resulted in a significant increase to 49.7% (±5.31) (P = 0.055) at 2 weeks. When the 6.5 folded concentration was used the mineralization reached 62.5% (±1.90) (P = 0.002). The bovine material alone achieved a mineralization of 43.1% (±5.51) at 2 weeks time. Adding PRP1 the mineralization reached 36.8% (±5.57). PRP2 led to 32.2% (±1.48) mineralization (Fig. 1). Material degradation. After placement of the bovine material alone, after 2 weeks it filled 28.2% (±3.50) of the defect. Adding PRP1 led to a value of 23.5% (±5.87), PRP2 to 22.3% (±2.00) (Fig. 1).

Mineralization content. After 12 weeks, autogenous bone led to a nearly complete defect filling with a mineralized compartment of 58.4% (±2.50). Autogenous bone plus PRP1 also showed an almost complete filling of the defect with a mineralized compartment of 56.9% (±3.21) compared to 60.2% (±5.79) when the higher concentration was used. The bovine material reached a mineralization of 43.4% (±1.90). After used of PRP1, the mineralization was 48.2% (±4.18), whereas the higher concentrated PRP reached 46.5% (2.33) mineralization (Fig. 1). In most of the samples of the DBBM groups, a central area of 20– 30% within the bone substitute was not yet filled with microradiographically visible bone tissue (Fig. 2). Material degradation. After 12 weeks, 19.8% (±4.02) of the defect was filled with the evaluated material. When PRP1 was

Fig. 1. Percentage of mineralized substance in the defect area (left) and percentage of bone augmentation material on this radiopaque structures in the defect (right) during the post-operative course, box plot diagrams giving the median and standard deviation; statistical data obtained by Wilcoxon rank test.

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Fig. 2. Microradiographic images group A—autogenous bone (top), and group D—deproteinized bone DBBM (bottom); from left to right: 2 weeks, 4 weeks, 12 weeks, 26 weeks; complete defect regeneration in both groups in the course of the study, note periosteal bridging of the DBBM starting at 4 weeks, high stability of the DBBM during the course of the study (original magnification ×3).

added initially, this value remained relatively unchanged at 19.6% (±1.26) compared to 24.3% (±2.63) when the higher concentration of PRP was used (Fig. 1).

while particles of the bone substitute exhibited signs of resorption on the surface indicated by the existence of giant cells.

26 weeks

4 weeks De novo bone formation in more of 2/3 of the defect was found after application of autogenous bone to the defect, leaving a residual superior defect area. Large osteon haversian canals and osteoclasts were seen in the newly formed bone tissue. In some regions, osteogenic connective tissue was identified. The addition of PRP1 resulted in reduced values in bone formation and broad areas with immature bone exhibiting signs of mineralization disturbances. Autogenous bone and PRP2 showed a more solid structure of the newly formed bone and a higher density in the central area. In the DBBM group, bone formation progressed beginning at the basal and lateral portions of the defect, toward the central regions of the defect. Bone growth into the defect region primarily took place at the surfaces of the DBBM. Areas with incomplete lining of the DBBM surface could be identified (Fig. 3, middle). Macrophages were present on the remaining uncovered ceramic surfaces. An almost comparable consolidation had taken place in the former defect after 4 weeks following the application of PRP in both concentrations to the DBBM.

Mineralization content. At the final observation period of 26 weeks, a bony consolidation of the complete cross-sectional area had taken place in all evaluated groups (Fig. 2). A mineralization rate of 59.2% (±2.97) was seen after placement of autogenous bone alone. Adding PRP1 57.4% (±3.92) of the defect was mineralized at 26 weeks. When the 6.5 folded concentration was used, the mineralization reached 56.3% (±3.88). The bovine material alone achieved a mineralization of 49.1% (±5.30) at the final observation period. Adding PRP1, the mineralization reached 51.0% (±5.73). PRP2 led to 47.4% (±3.65) mineralization (Fig. 1). Material degradation. At the final observation period of 26 weeks, the material filled 17.9% (±2.42) of the evaluated area. Adding PRP1 led to an almost similar level of 18.3% (±1.49). After use of PRP2 a value of 21.6% (±4.48) was found (Fig. 1). Light microscopy 2 weeks Defects filled with autogenous bone showed bone remnants, these particles were engaged in woven bone in the peripheral areas (Fig. 3, top). No differences were evident after use of PRP1. Following the addition of PRP2, there was an enhanced de novo bone formation and a bone particle differentiation was no longer possible at the base of the defect. After application of DBBM, the bone defects were filled by a large quantity of osteogenic connective tissue and by a few irregular, immature bone trabeculae, which were found mainly at the defect borders. Most of the ceramic particles were enclosed in fibrous tissue. The connective tissue showed a discrete inflammatory infiltrate,

12 weeks The complete cross-sectional area was consolidated with new bone following the use of autogenous bone. Even though a structured bone formation was seen, some areas with a disturbed mineralization were identified. Autogenous bone together with PRP1 showed numerous osteoids, the defect had not mineralized completely, and larger areas with disturbed mineralization were identified. PRP2 combined with autogenous bone showed a cortical rim but also areas of disturbed mineralization. In the DBBM group, the bone formation had reached the surface of the former defects bone formation. The identified bone trabeculae

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Immunohistochemistry A detailed description of the immunohistochemical data is given in Table 2 and Fig. 4. In summary, the results can be described as follows: Collagen I Enhanced values were found at 2 weeks in all autogenous bone groups but highest expression levels were seen without PRP. No beneficial effect was detected at that time in the DBBM group. At 4 weeks there was a distinct elevation in all autogenous bone groups and the DBBM group with the higher concentrated PRP (Fig. 5). Unspecific alterations were observed at 12 weeks. At the final observation period, a general decline was found for the collagen I expression in comparison to untreated bone. Osteonectin In this study, at 2 weeks there was no clear expression pattern of osteonectin. A clear increase in the expression was demonstrated for all groups at 4 weeks. Subsequently, there was a quick adjustment to regular expression levels in the further course of the study. Osteocalcin At the early evaluation time at 2 weeks only inconsistent values were found. Initial positive values were demonstrated after 4 weeks and a general expression peak was found at 12 weeks mirroring the way of de novo bone formation. The missing effect of PRP on defect regeneration using DBBM was also reflected by the osteocalcin expression. In these groups highest expression levels of osteocalcin were found without PRP. Fig. 3. Light microscopy: group A, 2 weeks (top): defects with bone remnants *, these particles are engaged in woven bone in the peripheral areas; group D, 4 weeks (middle): bone growth at the surfaces of the DBBM. Areas with incomplete lining of the DBBM surface **; group D, 12 weeks (bottom): areas without sufficient bone formation, particles of the hydroxyapatite enclosed in a well-vascularized fibrous tissue (original magnification ×25).

were mainly irregular and still immature. Enclosed in new bone, areas without sufficient bone formation were still found. In these areas, particles of the hydroxyapatite were enclosed in a wellvascularized fibrous tissue (Fig. 3, bottom). If PRP1 or PRP2 was added, bone formation did not differ significantly from that of group D. 26 weeks A regular bone structure was found in groups A, B, and C. The original preparation margins were no longer distinguishable. The histological image largely corresponded with that of local, untreated bone. The surface had a dense, almost compact, bone structure. In the DBBM groups, a small compacta layer had formed on the surface of the former defects. Resorption processes at bone-free ceramic surfaces were visible. There were no signs of a connective tissue separation layer between the bone replacement material and the local bone (Fig. 6, left).

Osteopontin In this study, increased expression values were found in all groups at 2 and 4 weeks. Especially, the addition of the higher concentrated PRP to DBBM seemed to stimulate the expression. Like osteonectin, an assimilation to the level of untreated bone had already taken place after 12 weeks, a point in time where collagen I and osteocalcin still expressed variable levels. Polychromatic fluorescence labeling In all specimens fluorochrome labels were clearly visible. The accumulation sequence of the labels revealed that initial bone formation had occurred starting at the defect margins. Under UV epifluorescence (λ = 365 nm), rolitetracycline fluoresced yellow-green and calcein was green, making differentiation problematic. The alizarin complexion appeared red, whereas the finally administered xylenol orange produced an orange mark (Fig. 6). Bone formation was homogeneous throughout the implanted bone mineral. The accumulation sequence of the labels revealed that initial bone formation started at the defect margins when the ceramic materials were administered. After application of autogenous bone, new bone formation also occurred in the

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Table 2 Results of the immunohistochemical staining, mean value, and standard deviation is given

2 weeks

4 weeks

12 weeks

26 weeks

AB AB + PRP 1 AB + PRP 2 DBBM DBBM + PRP 1 DBBM + PRP 2 AB AB + PRP 1 AB + PRP 2 DBBM DBBM + PRP 1 DBBM + PRP 2 AB AB + PRP 1 AB + PRP 2 DBBM DBBM + PRP 1 DBBM + PRP 2 AB AB + PRP 1 AB + PRP 2 DBBM DBBM + PRP 1 DBBM + PRP 2

Collagen I

Osteocalcin

Osteonectin

Osteopontin

13.0 (±3.54) 9.0 (±2.74) 5.0 (±1.58) −10.0 (±4.30) −4.0 (±2.62) −3.0 (±2.09) 8.0 (±2.00) 12.0 (±3.87) 8.0 (±2.55) −6.0 (±2.55) −1.0 (±1.58) 8.0 (±1.41) 6.0 (±1.58) −4.0 (±2.62) 10.0 (±3.32) −9.0 (±3.32) 1.0 (±1.66) 9.0 (±2.12) −12.0 (±2.24) −12.0 (±2.74) −12.0 (±2.85) −12.0 (±2.24) −12.0 (±3.20) −12.0 (±2.74)

−4 (±2.52) 12.0 (±3.20) −5.5 (±2.65) 6.0 (±1.58) −3.0 (±1.45) 1.0 (±1.66) 5.0 (±1.58) −1.0 (±1.58) 16.0 (±3.45) 9.0 (±2.45) 9.0 (±2.92) 0 (±2.45) 15.0 (±3.39) 9.0 (±2.74) 15.0 (±3.39) 13.0 (±3.54) 5.0 (±1.58) 4.0 (±1.41) −5.0 (±2.42) −5.0 (±1.77) −5.0 (±2.09) −5.0 (±0.71) −5.0 (±2.83) −5.0 (±1.90)

0 (±1.06) 4.5 (±2.24) 1.0 (±1.54) 0 (±1.02) 6.5 (±1.22) 0 (±1.17) 13.0 (±3.54) 10.0 (±4.30) 8.0 (±1.41) 5.0 (±1.58) 7.0 (±2.45) 5.0 (±1.46) 0 (±1.22) 4.0 (±1.58) 2.0 (±1.58) 1.5 (±1.58) 2.5 (±2.26) 1.0 (±1.90) −2.0 (±2.67) −2.0 (±1.84) −2.0 (±3.24) −2.0 (±2.55) −2.0 (±2.45) −2.0 (±1.46)

15.5 (±4.21) 6.5 (±1.94) 1.5 (±1.58) 4.0 (±2.12) 2.5 (±2.26) 24.5 (±10.83) 13.5 (±3.84) 18.5 (±3.98) 3.5 (±2.42) 9.5 (±3.20) 3.5 (±2.29) 16.0 (±2.55) −1.5 (±1.37) 1.5 (±1.84) 3.5 (±2.09) 6.5 (±1.66) 0.5 (±2.03) 6.5 (±1.94) −1.5 (±1.41) −2.0 (±1.70) −1.5 (±2.15) −1.5 (±1.41) −1.5 (±1.94) −1.5 (±1.58)

The ratio of the immunostained and unstained bone matrix area was calculated. The recorded percentages were correlated to the expression in untreated bone and applied to an open scale, the expression of untreated bone acting as neutral axis (0).

central areas of the defect, underlining the osteoinductive properties of the material. Polychromatic fluorescence labeling did not highlight any accelerating effect of PPR on bone formation. Discussion This study investigated the processes of bone formation following the application of deproteinized bovine bone matrix and autogenous bone used alone and in combination with platelet-rich plasma in two different concentrations. Within the same biopsy, an established evaluation procedure combined a microradiographic and histomorphometrical analysis with immunohistochemical staining of bone matrix proteins [26]. In this experimental setting, the use of PRP enhanced the healing process statistically significant only after use of autogenous bone at 2 weeks. It failed to have long-term effects on bone formation, although some alterations in the expression pattern of the bone matrix proteins were demonstrated. Addition of PRP to the DBBM resulted in a reduced bony regeneration at 2 weeks. During the initial period of proliferation and extracellular matrix biosynthesis, type I collagen is expressed. This early deposition is crucial for the regenerative process [29]. Reparative cells migrate on the collagenous matrix and subsequently organize and remodel the structure through cytoskeleton and matrix synthesis and degradation [30]. In the further course, collagen influences cell differentiation and morphogenesis [31–33]. In this study, this function was reflected by an early enhancement of collagen I after use of

autogenous bone. If DBBM was used for defect filling, this peak in the collagen expression was missing. In the formation of procollagen I and collagen I, a central role is fulfilled by TGF-β [34]. Predominantly, its TGF-β1 isoform is stored in the bone extracellular matrix (ECM). There it is maintained in a latent state by the non-covalent association of the bioactive peptide of TGF-β1 with the latency-associated peptide (LAP)*-β1 [35]. Normal function depends on activation from this latent state [36]. There may be several sequential steps by which ECM-bound TGF-β is rendered active but cell surface molecules or secreted extracellular molecules always contribute to this process. Matrix metalloproteinases (MMPs) for example activate latent TGF—by proteolysis [37,38]. MMPs can also release TGF-β from latent TGF-binding protein-1 [39]. Thus, MMPs may be closely involved in the control of TGF-β activation [40]. Both the limited number of target cells and the lack of such proteins within the DBBM at the early evaluation points are likely to result in a reduced TGF-β-activity. This may be one explanation for the low collagen expression in this group [8]. No beneficial effect of the PRP application to autogenous bone or DBBM was demonstrated for the collagen expression at 2 weeks although a statistically significant increase in defect mineralization was found at that time after use of the higher concentrated PRP2. However, it seems possible that an earlier evaluation could have demonstrated some alterations in the collagen expression after addition of PRP to autogenous bone. Microradiography indicated a limited early benefit of PRP on bone formation if placed together with osteoprogenitor cells. Missing long-term effects may be caused by the single shot application of PRP in this study. Whereas the single

M. Thorwarth et al. / Bone 38 (2006) 30–40

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Fig. 4. Expression of bone matrix proteins in the course of the study, box plot diagrams giving the median and standard deviation.

administration of differentiation factors like rhBMP2 can lead to differentiation of mesenchymal progenitor cells into bone forming cells and later results in accelerated bone healing [41]; multiple or continuous administration appeared to be necessary for growth factors like TGF-β to promote fracture repair in experimental studies [42,43].

Aghaloo et al. have described positive effects of PRP also for DBBM. Their study showed an increase in bone formation after administering PRP to the bone substitute in non-critical-sized defects in the rabbit cranium [44]. The own results are contradictory. One reason could be a delayed presence of cellular components within the DBBM. Further, pronounced giant cell

Fig. 5. Exemplary illustration of the performed immunohistochemical staining; group F after 4 weeks; left: corresponding microradiograph with beginning bone formation on DBBM starting at defect margins; center: staining with collagen I antibody (IgG, 1:10), overview indicates high amount of collagen synthesis in areas of local bone adjacent to defect (original magnification ×5); right: higher magnification (corresponds with green box of center image) demonstrates collagen synthesis on DBBM surface (magnification ×10).

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M. Thorwarth et al. / Bone 38 (2006) 30–40

Fig. 6. Group F (deproteinized bone DBBM + PRP2) at the end of the trial (26 weeks), left light microscopy * anorganic bovine bone, ** newly formed bone, center identical section using fluorescence microscopy, arrows indicate the sequence of the bone labeling at day 14 (rolitetracycline ∨), day 72 (alizarin complex ∨∨), and day 120 with (xylenol orange ∨∨∨), right identical section using polarization light microscopy, collagenous fibers within the new bone lining the DBBM surface (original magnification ×25).

formation around bone substitutes treated with PRP has been described [45]. This might be an additional explanation for the outcome in this study. Osteoblasts and, to a lesser extent, osteoclasts synthesize and secrete various non-collagenous proteins. Osteocalcin and osteonectin are mostly secreted by osteoblasts. Supporting collagen fibril organization and stabilization of hydroxyapatite molecules, osteonectin maintains newly formed bone structures [46,47]. In this study, this putative function was indicated by a clear expression increase at 4 weeks. These values resembled the mineralization content evaluated by microradiography at that time. An involvement of osteonectin especially in this early stage of bone formation may also explain the quick adjustment to regular expression levels in the further course of the study. The most abundant noncollagenous protein in bone is osteocalcin. It is well known as a marker of osteoblast function and its serum level correlates with bone formation [48,49]. The increased osteocalcin expression at four and especially at 12 weeks mirrored the microradiographically visible progress in bone formation and mineralization at that time. Whereas the addition of PRP did not seem to influence the osteocalcin expression in the autogenous bone groups, lower expression levels of this noncollagenous protein were detected if PRP was added to DBBM. One possible explanation may be given by the results of a recent in vitro study. In response to platelet-released supernatants, increased migration and proliferation of mesenchymal progenitor cells were detected while the indicators of osteogenic differentiation were decreased at the same time [50]. Osteopontin is secreted by both osteoblasts and osteoclasts. It is involved in processes of cell binding via their integrin receptors and indicates remodeling of mineralized tissues. Osteopontin expression is increased when cell-signaling pathways are activated [51]. The protein is expressed in resorption lacunae and in the osteoclasts at immediate resorption surfaces [52]. In this study, increased expression values were found in all groups at 2 and 4 weeks, which may point to an intense remodeling activity. Especially, the combination of DBBM and the higher concentrated PRP seemed to increase expression values. However, complete detection of osteopontin

may have been hampered because its production is almost undetectable in osteoblasts actively expressing osteocalcin [53]. Observed differences within the two PRP groups of this study may be due to the different thrombocyte levels obtained by the applied preparation methods [18,54]. The preparation of PRP has been presented as one critical aspect of PRP research. Particularly, advantageous biological effects were found using PRP with a platelet concentration of approximately 1,000,000/ μl. Only the PRP2 preparation of this study falls within this scope. Below this range, the effects are suboptimal; beyond this range, there may even be inhibitory effects [55]. This study is a cross-sectional analysis of densitometric and biochemical parameters and has several limitations. The first evaluation took place after 2 weeks and no detailed conclusions for immediate effects of PRP can be drawn. Further, the immunohistochemical staining identifies proteins present in the bone matrix, regardless of the timing when these proteins are expressed. In the autograft group, it is therefore impossible to distinguish proteins expressed before surgery versus those expressed after surgery. Thus, an error margin for the analysis of defects filled with autogenous bone must be considered. New experimental studies may quantify the expression of the markers of bone formation more closely (e.g., in a mRNA count). However, only the immunohistochemical analysis makes it possible to assess the expression of bone matrix proteins in relation to both static and dynamic parameters of bone turnover. Summary and conclusion Although bone immunohistology is a well-recognized technique for in vitro experiments, up to now there is no available data on quantitative immunohistology for in vivo experiments. The performed evaluation is a valuable method combining advantages of conventional techniques to demonstrate results of bone formation (e.g., microradiography, polychromatic fluorescence labeling) with the opportunity to explore the underlying biology (immunohistochemistry). A distinct expression pattern of bone matrix proteins became visible in this experimental setting. Even though PRP modulated the expression of bone matrix proteins, it only promoted early

M. Thorwarth et al. / Bone 38 (2006) 30–40

bone formation in combination with autogenous bone. No effects were discovered in conjunction with an anorganic bovine bone, demonstrating the lack of osteoinductive capacity in DBBM as well as in PRP. The osteoinductive properties of autogenous bone in terms of bone graft suitability were highlighted. The presented data is a supplementary source of information since it reflects the processes of bone formation on a qualitative level. We believe that these findings will be of growing interest. In order to distinguish proteins expressed before surgery versus those expressed after surgery future studies should verify the markers of bone formation in a mRNA count. Acknowledgments The authors express their gratitude to Prof. Dr. Mult. K. Donath for his support during the histological evaluation of the specimens. References [1] Moore WR, Graves SE, Bain GI. Synthetic bone graft substitutes. ANZ J Surg 2001;71(6):354–61. [2] Parikh SN. Bone graft substitutes: past, present, future. J Postgrad Med 2002;48(2):142–8. [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 (6):638–46. [4] Marx RE. Platelet-rich plasma: evidence to support its use. J Oral Maxillofac Surg 2004;62(4):489–96. [5] Fennis JP, Stoelinga PJ, Jansen JA. Mandibular reconstruction: a clinical and radiographic animal study on the use of autogenous scaffolds and platelet-rich plasma. Int J Oral Maxillofac Surg 2002;31 (3):281–6. [6] Shanaman R, Filstein MR, Danesh-Meyer MJ. Localized ridge augmentation using GBR and platelet-rich plasma: case reports. Int J Periodontics Restorative Dent 2001;21(4):345–55. [7] Roldan JC, Jepsen S, Miller J, Freitag S, Rueger DC, Acil Y, et al. Bone formation in the presence of platelet-rich plasma vs. bone morphogenetic protein-7. Bone 2004;34(1):80–90. [8] Schlegel KA, Donath K, Rupprecht S, Falk S, Zimmermann R, Felszeghy E, et al. De novo bone formation using bovine collagen and platelet-rich plasma. Biomaterials 2004;25(23):5387–93. [9] Hammerle CH, Chiantella GC, Karring T, Lang NP. The effect of a deproteinized bovine bone mineral on bone regeneration around titanium dental implants. Clin Oral Implants Res 1998;9(3):151–62. [10] Jensen SS, Aaboe M, Pinholt EM, Hjorting-Hansen E, Melsen F, Ruyter IE. Tissue reaction and material characteristics of four bone substitutes. Int J Oral Maxillofac Implants 1996;11(1):55–66. [11] Schlegel K, Donath K. Bio-Oss—A resorbable bone substitute? Int J Biomater Med Implants 1998;8(3–4):201–9. [12] Schortinghuis J, Ruben JL, Meijer HJ, Bronckers AL, Raghoebar GM, Stegenga B. Microradiography to evaluate bone growth into a rat mandibular defect. Arch Oral Biol 2003;48(2):155–60. [13] Sommer B, Bickel M, Hofstetter W, Wetterwald A. Expression of matrix proteins during the development of mineralized tissues. Bone 1996;19 (4):371–80. [14] Tadic D, Epple M. A thorough physicochemical characterisation of 14 calcium phosphate-based bone substitution materials in comparison to natural bone. Biomaterials 2004;25(6):987–94. [15] Laiblin C, Jaeschke G. Clinical study of bone- and muscle metabolism under stress conditions in the Goettingen minipig—An experimental study. Berl Münch Tierärztl Wschr 1979;92:124–8.

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