Expression of bone matrix proteins during de novo bone formation using a bovine collagen and platelet-rich plasma (prp)—an immunohistochemical analysis

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Biomaterials 26 (2005) 2575–2584 www.elsevier.com/locate/biomaterials

Expression of bone matrix proteins during de novo bone formation using a bovine collagen and platelet-rich plasma (prp)—an immunohistochemical analysis M. Thorwartha,, S. Rupprechta, S. Falkb, E. Felszeghyc, J. Wiltfangd, K.A. Schlegela a

Department of Maxillofacial Surgery, University of Erlangen-Nuermberg, Glueckstrasse 11, D-91054 Erlangen, Germany Department of Operative Dentistry and Periodontology, University of Erlangen-Nuermberg, Glueckstrasse 11, D-91054 Erlangen, Germany c Institute for Forensic Medicine of the Semmelweis Ovostudomanyi Egyetem, Ulloi ut 93, HU-1091 Budapest, Hungary d Department of Maxillofacial Surgery, University of Schleswig-Holstein/campus Kiel, Arnold-Heller-Strasse, 16, D-24105 Kiel, Germany

b

Received 23 April 2004; accepted 22 July 2004 Available online 11 September 2004

Abstract This animal study (domestic pig) examined the bone formation after filling defined defects with autogenous bone or a collagen lyophilisat in combination with Platelet-rich-plasma (PRP) by evaluating bone matrix proteins. Six groups, both materials with and without PRP in two concentrations (+1, +2) were compared to untreated bone by means of immunohistochemistry at 2, 4, 12 and 26 weeks. BMP-2 expression was increased at 2 weeks in the collagen+1 group and after 4 weeks in the collagen+1 and +2 group. Collagen-I expression was increased at 2 weeks in all collagen groups. After 4 weeks raised levels were observed after adding the higher concentrated PRP to bone and the collagen material. Osteocalcin expression was enhanced at 2 weeks in all collagen groups and the autogenous bone+PRP1 group, after 4 weeks in the bone and collagen +2 groups. At 12 weeks higher values were observed after adding higher concentrated PRP to bone. Osteonectin and especially osteopontin were confirmed to be effective markers of early bone formation in all specimens. The described setting allows to combine established techniques (microradiography, light microscopy) with approaches to explore the underlying biology (immunohistochemistry) on the same specimen. r 2004 Elsevier Ltd. All rights reserved. Keywords: Platelet-rich plasma; Bovine collagen; Bone regeneration; Prospective study; Bone substitutes; Bone matrix proteins

1. Introduction Bony defects of the maxillofacial region can be surgically treated with augmentation procedures. In these cases, the use of autogenous bone is still regarded as the ‘‘gold standard’’ [1–3]. Due to its osteogenic potential, it is the most effective bone graft material [4]. However, this graft is limited in quantity. Furthermore, there is an increased morbidity, as harvesting autoCorresponding author. Tel.: +49-9131-8533601; fax: +49-91318534219. E-mail address: [email protected] (M. Thorwarth).

0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.07.041

genous bone requires additional surgery [5,6]. To minimize these risks, bone substitute materials on their own or in combination with autogenous bone may be used [7–9]. Advantages of these materials are unlimited supply, easy sterilization, and storage. Despite the increase in the number of products, up to date there has not been a single ideal material [10]. Bone substitutes do not provide the cellular elements necessary for osteogenesis, and they cannot be considered osteoinductive. Providing only a scaffold for new bone deposition, they are osteoconductive instead [11]. Platelet-rich plasma (PRP) is an autologous concentration of platelets in a small volume of plasma.

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Because it is a concentration of platelets, it is also a concentration of the fundamental protein growth factors proved to be actively secreted by platelets to initiate all wound healing; isomeres of platelet-derived growth factor (PDGF), transforming growth factors (TGF1 and TGF2), vascular endothelial growth factor, and epithelial growth factor. It also contains proteins known to act as cell adhesion molecules for osteoconduction and as a matrix for bone, connective tissue, and epithelial migration [12]. Various publications report positive results in either or both bone and soft tissue healing and with composites of autogenous bone grafts and bone substitutes [12]. The collagen Collosss (Ossacur Medical Products GmbH & Co. KG, Oberstenfeld, Germany) is extracted after demineralization from the bony matrix of calf. It represents a completely resorbable collagen lyophilisat, which is, according to the manufacturer enhancing de novo bone formation in a osteoinductive way [13]. The material contains bovine bone morphogenetic protein (bBMP), transforming growth factor b1 (TGFb1) and epidermal growth factor (EGF) explaining its osteoinduktive capacities [14–18]. It can ectopically form bone [16–18]. Since 1997 Collosss is licensed in Germany as a medical product class 3 and CElabelled. In a recent study, the properties of this material were investigated in comparison to autogenous bone, in both groups with and without the addition of PRP [19]. In microradiography and light microscopy the mitogenetic cytokines found in PRP significantly changed the chronology of de novo bone formation in the autogenous group, signifying an earlier acceleration of maturation. The results also indicated the osteoinductive effect of the evaluated collagen lyophilisat. Whereas the regenerative performance could not be increased significantly with the addition of PRP over the whole observation period, an initial difference was observed in light microscopy. However, whereas microradiography and light microscopy allowed to evaluate the final results of the achieved de novo bone formation, this analysis did not explore the underlying biologic processes. The organic phase of tissue mineralization consists mainly of collagen and noncollagenous matrix proteins [20,21]. Their sequential expression depends on the location and differentiation stage of the producing cells. The control of the spacial and temporal expression probably reflects the specific roles the encoded proteins fulfil in the mineralization of hard tissues [22]. The aim of this experimental study was to examine the biology of bone formation more closely. Therefore, the expression of certain bone matrix proteins during the osseous regeneration of bony defects filled with autogenous grafts and a collagen lyophilisat in combination with PRP was evaluated.

2. Materials and methods 2.1. Selection of the study animal The adult domestic pig was the animal of choice since it is especially suitable for the evaluation of bone healing and bone remodelling [23]. 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) [23]. 24 adult female animals were included in this study.1 2.2. Fabrication of platelet-rich plasma 0.5 ml of PRP were available for each defect. Production of PRP was easy using both techniques. Application of the tube technique, further described as PRP1 (Curasan AG, Kleinostheim, Germany) rendered a thrombocyte concentration 4.1 times higher to a total of 483.8 with a standard deviation (SD) of 797.2 (thrombocytes 1000/ml) referring to untreated whole blood (118.0712.0 thrombocytes 1000/ml). Leukocytes were increased to 24.878.9 referring to the untreated whole blood (4.371.9 leucocytes 1000/ml) [24]. Applying the platelet concentration collection system (PCCS), a food and drug administration (FDA)-cleared device which is further described as PRP2 (3i, Hanau, Germany) we were able to increase the platelet concentration 6.5 times higher to a total of 767 with an SD of 7108.7 (thrombocytes 1000/ml). The leukocyte count in this group was raised to 14.776.8 [25]. Thus, using the PRP2 technique, we were able to obtain higher thrombocyte values in comparison with the PRP1 method. This is in accordance with studies of Weibrich & Kleis and Marx et al. who evaluated these two preparation methods [12,26]. In another comparative study Zimmermann et al. were able to demonstrate a correlation between thrombocyte counts and amount of growth factor levels [3]. In our investigation, the growth factor content differed significantly for TGF-beta1 (PRP1: 79.7 ng/ml vs. PRP2: 467.1 ng/ml) and PDGFAB (PRP1: 314.1 ng/ml vs. PRP 2: 251.8 ng/ml). This was less significant for IGF-I (PRP1: 69.5 ng/ml vs. PRP2: 91.0 ng/ml). 2.3. Test groups Six test groups were formed and examined at 4 different times (at 2, 4, 12 and 26 weeks). The following 1 The study was approved by the local animal committee of the government of Midfrankonia, Ansbach, Germany (approvalnr. 6212532.31-5/00).

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material combinations were randomly selected per defect. Group A: particulated autogenous bone, Group B: particulated autogenous bone and PRP 1 (0.5 ml per defect), Group C: particulated autogenous bone and PRP 2 (0.5 ml per defect), Group D: bovine collagen; 20 mg (Collosss), Group E: bovine collagen; 20 mg (Collosss) and PRP 1 (0.5 ml per defect), Group F: bovine collagen; 20 mg (Collosss) and PRP 2 (0.5 ml per defect). 2.4. Surgical procedure All surgical procedures were performed using intubation anaesthesia. The animals were given a perioperative antibiosis 1 h preoperatively and for 2 days postoperatively (Streptomycin, 0.5 g/d, 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 cm
1 cm, Roland Schmid, Fuerth, Germany) 6 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 [27,28]. 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 [29]. The defects were positioned at least 10 mm apart to avoid biological interactions. The forehead area was chosen because the 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 group A, B and C. To produce particles of a defined size, it was crushed in a bone mill (Quetin Dental Products, Leimen, Germany) [30]. The periosteum and skin over the defects was finally sutured in two layers (Vicryls 3.0; Vicryls 1.0; Ethicon GmbH & Co. KG, Norderstedt, Germany). 2.5. Sacrification procedure 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 a ear vein until cardiac arrest occurred. 2.6. Removal and preparation of the specimens The skull caps of the sacrificed animals were removed and immediately frozen at minus 80 1C. Before prepar-

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ing the specimens, an X-ray was taken (40–45 kV, 0.25 mA and 5 min) in a Faxitrons 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 1C 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 9100s (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 [31]. 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 [32].

2.7. Immunohistochemistry After the samples had been cut to 5 mm using a microtome saw (Leica microsystems, Heidelberg, Germany), they were dyed using antibodies for BMP-2 (Santa Cruz Biotechnologies Inc., CA, USA), collagen I (Novacastra Laboratories Ltd., England), osteocalcin (Takara Biomedicals Europe, France), osteonectin (Takara Biomedicals Europe, France) and osteopontin (ChemIcon Inc., Pittsburgh, USA) (Table 1). In advance histologic sections were partly pretreated in citrate buffer (collagen I staining) and trypsin (BMP-2 staining) respectively. Then endogenous peroxidase was blocked by incubation in 3% H2O2 solution for 15 min. Subsequently, the samples had to be blocked to prevent unspecific staining using protein-block serum free (DAKO Diagnostics GmbH, Germany). Then the primary antibodies against BMP-2, collagen I, osteocalcin, osteonectin and osteopontin were added (Concentrations BMP-2: 1:10; collagen I 1:10; osteocalcin 1:1500; osteonectin 1:500 and osteopontin 1:100). To enable a colored presentation, a secondary antibody (DAKO) had to be added. Finally, the addition of StreptAB/HRP (DAKO) made it possible to bind the actual dye AEC+ (DAKO). The procedure was completed by hematoxylin–eosin counterstaining. 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).

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Table 1 Specifications of the antibodies used for immunohistochemical staining Isotype

Manufacturer

Concentration

Source

Specifity

BMP 2

IgG

Santa Cruz biotechnologies Inc., USA

1:10

Polyclonal rabbit anti human

Collagen 1

IgG

1:10

Polyclonal goat anti human

Osteocalcin

IgG

Novacastra laboratories Ltd., England Takara biomedicals, Europe, France

1:1500

Monoclonal mouse anti human

Osteonectin

IgG

1:500

Osteopontin

IgG

Monoclonal mouse anti human Polyclonal rabbit anti human

Amino acids 300–350 mapping within an internal region of BMP Carboxy terminus of mature collagen a1 type I Epitope including gcarboxylated residue at position 17 Bone derived and platelet derived osteonectin Amino acids 75–90 of osteopontin

Takara biomedicals, Europe, France ChemIcon Inc., USA

1:100

2.8. Analyzing the images

3.1. BMP-2

Osiris image analysis software (Digital Imaging Unit, University Hospital of Geneva, Switzerland) was used for analyzing the images. Staining with an antibody in the described manner produces a coloration of defined areas of the specimens. With the used software it is possible to recognize the dimensions of stained areas in a region of interest (ROI). This allows to determine the percentage of a specific color in the evaluated cross section. The recorded percentages were then compared with one another and correlated to the expression in untreated bone. Finally all values were applied to an open scale, the expression of untreated bone acting as neutral axis (¼ 0), respectively.

2 weeks: An increase of 0.5 (71.12) in the BMP-2 expression could be found in the defect filled solely with autogenous bone (Group A) compared to untreated bone (0). Adding PRP 1 (Group B) produced a value of 1.8 (71.15), whereas after use of PRP 2 group (Group C) an ascent to 7.8 (71.79) in the expression of BMP-2 was seen. For the bovine collagen (Group D) a level of 5.0 (71.73) was found. The material plus PRP 1 (Group E) had an incline to 13.8 (73.42) and to 7.8 (71.48) after use of PRP2 (Group F) (Fig. 1a). 4 weeks: A value of 4.8 (71.30) in BMP-2 expression was found in the defect filled solely with autogenous bone. The PRP 1 group had an amplification to 1.8 (71.15) compared to 4.8 (71.30) in the PRP 2 group. The bone substitute material at 4 weeks showed a slight increase to 0.8 (71.64). In combination with PRP 1 an increase to 6.5 (72.78) was found compared to an incline to 6.8 (72.77) in the PRP 2 group (Fig. 1a,b). 12 weeks: At 12 weeks BMP-2 expression exhibited a level of 6.8 (71.10). Adding PRP 1 lead to a value of 6.8 (71.92) compared to 4.8 (71.30) in the PRP 2 group. The bovine material at 12 weeks showed a value of 2.8 (71.35). Adding PRP 1 led to a decrease to 1.0 (72.76) in the expression of BMP-2, a 2.0 (72.92) value was seen after use of PRP 2 (Fig. 1a). 26 weeks: At the final observation period a decline to 2.0 (71.70) was found in the autogenous bone group. Adding PRP 1 led to a decrease to 3.0 (71.46), whereas after use of PRP 2 an increased level of 1.8 (71.82) was seen. Using the bovine collagen alone a lower expression of 2 (71.70) was measured, the addition of PRP 1 produced a value of 3.0 (71.46). The use of PRP 2 led to a incline to 1.8 (71.82) in the expression of BMP-2 at the final observation period (Fig. 1a).

2.9. Statistics The images were each analyzed by two test persons and the values were aggregated for each specimen. Then the mean, minimum and maximum of these values were given for each material group. The AXUM programme (Math-Software, Cambridge/MA, USA) was used for the graphic images. Box plots were created, each covering all groups at a certain point in time. Due to the small number of individual probes (N ¼ 5 per group per time) only absolute numbers representing the mean findings and standard deviations are shown.

3. Results All the test animals survived the surgical procedure and each of the postoperative examinations. All animals could finally be evaluated. The given numbers represent relative values compared to the expressions in untreated bone using the identical evaluation method.

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Collagen Expression

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(b) Fig. 1. (a) Expression of BMP-2 (bars from left to right: group A=autogenous bone; Group B—autogenous bone+PRP 1, Group C—autogenous bone+PRP 2, Group D—xenograft, Group E— xenograft+PRP 1, Group F—xenograft+PRP 2, (box plot diagram giving median and standard deviation). (b) Expression of BMP-2 group—Group F—xenograft+PRP 2 at 4 weeks, green line marks bottom of former defect (staining with BMP-2 antibody/Santa Cruz Biotechnologies/magnification
2.5).

3.2. Collagen I 2 weeks: Expression in the defect filled solely with autogenous bone showed an increase to 13.0 (73.54) compared to a rise to 9.0 (72.74) in the PRP 1 group and 5.0 (71.58) in the PRP 2 group. The collagen lyophilisat expressed a level of 2.0 (72.92). In the PRP 1 group a value of 12.0 (72.74) was found compared to 13.0 (75.48) in the PRP 2 group (Fig. 2a,b). 4 weeks: Expression in the defect filled solely with autogenous bone showed a level of 8.0 (72.00). Adding PRP 1 a rise to 12.0 (73.87) was found. PRP 2 produced a value of 8.0 (72.55). The xenograft had a value of 8.0 (72.35) compared to 6.0 (73.06) in the PRP 1 group. In the PRP 2 group there was an incline to –3.0 (71.41) (Fig. 2a).

(b) Fig. 2. (a) Expression of collagen I (bars from left to right: Group A=autogenous bone; Group B—autogenous bone+PRP 1, Group C—autogenous bone+PRP 2, Group D—xenograft, Group E— xenograft+PRP 1, Group F—xenograft+PRP 2, (box plot diagram giving median and standard deviation). (b) Expression of collagen I group E—xenograft+PRP 1 at 2 weeks, green line marks bottom of former defect (staining with collagen I antibody/Novacastra laboratories/magnification
2.5).

12 weeks: A level of 6.0 (71.58) was seen in the autogenous group, adding PRP 1 the expression dropped to 4.0 (72.62). A level of 10.0 (73.32) was found in the PRP 2 group. The bovine collagen showed a level of 18.0 (74.85), plus PRP 1 16.0 (75.87) compared to decline to 7.5 (75.50) in the PRP 2 group (Fig. 2a). 26 weeks: At the final observation period the expression of collagen I had equalized at 12 in all groups compared to untreated bone (standard deviation given in the diagram; Fig. 2a).

3.3. Osteocalcin 2 weeks: The defect filled with autogenous bone at 2 weeks showed a lower expression compared to untreated bone, 4 (72.52). After adding PRP 1 the expression

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was 12.0 (73.20) compared to 5.5 (72.65) found in the PRP 2 group. The xenograft alone showed an unchanged average expression of 0 (72.57), adding PRP 1 led to an increase to 1.0 (72.55). In contrast, PRP 2 showed a clear incline to 14.0 (74.57) in the osteocalcin expression (Fig. 3a,b). 4 weeks: Autogenous bone showed a expression of 5.0 (71.58), PRP 1 led to a value of 1.0 (71.58) compared to 16.0 (73.45) after use of PRP 2. The bovine collagen showed a value of 3.0 (72.24) regarding the osteocalcin expression in contrast to 5.0 (72.35) seen in the PRP 1 group. The use of PRP 2 resulted in average expression of 15.0 (76.67) (Fig. 3a). 12 weeks: Osteocalcin expression in the autogenous bone expressed a value of 15.0 (73.39). PRP 1 led to 9.0 (72.74) compared to 15.0 (73.39) in the PRP 2 group.

Expression (neutral axis represents untreated bone)

Osteocalcin Expression

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The collagen showed a osteocalcin expression of 4.0 (71.17). Adding PRP 1 led to an unchanged average value of 0 (72.42) whereas the PRP 2 group also expressed a value of 4.0 (73.00) (Fig. 3a). 26 weeks: At the final observation period of 26 weeks the expression of osteocalcin had equalized at a 5.0 in all groups compared to untreated bone (standard deviation given in the diagram; Fig. 3a). 3.4. Osteonectin 2 weeks: No incline 0 (71.06) in the average osteonectin expression could be found in the defect filled with autogenous bone. Adding PRP 1 produced an increase to 4.5 (72.24), whereas after use of PRP 2 (Group C) a small incline in the average expression to 1 (71.54) was found. The bone substitute presented an incline in the average osteonectin expression to 7.0 (72.00). The material plus PRP 1 produced a value of 3.5 (73.57), only a small effect was present 1.0 (73.66) after use of PRP 2 (Fig. 4a). 4 weeks: A rise to 13.0 (73.54) in osteonectin expression was found in the defect filled solely with autogenous bone. The PRP 1 group expressed an increase to 10.0 (74.30) compared to 8.0 (71.41) in the PRP 2 group. The collagen showed a value of 8 (75.24). In combination with PRP 1 a level of 8.0 (72.00) was found compared to 7.0 (7 3.08) seen in the PRP 2 group (Fig. 4a,b). 12 weeks: No differences in the osteonectin expression were seen after transplantation of autogenous bone compared to untreated bone 0 (71.22). Adding PRP 1 lead to an increase to 4.0 (71.58) compared to a value of 2.0 (71.58) in the PRP 2 group. The collagen at 12 weeks showed an average level of 0 (71.97). Adding PRP 1 led to an increase to 8.0 (72.35) in expression of osteonectin. A value of 2.5 (73.28) was seen after use of PRP 2 (Fig. 4a). 26 weeks: At the end of the observation period of 26 weeks, the expression of osteonectin had equalized in all groups at a level of 2 times compared to untreated bone (standard deviation given in the diagram; Fig. 4a). 3.5. Osteopontin

(b) Fig. 3. (a) Expression of osteocalcin (bars from left to right: Group A=autogenous bone; Group B—autogenous bone+PRP 1, Group C—autogenous bone+PRP 2, Group D—xenograft, Group E— xenograft+PRP 1, Group F—xenograft+PRP 2, (box plot diagram giving median and standard deviation). (b) Expression of osteocalcin group F—xenograft+PRP 2 at 2 weeks, green box marks border of former defect (staining with osteocalcin antibody/Takara Biomedicals/ magnification
1.25).

2 weeks: Expression in the defect filled with autogenous bone showed a rise to 15.5 (74.21) compared to 6.5 (71.94) in the PRP 1 group and 1.5 (71.58) in the PRP 2 group. The bovine material showed a level of 21.5 (78.59). In the PRP 1 group 4.5 (73.39) was found compared to an equal value of 4.5 (74.21) in the PRP 2 group (Fig. 5a,b).

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Osteonectin Expression

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30

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(b)

Fig. 4. (a) Expression of osteonectin (bars from left to right: Group A=autogenous bone; Group B—autogenous bone+PRP 1, Group C—autogenous bone+PRP 2, Group D—xenograft, Group E— xenograft+PRP 1, Group F—xenograft+PRP 2, (box plot diagram giving median and standard deviation). (b) Expression of osteonectin group D—xenograft at 4 weeks, green line marks bottom of former defect (staining with osteonectin antibody/Takara biomedicals/ magnification
5.0).

Fig. 5. (a) Expression of osteopontin (bars from left to right: Group A=autogenous bone; Group B—autogenous bone+PRP 1, Group C—autogenous bone+PRP 2, Group D—xenograft, Group E— xenograft+PRP 1, Group F—xenograft+PRP 2, (box plot diagram giving median and standard deviation). (b) Expression of osteopontin group A—autogenous bone at 2 weeks, green line marks bottom of former defect (staining with osteopontin antibody/ChemIcon Inc./ magnification
2.5).

4 weeks: Expression in the defect filled with autogenous bone showed a level of 13.5 (73.84). Adding of PRP 1 indicated an expression value of 18.5 (73.98); while adding PRP 2 produced a value of 3.5 (72.42). The collagen expressed a level of 7.5 (72.12) and also 7.5 (72.55) in the PRP 1 group. In PRP 2 group a slight increase to 1.5 (71.87) was found (Fig. 5a). 12 weeks: A decreased value of 1.5 (71.37) was seen in the autogenous group, adding PRP 1 lead to an expression of 1.5 (71.84) compared to 3.5 (72.09) in the PRP 2 group. The bone xenograft exhibited 1.5 (72.03), plus PRP 1 2.5 (72.57) and plus PRP 2 an unchanged value of 0 (73.24) (Fig. 5a). 26 weeks: Expression in the defect filled with autogenous bone showed a 1.5 (71.41) times lower expression, adding PRP 1 the expression dropped to 2 (71.70) compared to 1.5 (72.15) in the PRP 2 group.

The bone xenograft exhibited 2.0 (71.66), plus PRP 1 1.5 (72.45) and plus PRP 2 and also 1.5 (71.41) (Fig. 5a).

4. Discussion This study compared the regenerative potential of autogenous bone with a bovine collagen (Collosss) used alone and in combination with platelet-rich plasma in two different concentrations by means of immunohistochemistry. The evaluated collagen is assumed to have osteoinductive properties [14]. The adult pig was selected as the test model, as it has virtually the same bone reparative capacity as humans [33]. Defects of a defined size were created in ossea frontalia of adult female animals and randomly filled with the different materials. PRP ad modum CurasanTM and ad modum

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3iTM were added to both materials. Using the 3i technique, we were able to obtain higher thrombocyte values in comparison to the Curasan method. This may explain differences in the effects between the two PRP preparations, since the existence of a correlation between thrombocyte counts and amount of growth factor levels was shown in previous studies [3,26]. Multiple cytokines affecting all stages of de novo bone formation have been identified. Displaying their expression by immunohistochemistry makes it possible to evaluate the time sequence in the bone regeneration process [21,34,35]. BMP-2 stimulates proliferation of both chondrocytes and osteoblasts and causes increased matrix production in each cell type. BMPs have also been found to induce mesenchymal stem cells differentiation to osteoblasts [36,37]. When looking at the immunhistochemical results of the autogenous bone, a morphogene itself, it showed differences in expression of BMP-2 in presence or absence of PRP particularly at an early stage. Whereas after use of the higher concentrated PRP a distinct increase in the expression was found after 2 weeks, the addition of PRP lead to rather unchanged or reduced expression values after 4 and 12 weeks. Increased levels in expression of BMP-2 after use of the bovine collagen in presence or absence of PRP underline the osteoinductive properties of the evaluated material within a bony environment. As expected, the results had almost converged to the level of untreated bone at the final observation period of 26 weeks in all 6 groups due to the termination of de novo bone formation. Collagen I is considered as being the basic initial bone matrix protein in bone formation [38–40]. It forms a scaffold for cell attachment and migration in tissues and modulates cell differentiation and morphogenesis by mediating the flux of chemical and mechanical stimuli [41]. During the remodelling of skeletal structures, reparative cells migrate on this matrix, and subsequently organize and remodel the matrix through cytoskeleton and matrix synthesis and degradation [42]. The expression of Collagen I is described to be an early, but unspecific indicator of de novo bone formation. In our setting Collagen I expression at 2 weeks was enhanced in all but the xenograft +PRP 1 group. At 4 weeks there was still a distinct elevation in all autogenous bone groups and the xenograft group without PRP. The addition of PRP to the bovine collagen lead to marked decreased values after 4 weeks. At the final observation period, a general decline was found for the collagen I expression in comparison to untreated bone. Extracellular matrix proteins, such as osteocalcin, osteonectin and osteopontin are synthesized and secreted during the process of osteoblasts differentiation and mineralization. They form the bone matrix, and are

known to play an important role in the process of ossification [43]. Osteocalcin or bone gliaprotein is a small noncollagenous protein of molecular weight 5800 kDa. It is a unique product of osteoblasts and odontoblasts [38]. Osteocalcin is the most abundant noncollagenous protein in bone and is mainly incorporated into the bone matrix where it is bound to hydroxyapatite. Only a small proportion of osteocalcin is released into circulation. Its serum concentration is a sensitive marker of bone formation and correlates with histomorphometric indices of bone formation [38–40]. Osteocalcin indicates the mineralization process in bone formation implemented by the calcification of the osteocyts in the collagen layer. Therefore its appearance in the process of bone maturation is known to be delayed to collagen I. In principle this was affirmed by the presented results. Autogenous bone alone showed a initial positive expression value at 4 weeks, with an expression peak at 12 weeks mirroring the way of de novo bone formation. Autogenous bone with PRP showed higher values at 2 and 4 weeks indicating an earlier calcification in these groups. In comparison to untreated bone, osteocalcin expressions in the xenograft groups was elevated after 2 weeks only when the higher concentrated PRP was added. A general incline was seen at 4 weeks. Again, equal levels slightly below the expression of untreated bone were found after 26 weeks. Osteonectin, a 43 kDa phospho-glycoprotein is a noncollagenous protein synthesized by human osteoblasts [44,45]. It represents 2–3% of the total amount of protein in developing bone tissue. Although it is not only found in bone tissue, it is 1000 times more abundant in bone than in any other tissue [45,46]. Osteonectin shows strong affinity for calcium and hydroxyapatite, probably linking them to collagen. In the described setting osteonectin showed increased values at 2 weeks after addition of the lower concentrated PRP to autogenous bone. An obvious increase was also found when the bovine collagen was used alone. At 4 weeks increased values in all groups occurred. This may reflect the putative function of osteonectin which is to maintain bone structure by helping in the stabilization of hydroxyapatite molecules and the collagen fibril organization [47,48]. Osteopontin is a sialoprotein found in the bone matrix. Osteopontin is widely expressed and is one of the major noncollagenous proteins in bone [49]. It is involved in many diverse functions, such as binding to cells via their integrin receptors or regulation of the formation and remodelling of mineralized tissues. Osteopontin expression is increased when cell-signaling pathways are activated [50]. Previously osteopontin was shown to be an early marker of cells of the osteoblast lineage [51]. These findings were affirmed by the observed results. Higher expression values were found

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in all groups at two and 4 weeks. An assimilation to the level of untreated bone had already taken place after 12 weeks, a point in time where the other extracellular matrix proteins still expressed variable levels.

5. Conclusion The described experimental setting is a valuable method that combines established techniques of evaluating the result of bone formation (microradiography, light microscopy) with a possibility to explore the underlying biology (immunhistochemistry). 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. Thus the observed data and the deduced conclusions should be handled cautiously. The results indicate the participation of BMP-2 in all phases of bone formation. The expression of collagen I proved to be a sufficient marker of early bone formation. Osteocalcin indicated the later phase of bone formation, especially after addition of PRP. Osteonectin and especially osteopontin were confirmed to be early and effective markers of bone formation. Over the whole observation period the results of the various groups adapted when de novo bone formation had terminated. Effects of PRP on the expression of bone matrix proteins were identified particularly in the early phase. These results indicate that PRP has an osseopromotive effect due to its mitogenic properties if placed together with bone as morphogenic responder cells. This confirms the results obtained by microradiographic and histologic evaluation, where a significant accelerating effect on bone formation was demonstrated at 2 weeks [19]. The increased initial expression of BMP-2 in the group treated with the bovine collagen is of special interest. Together with the subsequently increased osteocalcin levels it may indicate osteoinductive properties of the material within a bony environment. The evaluation of the cytokine expression described in this article is based on comparing the cytokine expression measured in our experimental setting to primary expression found in identical portions of untreated bone of same origin. A validation of these measurements can only be achieved by numerous repetitive experiments using the same method. However, in the light of the results achieved by microradiography and light microscopy, this data can be seen as a supplementary source of information. Although it marks a rather new development we believe that the obtained data will be of growing interest. Considering the presented results, different observation times may be chosen in new prospective studies to follow the protein expression pattern during the early stages of de novo bone formation more closely.

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