Comparative evaluation of heat-treated bone allograft combined with platelet rich plasma, recombinant human bone morphogenetic protein 2 and zoledronic acid: An experimental study on the histopathological and histomorphometric aspects of bone healing

Comparative evaluation of heat-treated bone allograft combined with platelet rich plasma, recombinant human bone morphogenetic protein 2 and zoledronic acid: An experimental study on the histopathological and histomorphometric aspects of bone healing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative evaluation of heat-treated bone allograft combined with platelet rich plasma, recombinant human bone morphogenetic protein 2 and zoledronic acid: An experimental study on the histopathological and histomorphometric aspects of bone healing Dina Saginova, Elyarbek Tashmetov, Yevgeniy Kamyshanskiy, Berik Tuleubaev, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2813335/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The establishment of a reliable technique for promoting bone formation without resorting to autografts remains an unresolved challenge. Currently, platelet-rich plasma (PRP), bone morphogenetic protein (BMP), and bisphosphonates are under intense scrutiny for their potential clinical utility in such instances. Despite their widespread investigation, the effect of these agents on bone formation is still a matter of debate, and the observed variations in their efficacy are influenced by multiple factors, including the biomaterials employed. The aim of this research was to assess the impact of bone allograft combined with platelet rich plasma, recombinant human bone morphogenetic protein-2 (rhBMP-2), and zoledronic acid on bone formation in rabbit femur defects. Materials and methods A total of 96 rabbits were used, and femoral bone defects with a diameter of 5mm and depth of 10mm were created. The bone allografts used were prepared from femoral heads according to the Marburg system. The rabbits were divided into four groups: (1) bone allograft with PRP (AG + PRP), (2) bone allograft with rhBMP-2 5µg (AG + BMP-2), (3) bone allograft with zoledronic acid 5µg (AG + ZA), and (4) bone allograft only (AG) as the control group. Histopathological and histomorphometric analyses were performed to evaluate bone defect healing after 14, 30, and 60 days. Results The findings from the histomorphometric analysis showed that the new bone formation inside the bone allograft was significantly greater in the AG + PRP group compared to AG and AG + Zol groups after 14- and 30 days (p < 0.000). The use of bone allograft with rhBMP-2 induced higher bone formation compared to AG group on days 14 and 30 (p < 0,000), but excessive osteoclast activity was observed on day 60. The local co-administration of ZOL with heat-treated allograft inhibits allograft resorption as well as new bone formation in the bone defect at all periods. Conclusion In conclusion, the study demonstrated that PRP and rhBMP-2, combined with a Marburg bone allograft, can significantly promote bone formation in the early stage of bone defect healing. Bone regeneration bone graft platelet-rich plasma BMP-2 zoledronic acid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background The replacement of bone defects is a pressing issue in modern traumatology and orthopedics. Osteogenesis can be stimulated by autografts and bone substitutes, with over 2 million bone graft surgeries being performed worldwide each year [1,2]. Autologous bone is considered the "gold standard" in orthopedics for replacing bone defects caused by various factors. However, the use of autologous bone has its own drawbacks, including filling large bone defects, pain in the donor site, increased operation time, and cosmetic defects [3,4]. This led to the development of various bone substitute materials, which are structurally similar to bone tissue, as an alternative to autologous bone [5–7]. Commercial tissue banks currently offer bone substitute materials, such as cortical cancellous ilium, femoral head, freeze-dried bone substitutes, and decalcified freeze-dried bone, which are sterilized using chemical or physical methods [3,8]. The Marburg Bone Bank prepared bone graft is a type of bone allograft widely used in orthopedic surgery. The Marburg Bone Bank system is based on thermal disinfection of the femoral bone head and is considered safe, as the bone matrix in the graft provides the necessary osteoconductive properties required for successful bone repair [9–11]. However, after being extensively processed, bone allografts lose their innate osteoinductive properties and, as a result, are unable to produce the same clinical outcomes as autologous bone grafts. [12,13]. Therefore, there has been a growing interest in using growth factors and morphogens as substances that can provide osteoinductivity to bone substitutes. [14–17]. The addition of bone morphogenetic proteins (BMPs) is an example of such incorporation in bone graft substitutes [18,19]. BMPs are a naturally occurring group of proteins belonging to the transforming growth factor beta (TGF-β) family. They act as cytokines that facilitate the differentiation of mesenchymal cells into bone- and cartilage-forming cells. Among them, recombinant human bone morphogenetic protein-2 (rhBMP-2) is known to be critical in bone formation and healing as it has the ability to induce osteoblast differentiation [18–20]. Platelet-rich plasma (PRP) has been used as an autologous blood product in clinical settings to promote tissue regeneration in various types of bone defects and guide bone regeneration with bone grafts [21,22]. PRP is believed to possess the ability to stimulate bone regeneration due to the growth factors released from activated platelets, which have a stimulatory effect on progenitor cells and vascularization at local sites. Platelets may contain unique activators of bone morphogenetic proteins (BMPs) that stimulate the differentiation of progenitor cells into bone-forming cells in laboratory settings [21,23,24]. Bisphosphonates (BP) prevent and treat increased bone resorption in skeletal diseases [25]. Zoledronic acid (Zol) is considered the most potent bisphosphonate in terms of its pharmacological activity and affinity to bone, especially in areas of active bone metabolism [26]. While the effects of Zol on bone resorption have been extensively studied both in vivo and in vitro, its impact on bone formation is still not completely understood and is currently a subject of debate [26,27]. Over the past few decades, some studies have employed specific concentrations of ZA to stimulate osteoregeneration, leading to different findings and varying conclusions [27]. Due to the lack of relevant literature on the combined use of a bone allograft prepared according to the Marburg system and osteoinductive substances, this study aims to evaluate the effect on the bone formation of a bone allograft in combination with platelet-rich plasma, recombined human bone morphogenetic protein-2 (rhBMP-2) and zoledronic acid in rabbit femur defects using histopathological and histomorphometric analyses. Materials And Methods Preparation of Marburg bone graft In this study, heat-treated femoral heads were utilized as bone allograft (Fig. 1). Femoral heads were acquired from a living donor who had undergone hip joint arthroplasty surgery, in accordance with national regulations [28,29]. In the context of endoprosthetics of the hip joint, a bone transplant is extracted from the operating room, specifically the head of the femur, and subjected to a series of mechanical cleaning procedures in sterile conditions. These procedures involve removing any soft tissue, cartilage, and ligaments from the bone allograft. Once cleaned, the femoral bone allografts were then perforated using a specially developed device at equal intervals [30]. Then, the femoral head were placed in a disposable, sterile container and filled with 0.9% NaCl solution in a volume of 300 ml. The container were then sealed and processed in a Lobator SD-2 (Telos Company, Germany) heat treatment device for a total of 94 minutes, maintaining a temperature of 82.5°C in the femoral head for at least 15 minutes, as per the established protocol. At the end of the cycle, the sterility of the container is ensured through a special opening, after which the liquid is completely drained. The bone allografts were then stored in a freezer at a temperature of -80°C, as per the prescribed protocol [9]. Two hours before the experiment, the femoral head was unfrozen at room temperature and cut into chips. Then, to standardize the mixture of bone allograft with platelet-rich plasma, rhBMP-2, and zoledronic acid, a specific weight was used to ensure a consistent ratio of ingredients: 0.5 g bone allograft/0.5 ml PRP, 0.5 g bone allograft/5 µg rhBMP-2, 0.5 g bone allograft/5 µg Zol. Preparation of platelet-rich plasma Prior to each transplantation procedure, approximately 5 ml of blood was collected from the heart and placed in siliconized tubes containing 3.8% sodium citrate at a blood-to-citrate ratio of 9:1[31]. Platelet-rich plasma was obtained through a two-step centrifugation process [24]. The collected blood was initially centrifuged at 900 g for 8 minutes, separating the blood cell component (BCC) in the lower fraction and the serum component (SC) in the upper fraction. The BCC fraction was removed, and the remaining material was centrifuged again at 1500 g for 5 minutes to yield platelet-poor plasma (PPP) and PRP. The PRP was obtained by isolating approximately 0.5 ml of the PPP fraction, and subsequently used for impregnation of the bone allograft. Preparation of rhBMP-2 A total of 150 µ g of recombinant human bone morphogenetic protein-2 (rhBMP-2) (CUSABIO, USA) was mixed with 3 ml of saline solution to form the rhBMP-2 solution, which was mixed with bone chips to achieve 0,5 g of containing 5 µ g of rhBMP-2 per graft [21]. Preparation of zoledronic acid Zoledronic acid (Sun Pharmaceutical Industries Ltd, India) with a concentration of 0.05 mg/ml (100 µl ) [25] was added to 0.5 g of bone chips by soaking and kept in a sterile container. Animals surgery For this study, 96 adult rabbits weighing 3078 ± 87 g were procured and placed in cages for two weeks to acclimate. All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals [32] and were approved by the University Animal Care Committee (UACC) under protocol № 27 27.09.2020. During the study, the rabbits were housed at a room temperature of 22 ± 2°C and maintained at 40%-50% humidity under a 12-hour light-dark cycle. The rabbits were provided with standard rabbit pellets and tap water throughout the study. The sample size for the animal experimentation in this study was determined in accordance with Russell and Burch's bioethical principles of replacement, reduction, and refinement (1959). These principles aim to minimize animal use by using the minimum number of animals necessary to obtain statistically significant results [33]. The hip area was prepared for surgery by shaving and cleaning with an iodine solution. Aseptic techniques were employed, and sterile instruments were used. A 2% lidocaine + epinephrine 1:100000 solution was diluted to 1% and injected for infiltration. The skin was incised distally, and the muscles were dilated bluntly. A 5 mm drill was used to create bone defects in the metaphysis of the femur to a depth of 10 mm (Fig. 2) [34]. Following this procedure, the rabbits were randomly assigned to one of the four experimental groups using simple randomization. The first group received a bone allograft with platelet-rich plasma (PRP) filling in the bone defects (AG + PRP). The second group received bone allograft with recombinant human bone morphogenetic protein-2 (rhBMP-2) filling in the bone defects (AG + rhBMP-2). In the third group, the bone defects were filled with a bone allograft with zoledronic acid (AG + ZA). Finally, the fourth group (control) received only bone allograft filling in the bone defects (AG). All surgical procedures were carried out by a trained operator. The surgical incision was closed using absorbable sutures (4 − 0 Vicryl, Ethicon, Johnson & Johnson, USA). To prevent wound infection following surgery, the rabbits were given intramuscular injections of the antibiotic gentamicin 0.1 ml/kg (MAPICHEM, Switzerland) two times daily for three days postoperatively. Pain relief was provided by administering ketonal 0.04 ml/kg (Sandoz, Slovenia). The healing process was observed daily after surgery based on a predetermined schedule for several days. At 14-, 30-, and 60-days, the rabbits were euthanized with a lethal dose of Zoletil 50 mg/ml, and the distal femur was collected for histological analysis. Histopathological and histomorphometric examination The bone fragment exhibiting a formed defect underwent histopathological examination subsequent to fixation in 10% neutral buffered formalin for 24 hours, followed by decalcification in Biodec R solution for 24 hours. The resultant samples were rinsed in phosphate buffer (pH = 7.4) and processed for optimal decalcification. After a bone incision, the tissue was fixed in 10% formalin at 4°C for 24 hours, washed in tap water, dehydrated in graded alcohol concentrations (70%, 90%, 95%, 100%), cleared in xylene, and finally embedded in paraffin blocks. Subsequently, serial longitudinal sections of 5µm thickness were prepared using a rotary microtome parallel to the sagittal plane and stained using hematoxylin and eosin (for determining the general tissue morphology and cellular composition of the bone defect) and Masson's trichrome staining (for identifying the percentage of fibrous tissue, cartilage tissue, and bone tissue) [35]. The microscopic evaluation of the preparations was performed using a Zeiss AxioLab 4.0 microscope at a magnification of x400. AxioVision 7.2 software was utilized for analyzing and capturing the images. The cellular composition of the bone defect, including osteoclasts, osteoblasts, and osteocytes, was determined by enumerating these cells in each section stained with hematoxylin and eosin. The calculation was based on the enumeration of cells per 1000 cells around the defect zone, and the mean values were expressed to two decimal places for each group. The morphometric measurements of fibrous, cartilaginous, and bone tissue were conducted on the area delimited radially by the defect ends and laterally by the original femur and the outer boundary of the allograft and/or newly formed bone as a percentage of the total area of the defect zone. Each bone defect was evaluated by three slices, and the arithmetic mean was determined. The proportion of the closure of the defect area with bone and cartilage tissue was determined by plotting a horizontal line across the outer part of the inner and outer cortical layer of bone at the edges of the defect. Blood vessels were characterized by the presence of erythrocytes in the lumen and endothelial cell lining, and the number of vessels per area of the formed defect was estimated based on 10 fields of view at x200 magnification. Two certified histologists, who were blinded to the group distribution, performed the histological analysis using a histological bone defect healing score (Table 1 ). Table 1 Histopathological parameters of bone defect healing score Histological score Inflammation Polymorphonuclear leukocytes* Lymphocytes* Macrophages/Histiocytes* Сellular composition Osteoblasts** Osteocytes ** Osteoclasts** Bone defect healing rate Bone defect closure area (%) Tissue composition Fibrous tissue (%) Cartilage (%) Bone (%) Neovascularization *** * - assessment of cellular infiltrate was carried out on 100 cells by summing the average values of different cell types in the area of the defect zone ** - assessment of the cellular composition was carried out for 1000 cells by summing the average values of different types of cells in the area of the defect zone *** - assessment of the number of newly formed vessels was carried out on the area of the formed defect calculated for 10 fields of view Statistical analysis The experimental data were presented as the median and interquartile range (Q1-Q3). The Chi-Squared Test with Yates Continuity Correction and Mann-Whitney test were applied for comparing the two groups, while Pearson's Chi-Squared Test and Kruskal-Wallis Test were used for multiple comparisons. IBM SPSS Statistics 20.0 and STATISTICA 10 were used for statistical analysis of the research results. A p-value less than 0.05 was considered statistically significant. Results And Discussion The postoperative period in 93 animals of all experimental groups proceeded without visible complications (Fig. 3). The animals remained active and maintained their appetite. Histological analysis showed no evidence of inflammatory cell infiltration near of the allograft in any of the cases. On day 14, the number of osteoblasts in the AG + PRP group was significantly higher than in the AG, AG + rhBMP-2, and AG + ZA groups (p 0.05). On day 30, the number of osteoblasts was significantly higher in the AG + PRP and AG + rhBMP-2 groups than in the AG and AG + ZA groups (Table 3 ). There was no significant difference in osteoblast numbers between the AG + PRP and AG + rhBMP-2 groups (p = 0.819). On day 60, there was no significant difference in osteoblast number among all groups (p = 0.730) (Table 4 ). Table 2 Histopathological evaluation of bone defect healing after 14 days. 1 group (AG + PRP) 2 group (AG + rhBMP-2) 3 group (AG + Zol) 4 group (AG) 14 days Osteoblasts 412.0 (410.0; 465.8) p1 = 0.0000 p2 = 0.0000 p3 = 0.0000 p4 = 0.0000 273.0 (267.0; 304.0) p5 = 0.5059 p6 = 0.1975 268.0 (262.8; 289.0) p7 = 0.1134 298.0 (281.5; 310.5) Osteocytes 335.0 (317.8; 366.8) p1 = 0.0000 p2 = 0.0001 p3 = 0.0000 p4 = 0.0001 250.0 (241.3; 251.8) p5 = 0.6929 p6 = 0.0000 265.5 (240.8; 287.0) p7 = 0.0001 217.0 (193.0; 229.5) Osteoclasts 8.0 (5.0; 11.0) p1 = 0.4196 p2 = 0.1767 p3 = 0.2582 p4 = 0.5591 10.5 (7.3; 13.5) p5 = 0.5605 p6 = 0.2444 11.0 (5.8; 11.3) p7 = 0.5217 8.5 (4.5; 11.0) Fibrous tissue 35.0 (32.8; 38.0) p1 = 0.0000 p2 = 0.0000 p3 = 0.0001 p4 = 0.0001 50.0 (45.8; 51.8) p5 = 0.8192 p6 = 0.0235 48.5 (42.8; 55.0) p7 = 0.1134 47.0 (40.8; 49.0) Cartilage tissue 5.5 (3.8; 9.0) p1 = 0.0000 p2 = 0.0000 p3 = 0.0001 p4 = 0.0001 12.5 (12.0; 14.5) p5 = 0.0001 p6 = 0.0078 16.0 (16.0; 18.8) p7 = 0.0000 11.5 (10.8; 12.3) Bone tissue 58.0 (54.5; 62.8) p1 = 0.0000 p2 = 0.0003 p3 = 0.0001 p4 = 0.0001 38.0 (36.3; 40.0) p5 = 0.1400 p6 = 0.0178 36.0 (27.5; 38.0) p7 = 0.0009 41.5 (38.8; 48.5) Vessels 39.0 (33.8; 41.3) p1 = 0.0000 p2 = 0.0000 p3 = 0.0000 p4 = 0.0001 20.0 (18.0; 27.3) p5 = 0.0001 p6 = 0.0306 5.0 (5.0; 10.3) p7 = 0.0000 16.0 (14.3; 20.0) Note: n is the number of samples in a group; p is the significance level; p1 < 0.05 - statistically significant difference compared between all groups; p2 < 0.05 - statistically significant difference compared to AG + PRP and AG + rhBMP-2; p3 < 0.05 - statistically significant difference compared to baseline AG + PRP and AG + ZA; p4 < 0.05 - statistically significant difference compared to the corresponding values in control samples AG + PRP and AG; p5 < 0.05 - statistically significant difference compared to the corresponding values in control samples AG + rhBMP-2 and AG + ZA; p6 < 0.05 - statistically significant difference compared to baseline AG + rhBMP-2 and AG; p7 < 0.05 - statistically significant difference compared to the corresponding values in control samples AG + ZA and AG. Table 3 Histopathological evaluation of bone defect healing after 30 days. 1 group (AG + PRP) 2 group (AG + rhBMP-2) 3 group (AG + Zol) 4 group (AG) 30 days Osteoblasts 410.5 (401.5; 446.8) p1 = 0.0000 p2 = 0.8192 p3 = 0.0000 p4 = 0.0129 417.0 (337.3; 457.5) p5 = 0.0047 p6 = 0.2124 276.5 (233.3; 354.0) p7 = 0.0104 364.5 (316.5; 398.5) Osteocytes 438.5 (428.5; 455.3) p1 = 0.0000 p2 = 0.0014 p3 = 0.0001 p4 = 0.0001 425.5 (396.3; 427.8) p5 = 0.0000 p6 = 0.2987 255.0 (230.0; 371.0) p7 = 0.0000 408.5 (389.5; 421.5) Osteoclasts 6.5 (5.8; 7.0) p1 = 0.0000 p2 = 0.0000 p3 = 0.2278 p4 = 0.8653 22.5 (17.5; 24.0) p5 = 0.0000 p6 = 0.0000 11.0 (2.0; 11.0) p7 = 0.5465 6.5 (2.8; 11.0) Fibrous tissue 6.5 (6.0; 8.0) p1 = 0.0001 p2 = 0.9008 p3 = 0.0000 p4 = 0.0018 6.0 (2.0; 11.0) p5 = 0.0001 p6 = 0.0306 51.0 (35.0; 60.0) p7 = 0.000 12.0 (8.5; 14.5) Cartilage tissue 8.5 (6.8; 9.3) p1 = 0.0001 p2 = 0.0026 p3 = 0.0007 p4 = 0.7063 5.5 (1.0; 7.8) p5 = 0.0000 p6 = 0.0199 12.0 (11.0; 13.5) p7 = 0.1134 9.0 (5.5; 13.0) Bone tissue 84.5 (83.8; 85.3) p1 = 0.0000 p2 = 0.1559 p3 = 0.0001 p4 = 0.0006 89.5 (85.0; 93.3) p5 = 0.0000 p6 = 0.0018 34.0 (29.0; 53.8) p7 = 0.0000 80.5 (76.0; 82.5) Vessels 31.0 (25.0; 41.3) p1 = 0.0000 p2 = 0.0003 p3 = 0.0000 p4 = 0.0041 19.0 (14.8; 21.8) p5 = 0.0019 p6 = 0.0673 10.0 (8.0; 11.0) p7 = 0.000 20.5 (19.0; 23.3) Note: n is the number of samples in a group; p is the significance level; p1 < 0.05 - statistically significant difference compared between all groups; p2 < 0.05 - statistically significant difference compared to AG + PRP and AG + rhBMP-2; p3 < 0.05 - statistically significant difference compared to baseline AG + PRP and AG + ZA; p4 < 0.05 - statistically significant difference compared to the corresponding values in control samples AG + PRP and AG; p5 < 0.05 - statistically significant difference compared to the corresponding values in control samples AG + rhBMP-2 and AG + ZA; p6 < 0.05 - statistically significant difference compared to baseline AG + rhBMP-2 and AG; p7 < 0.05 - statistically significant difference compared to the corresponding values in control samples AG + ZA and AG. Table 4 Histopathological evaluation of bone defect healing after 60 days. 1 group (AG + PRP) 2 group (AG + BMP-2) 3 group (AG + Zol) 4 group (AG) 60 days Osteoblasts 405.0 (350.8; 421.3) p1 = 0.7302 p2 = 0.8191 p3 = 0.6511 p4 = 0.4286 383.0 (341.0; 427.3) p5 = 0.3286 p6 = 0.3714 396.0 (370.0; 424.0) p7 = 0.9989 402.5 (357.0; 441.3) Osteocytes 422.0 (411.0; 435.0) p1 = 0.0000 p2 = 0.8679 p3 = 0.0000 p4 = 0.6511 412.5 (412.0; 453.0) p5 = 0.0000 p6 = 0.6511 301.0 (212.0; 314.3) p7 = 0.0001 422.5 (413.8; 431.5) Osteoclasts 5.5 (4.8; 7.0) p1 = 0.0000 p2 = 0.0000 p3 = 0.7063 p4 = 0.9399 19.0 (14.3; 21.8) p5 = 0.0000 p6 = 0.0001 6.0 (3.5; 8.3) p7 = 0.7630 6.0 (3.8; 7.0) Fibrous tissue 5.5 (4.8; 8.3) p1 = 0.0000 p2 = 0.0142 p3 = 0.0000 p4 = 0.5465 14.0 (8.8; 41.0) p5 = 0.0004 p6 = 0.0258 57.0 (47.0; 64.0) p7 = 0.0000 6.5 (4.8; 8.8) Cartilage tissue 11.5 (10.0; 13.3) p1 = 0.3017 p2 = 0.0964 p3 = 0.8802 p4 = 0.4509 5.0 (2.5; 10.0) p5 = 0.2279 p6 = 0.1345 11.5 (9.5; 12.5) p7 = 0.4509 10.5 (9.8; 11.8) Bone tissue 81.5 (80.8; 84.3) p1 = 0.0000 p2 = 0.7711 p3 = 0.0000 p4 = 0.8081 81.0 (49.3; 89.3) p5 = 0.0009 p6 = 0.9337 32.5 (26.8; 49.0) p7 = 0.0000 81.0 (80.8; 82.3) Vessels 14.5 (10.3; 15.8) p1 = 0.4180 p2 = 0.6475 p3 = 0.4660 p4 = 0.5316 15.5 (9.0; 17.0) p5 = 0.2617 p6 = 0.6776 12.5 (8.0; 15.0) p7 = 0.7063 13.0 (11.0; 15.8) Note: n is the number of samples in a group; p is the significance level; p1 < 0.05 - statistically significant difference compared between all groups; p2 < 0.05 - statistically significant difference compared to AG + PRP and AG + rhBMP-2; p3 < 0.05 - statistically significant difference compared to baseline AG + PRP and AG + ZA; p4 < 0.05 - statistically significant difference compared to the corresponding values in control samples AG + PRP and AG; p5 < 0.05 - statistically significant difference compared to the corresponding values in control samples AG + rhBMP-2 and AG + ZA; p6 < 0.05 - statistically significant difference compared to baseline AG + rhBMP-2 and AG; p7 < 0.05 - statistically significant difference compared to the corresponding values in control samples AG + ZA and AG. On day 14, the number of osteocytes in the AG + PRP group was significantly higher than in the AG, AG + rhBMP-2, and AG + ZA groups (p 0.05), but both were significantly higher compared to the osteocyte numbers in the AG group (p < 0.001). On day 30, the osteocyte numbers in the AG, AG + PRP, and AG + rhBMP-2 groups were significantly higher than in the AG + ZA group (p < 0.001). There was no significant difference between AG and AG + rhBMP-2 (p = 0.29), although both were significantly lower than in the AG + PRP group (p < 0.05) (Table 3 ). On day 60, osteocyte cell numbers in the AG + PRP, AG + rhBMP-2, and AG groups were significantly higher than in the AG + ZA group (p 0.05). On day 14, there was no significant difference in osteoclast numbers among the AG, AG + PRP, AG + rhBMP-2, and AG + ZA groups (p > 0.05) (Table 2 ). On days 30 and 60, the osteoclast numbers were significantly higher in the AG + rhBMP-2 group than in the AG, AG + PRP, and AG + ZA groups (p 0.05). At the 14-day interval, new bone formation was observed at the site of the bone defect in three groups: AG + PRP, AG + rhBMP-2, and AG (Table 2 ) (Fig. 4 ). The AG + PRP group exhibited significantly greater closure of the bone defect area compared to the other groups, as depicted in Fig. 5 . The newly formed bone adjacent to the graft particles was composed of bone and lacunae containing osteocytes and numerous vascular channels, which were more abundant in the AG + PRP group (Fig. 4 a). Histologically, the newly formed bone trabecular meshwork was connected to the allograft bone. The bone beams of the newly formed tissue were mostly thin and heterogeneous, with focal bridge-like areas and single contacts, mainly at the poles of the bone beams. In contrast, the AG + Zol group displayed a predominance of fibrous tissue covering the defect area with minimal resorbed allograft fragments (Table 2 ) (Fig. 4 c). The fibrous tissue contained single thin-walled vessels and scant infiltrate, with single bone trabeculae found along the edges of the bone plate in a chaotic pattern. On day 30, the reparative process in the cortical layer of the bone among groups AG + PRP, AG + rhBMP-2, and AG was characterized by a progressive increase in mature bone tissue with minimal fibrosis (Fig. 4 and Fig. 6 ). In particular, the AG + PRP and AG + rhBMP-2 groups exhibited a notable prevalence of increased bone defect closure and new bone formation compared to the AG and AG + Zol groups (Fig. 5 b). The bone tissue in the affected area was observed as randomly located bone beams and strands, which formed lamellar structures. Additionally, the bone beams had a high degree of mineralization and demonstrated active longitudinal growth. In terms of newly formed vessels, the AG + PRP group exhibited a higher prevalence compared to the AG + rhBMP-2, AG + Zol, and AG groups (Table 3 ). At this point in time, the AG + Zol group still demonstrated a predominance of coarse fibrous connective tissue within the defect zone, as was observed on day 14 (Fig. 4 g and Fig. 6 g). At day 60, in groups AG + PRP and AG, complete trabecular bone tissue was observed at the defect site with normal development of bone trabeculae, which were predominantly composed of spindle-shaped osteocytes (Table 4 ) (Fig. 4 and Fig. 6 ). However, in the AG + rhBMP-2 group, the thickness of the newly formed bone tissue was less than that of the cortical plate outside the defect zone, and a U-shaped depression was formed in the defect area. The newly formed bone tissue was located within the cortical plate and did not spread into the intramedullary space. Furthermore, multinucleated giant cells (osteoclasts) were detected in small lacunae and spaces between allograft fragments and bone beams, which were resorbing the bone tissue (Fig. 4 j). The intervals between the bone tissues were filled with fibrous tissue without any signs of inflammatory cell infiltration. In contrast, the defect area in the AG + Zol group was primarily covered with fibrous tissue and bone formation were limited (Table 4 ). Thin, randomly located newly formed bone trabeculae extended from the edge of the bone plate into the intramedullary space (Fig. 4 k and Fig. 6 k). The boundary between the bone plate and the newly formed bone was evident. The newly formed bone tissue was primarily composed of randomly located, thin bone trabeculae extending into the intramedullary space. The surface of the newly formed bone beams and the edge of the cortical plate defect were surrounded by a fibrous layer without reactive infiltration. Discussion This study examined the use of heat-treated Marburg bone in combination with PRP, rhBMP-2, and zoledronic acid to fill bone defects in an experimental rabbit femur model. Our research findings reveal that the application of a PRP-perforated allograft combination resulted in superior bone regeneration after 14 days in comparison to the use of rhBMP-2, zoledronic acid or bone allograft alone to fill the defect. Furthermore, after 30 days, this combination demonstrated better results compared to the usage of zoledronic acid in combination with allograft and filling the defect with allograft alone. This fact was confirmed by the analysis of histological and histomorphometric data of osteogenesis and angiogenesis in the groups. The results obtained are consistent with clinical and experimental studies demonstrating the positive effect of PRP on bone regeneration at an early stage due to the release of several growth factors, such as PDGF, TGF-β and VEGF [22–24]. Furthermore, growth factors contained in PRP stimulate angiogenesis and proliferation of osteoprogenitor cells only in the very early period after transplantation [36,37]. In our study, the use of PRP resulted in faster bone formation compared to the other groups in the early stages. Concerning the acceleration of bone formation when using a bone allograft with rhBMP-2, it is well-known that allografts function as carriers of rhBMP2 [20,38,39]. Previous studies, as well as our results, have shown that the incorporation of rhBMP2 into the allograft significantly accelerates bone formation from an early stage up to 4 weeks after transplantation [40,41]. The combined use of a bone allograft with 5 µg of rhBMP2 induced bone formation was higher than that with the allograft alone at day 30. On the contrary, at 60 days, there was a thinning of the cortical plate of the femur at the site of the defect and the presence of many giant cells. This phenomenon is possibly related to the fact that BMPs, in particular rhBMP-2, not only accelerate bone formation but also cause premature bone resorption mediated by osteoclasts through RANKL-RANK signaling, a side effect that is often overlooked [39, 43]. Further study is underway to investigate if bone resorption and formation occur simultaneously at different dosages and when combined with bisphosphonates. Pre-treatment of bone grafts in a bisphosphonate (BP) solution may offer a potential preventive measure against bone graft resorption. The direct influence of BPs on osteoclasts is well established, and this effect on bone formation is noteworthy. The administration of BPs can alter the function of osteoclasts by inducing the secretion of an osteoclastic inhibitory factor by osteoblasts. The regulation of osteoclastic activity is primarily controlled by osteoblastic cells during bone remodeling. BPs enhance the proliferation and differentiation of osteoblasts and reduce apoptotic cell death [25,44,45]. The dosage and application technique of BPs are critical factors that determine the interaction between BPs and bone tissue. Research has revealed that BPs exhibit a biphasic effect on bone cells, with low concentrations stimulating cell proliferation and tissue formation, while high concentrations restrict these processes [26, 46–49]. This study used zoledronic acid 0.05 mg/ml in combination with a bone allograft. We found a decrease in resorption, as expected, and a decrease in the amount of new bone in the bone defect at all follow-up periods compared with other groups. In this group, the bone defect was filled mainly with fibrous tissue at all stages (Fig. 6 ). During the remodeling phase, our data support the idea that bisphosphonates may reduce bone resorption and inhibit osteogenic cell activity. Although in previous studies [26,27], the authors showed that the use of zoledronic acid 0.05 mg/ml promotes osteogenesis, in this study, the use of zoledronic acid 0.05 mg/ml led to the inhibition of both resorption and bone formation. The application of a thermally treated bone allograft in combination with zoledronic acid has potential applications in joint revision surgery, specifically in scenarios necessitating impaction with bone tissue and prevention risk of endoprosthesis loosening. The strength of this study is the comparative characterization of the use of a bone allograft prepared according to the Marburg system in combination with PRP, rhBMP-2, and zoledronic acid both in the early stages and in the late stages of bone defect healing. This made it possible to reveal the stimulation of osteoregeneration in the early stages with the use of PRP and rhBMP-2, as well as the inhibition of bone allograft resorption with the use of zoledronic acid. The use of a standardized rabbit femoral defect model and histological and morphometric analysis is also a strength of the study. Limitations are the absence of an empty defect in the model, which could demonstrate the initial efficiency of the regenerative potential of the bone without any bone grafts, as well as the need to draw blood from the heart before surgery in the group with PRP, which could affect the recovery process after surgical intervention. Conclusions In conclusion, the present study showed that PRP and rhBMP-2, in combination with a Marburg bone allograft, can markedly promote bone formation in bone defects at early stages. Although the mechanisms that lead to the stimulation of bone regeneration remain unclear, enhancing the osteoinductivity of bone substitutes may provide a highly relevant approach to clinical bone reconstruction in the near future. In this study, we found a strong effect of bisphosphonates on graft resorption as well as inhibition of new bone ingrowth into grafts. However, due to the impossibility of direct extrapolation of the results to human conditions, further studies are ongoing in controlled clinical trials. Declarations Funding Statement This research has been funded by the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan (Grant №AP09260954). Author Contributions Conceptualization, D.S., Y.K. and E.T.; methodology, D.S. and E.T.; software, E.T.; validation, B.T., D.R. and Y.K.; formal analysis, D.S.; investigation, E.T. and Y.K.; resources, B.T.; data curation, E.T.; writing—original draft preparation, E.T. and Y.K.; writing—review and editing, S.D. and D.R.; visualization, E.T.; supervision, S.D.; project administration, S.D.; funding acquisition, Y.K. Acknowledgments Not applicable Data Availability The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate The animal study protocol was approved by the University Animal Care Committee (UACC) of Karaganda Medical University (protocol № 27 27.09.2020). The standard principles of animal experiment ethics were followed in all the animal experiments, and they adhered to the protection guidelines of DIRECTIVE 2010/63/EU for animals used in scientific research. The study was conducted while adhering to the ARRIVE guidelines. Consent for publication Not applicable Competing interests The authors declare no competing interests. References Winkler T, Sass FA, Duda GN, et al. A review of biomaterials in bone defect healing, remaining shortcomings and future opportunities for bone tissue engineering: The unsolved challenge. 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Biomaterials. 2005 Nov;26(32):6941-9. doi: 10.1016/j.biomaterials.2005.04.056. PMID: 15990113. Gao Y, Liu X, Gu Y, Song D, Ding M, Liao L, Wang J, Ni J, He G. The Effect of Bisphosphonates on Fracture Healing Time and Changes in Bone Mass Density: A Meta-Analysis. Front Endocrinol (Lausanne). 2021 Aug 30;12:688269. doi: 10.3389/fendo.2021.688269. PMID: 34526966; PMCID: PMC8435630. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2813335","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":193140772,"identity":"6bfcd584-ddbf-478e-b85b-9ec5fecba824","order_by":0,"name":"Dina Saginova","email":"","orcid":"","institution":"National Scientific center of Traumatology and Orthopaedics named after academician N.D.Batpenov","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dina","middleName":"","lastName":"Saginova","suffix":""},{"id":193140773,"identity":"814b0ef6-b2a7-4329-a0b9-57c438e3b6e2","order_by":1,"name":"Elyarbek Tashmetov","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYHACNhiD8QGQ4OEjpJ4HosUAxGYGkTxs+DWgamGTQLEUF7BnP/7s0Y2KP3IGB5iPVX7NsZNhY2B++OgGPlt4csyNc84YGBscYEu7LbstGegwNmPjHLwOy2GTzm0zSJzZwGN2W3IbM1ALD5s0Xi38z59J5/4DaeH/Viy5rZ4ILRIJZtK5DQaJ/UCVjB+3HSZCy403ZtI5x4yN+ZnZjKUZtx3nYWMm4Bf2/vRn0jk1cnJs7M0PP/7cVm3PD2Q8xqcFAZiBiAfKIB4w/iBF9SgYBaNgFIwYAAAl0DtQsi5BLQAAAABJRU5ErkJggg==","orcid":"","institution":"Karaganda Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Elyarbek","middleName":"","lastName":"Tashmetov","suffix":""},{"id":193140774,"identity":"aa140a8e-09c8-4e25-b7b9-928bc2b1f13d","order_by":2,"name":"Yevgeniy Kamyshanskiy","email":"","orcid":"","institution":"Karaganda Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yevgeniy","middleName":"","lastName":"Kamyshanskiy","suffix":""},{"id":193140775,"identity":"6018b2c7-bd8d-4833-b7f4-12593d80d36c","order_by":3,"name":"Berik Tuleubaev","email":"","orcid":"","institution":"Karaganda Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Berik","middleName":"","lastName":"Tuleubaev","suffix":""},{"id":193140776,"identity":"4b5c0f93-d082-49cd-b2b6-d23143a2d7bd","order_by":4,"name":"Denis Rimashevskiy","email":"","orcid":"","institution":"Peoples’ Friendship University of Russia","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Denis","middleName":"","lastName":"Rimashevskiy","suffix":""}],"badges":[],"createdAt":"2023-04-13 14:14:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2813335/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2813335/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":36079173,"identity":"e0497a56-61b7-431d-aceb-a1ecfefc0d6e","added_by":"auto","created_at":"2023-04-20 17:42:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106283,"visible":true,"origin":"","legend":"\u003cp\u003eHeat-treated femoral head\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2813335/v1/8e33824c77282bac27898a2b.png"},{"id":36079175,"identity":"5c6adfdd-c696-4691-9748-ff2b451c4445","added_by":"auto","created_at":"2023-04-20 17:42:09","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":393100,"visible":true,"origin":"","legend":"\u003cp\u003eCreation of bone defect in rabbit femur: a - intact bone; b - created bone defect.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2813335/v1/204fb2667ab58b73ff9b66ae.jpeg"},{"id":36079994,"identity":"57d66203-8772-4589-9e74-e42e2804fac6","added_by":"auto","created_at":"2023-04-20 17:50:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":153105,"visible":true,"origin":"","legend":"\u003cp\u003eFlow diagram of experiment\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2813335/v1/673ab6efbabba3e6a63b4440.png"},{"id":36080389,"identity":"b916dcff-9f07-49ce-9012-29aa84fb060e","added_by":"auto","created_at":"2023-04-20 17:58:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":555966,"visible":true,"origin":"","legend":"\u003cp\u003eHistological sections 14, 30, and 60 days after implantation: a, d - osteoblasts (black arrow) and giant multinucleated osteoclasts (yellow arrow) are observed on the bone surface; also, osteocytes (arrowhead) surrounded by bone matrix (HE × 200); b - section showing bone tissue presented as chaotically arranged bone beams and ties forming lamellar structures (black asterisk). Bone beams with a high degree of mineralization and active longitudinal growth. Polarized osteoblasts (black arrows) and giant multinucleated osteoclasts (red arrows) are observed on the bone surface; osteocytes surrounding the bone matrix are also observed (HE × 100); c - The area of the defect was partially closed by fibrous tissue and minimally resorbed fragments of allograft (red asterisk). Forming fibrous tissue with single thin-walled vessels and scanty infiltrate. Along the edge of the bone plate (black asterisk), single chaotically located bone trabeculae (black arrows) are defined (HE × 100); e, f, h - osteoblasts total and circularly lined allograft bone fragments with newly formed bone tissue and formation of multiple bundles of osteoblast cell clusters with multidirectional growth of bone tissue. Polarized osteoblasts and giant multinucleated osteoclasts are observed on the bone surface; osteocytes surrounded by bone matrix are also observed; (HE × 100, × 200) g - \u0026nbsp;\u0026nbsp;allograft fragments lysed by osteoclasts, located in the fibrous stroma with single microenvironment cells (HE × 200); i - \u0026nbsp;Newly formed bone tissue around the defect (black arrows). Haversian canals of various sizes and shapes, which is typical for the site of bone tissue restoration (HE × 400); j - between the bone beams, giant multinucleated cells (osteoclasts) (arrows) resorbing bone tissue are determined (HE × 200); k - the defect area is closed by fibrous tissue without inflammatory cellular infiltration. Newly formed bone tissue extending into the intramedullary space and interning with a minimally resorbed allograft (black arrows) is identified around the bone plate margin. The boundary between the bone plate, and the newly formed bone tissue is well defined (red arrows) (HE × 100); l - The defect area is closed by bone (black arrows) and cartilage tissue; active longitudinal and transverse growth of bone beams (black arrows) is noted. In the adjacent intramedullary canal to the plate there is adipose bone marrow tissue with pleomorphic bone marrow cells (black asterisk) (HE × 400)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2813335/v1/956852ba49905d0640de9f07.png"},{"id":36079174,"identity":"bb8aa660-80f9-4dad-b626-56abfb0bb778","added_by":"auto","created_at":"2023-04-20 17:42:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":236355,"visible":true,"origin":"","legend":"\u003cp\u003eBone defect healing rate (%) in femoral defects: a – 14 days; b – 30 days; с – 60 days. AG+PRP, bone allograft with platelet rich plasma; AG+rhBMP-2, bone allograft with recombinant human\u003cstrong\u003e \u003c/strong\u003ebone morphogenetic protein-2; AG+Zol, bone allograft with zoledronic acid; AG, bone allograft. (*p\u0026lt;0,05; **p\u0026lt;0,01; ***p\u0026lt;0,001)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2813335/v1/9e9ae8f88f1781911131f598.png"},{"id":36079176,"identity":"15e0137d-07cb-43af-b0bc-2810b5423539","added_by":"auto","created_at":"2023-04-20 17:42:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":941290,"visible":true,"origin":"","legend":"\u003cp\u003eHistological sections 14, 30, and 60 days after implantation:\u003cstrong\u003e \u003c/strong\u003ea, b, d - the fibrous layer surrounds the surface of the allograft with varying degrees of remodeling and newly formed bone tissue (Masson's trichrome × 40, × 100); c - \u0026nbsp;The defect site is covered with fibrous tissue, proliferating fibroblasts (yellow arrows) surround allograft fragments (Masson's trichrome × 40); e,f,h - \u0026nbsp;the defect zone is closed by formed bone beams with a minimum amount of fibrous tissue (Masson's trichrome × 40); g- \u0026nbsp;\u0026nbsp;the site of the defect is covered with fibrous tissue and newly formed bone trabeculae extending from the edge of the defect in the bone plate (Masson's trichrome × 40);\u003cem\u003e \u003c/em\u003ei - \u0026nbsp;fragment of newly formed bone tissue in the area of the defect, mineralized bone tissue with the presence of chaotically located Haversian canals (Masson's trichrome × 40); j - newly formed bone with a large number of osteocytes and osteoblasts integrates with the allograft (Masson's trichrome × 40); k - \u0026nbsp;\u0026nbsp;The defect zone is covered with fibrous tissue and allograft fragments with minimal resorption and remineralization. The emerging bone tissue extends perpendicular to the cortical plate into the intramedullary space (Masson's trichrome × 40); l - area of the defect showing allograft fragments surrounded by less mature new bone. Newly formed bone tissue around the defect with integrated fragments of the allograft (Masson's trichrome × 40).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2813335/v1/f7e0766b880eba8802654025.png"},{"id":49101341,"identity":"d19601db-bf04-423d-bc72-a2801aaa7d30","added_by":"auto","created_at":"2024-01-03 05:52:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3778524,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2813335/v1/d2ea1cfc-f992-48c4-a280-dadae9174efb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative evaluation of heat-treated bone allograft combined with platelet rich plasma, recombinant human bone morphogenetic protein 2 and zoledronic acid: An experimental study on the histopathological and histomorphometric aspects of bone healing","fulltext":[{"header":"Background","content":"\u003cp\u003eThe replacement of bone defects is a pressing issue in modern traumatology and orthopedics. Osteogenesis can be stimulated by autografts and bone substitutes, with over 2\u0026nbsp;million bone graft surgeries being performed worldwide each year [1,2]. Autologous bone is considered the \"gold standard\" in orthopedics for replacing bone defects caused by various factors. However, the use of autologous bone has its own drawbacks, including filling large bone defects, pain in the donor site, increased operation time, and cosmetic defects [3,4]. This led to the development of various bone substitute materials, which are structurally similar to bone tissue, as an alternative to autologous bone [5\u0026ndash;7]. Commercial tissue banks currently offer bone substitute materials, such as cortical cancellous ilium, femoral head, freeze-dried bone substitutes, and decalcified freeze-dried bone, which are sterilized using chemical or physical methods [3,8].\u003c/p\u003e \u003cp\u003eThe Marburg Bone Bank prepared bone graft is a type of bone allograft widely used in orthopedic surgery. The Marburg Bone Bank system is based on thermal disinfection of the femoral bone head and is considered safe, as the bone matrix in the graft provides the necessary osteoconductive properties required for successful bone repair [9\u0026ndash;11]. However, after being extensively processed, bone allografts lose their innate osteoinductive properties and, as a result, are unable to produce the same clinical outcomes as autologous bone grafts. [12,13]. Therefore, there has been a growing interest in using growth factors and morphogens as substances that can provide osteoinductivity to bone substitutes. [14\u0026ndash;17].\u003c/p\u003e \u003cp\u003eThe addition of bone morphogenetic proteins (BMPs) is an example of such incorporation in bone graft substitutes [18,19]. BMPs are a naturally occurring group of proteins belonging to the transforming growth factor beta (TGF-β) family. They act as cytokines that facilitate the differentiation of mesenchymal cells into bone- and cartilage-forming cells. Among them, recombinant human bone morphogenetic protein-2 (rhBMP-2) is known to be critical in bone formation and healing as it has the ability to induce osteoblast differentiation [18\u0026ndash;20].\u003c/p\u003e \u003cp\u003ePlatelet-rich plasma (PRP) has been used as an autologous blood product in clinical settings to promote tissue regeneration in various types of bone defects and guide bone regeneration with bone grafts [21,22]. PRP is believed to possess the ability to stimulate bone regeneration due to the growth factors released from activated platelets, which have a stimulatory effect on progenitor cells and vascularization at local sites. Platelets may contain unique activators of bone morphogenetic proteins (BMPs) that stimulate the differentiation of progenitor cells into bone-forming cells in laboratory settings [21,23,24].\u003c/p\u003e \u003cp\u003eBisphosphonates (BP) prevent and treat increased bone resorption in skeletal diseases [25]. Zoledronic acid (Zol) is considered the most potent bisphosphonate in terms of its pharmacological activity and affinity to bone, especially in areas of active bone metabolism [26]. While the effects of Zol on bone resorption have been extensively studied both in vivo and in vitro, its impact on bone formation is still not completely understood and is currently a subject of debate [26,27]. Over the past few decades, some studies have employed specific concentrations of ZA to stimulate osteoregeneration, leading to different findings and varying conclusions [27].\u003c/p\u003e \u003cp\u003eDue to the lack of relevant literature on the combined use of a bone allograft prepared according to the Marburg system and osteoinductive substances, this study aims to evaluate the effect on the bone formation of a bone allograft in combination with platelet-rich plasma, recombined human bone morphogenetic protein-2 (rhBMP-2) and zoledronic acid in rabbit femur defects using histopathological and histomorphometric analyses.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Marburg bone graft\u003c/h2\u003e \u003cp\u003eIn this study, heat-treated femoral heads were utilized as bone allograft (Fig.\u0026nbsp;1). Femoral heads were acquired from a living donor who had undergone hip joint arthroplasty surgery, in accordance with national regulations [28,29]. In the context of endoprosthetics of the hip joint, a bone transplant is extracted from the operating room, specifically the head of the femur, and subjected to a series of mechanical cleaning procedures in sterile conditions. These procedures involve removing any soft tissue, cartilage, and ligaments from the bone allograft. Once cleaned, the femoral bone allografts were then perforated using a specially developed device at equal intervals [30]. Then, the femoral head were placed in a disposable, sterile container and filled with 0.9% NaCl solution in a volume of 300 ml. The container were then sealed and processed in a Lobator SD-2 (Telos Company, Germany) heat treatment device for a total of 94 minutes, maintaining a temperature of 82.5\u0026deg;C in the femoral head for at least 15 minutes, as per the established protocol. At the end of the cycle, the sterility of the container is ensured through a special opening, after which the liquid is completely drained. The bone allografts were then stored in a freezer at a temperature of -80\u0026deg;C, as per the prescribed protocol [9]. Two hours before the experiment, the femoral head was unfrozen at room temperature and cut into chips. Then, to standardize the mixture of bone allograft with platelet-rich plasma, rhBMP-2, and zoledronic acid, a specific weight was used to ensure a consistent ratio of ingredients: 0.5 g bone allograft/0.5 ml PRP, 0.5 g bone allograft/5 \u0026micro;g rhBMP-2, 0.5 g bone allograft/5 \u0026micro;g Zol.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of platelet-rich plasma\u003c/h2\u003e \u003cp\u003ePrior to each transplantation procedure, approximately 5 ml of blood was collected from the heart and placed in siliconized tubes containing 3.8% sodium citrate at a blood-to-citrate ratio of 9:1[31]. Platelet-rich plasma was obtained through a two-step centrifugation process [24]. The collected blood was initially centrifuged at 900 g for 8 minutes, separating the blood cell component (BCC) in the lower fraction and the serum component (SC) in the upper fraction. The BCC fraction was removed, and the remaining material was centrifuged again at 1500 g for 5 minutes to yield platelet-poor plasma (PPP) and PRP. The PRP was obtained by isolating approximately 0.5 ml of the PPP fraction, and subsequently used for impregnation of the bone allograft.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of rhBMP-2\u003c/h2\u003e \u003cp\u003eA total of 150 \u003cb\u003e\u0026micro;\u003c/b\u003eg of recombinant human bone morphogenetic protein-2 (rhBMP-2) (CUSABIO, USA) was mixed with 3 ml of saline solution to form the rhBMP-2 solution, which was mixed with bone chips to achieve 0,5 g of containing 5 \u003cb\u003e\u0026micro;\u003c/b\u003eg of rhBMP-2 per graft [21].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of zoledronic acid\u003c/h2\u003e \u003cp\u003eZoledronic acid (Sun Pharmaceutical Industries Ltd, India) with a concentration of 0.05 mg/ml (100 \u003cb\u003e\u0026micro;l\u003c/b\u003e) [25] was added to 0.5 g of bone chips by soaking and kept in a sterile container.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAnimals surgery\u003c/h2\u003e \u003cp\u003eFor this study, 96 adult rabbits weighing 3078\u0026thinsp;\u0026plusmn;\u0026thinsp;87 g were procured and placed in cages for two weeks to acclimate. All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals [32] and were approved by the University Animal Care Committee (UACC) under protocol № 27 27.09.2020. During the study, the rabbits were housed at a room temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and maintained at 40%-50% humidity under a 12-hour light-dark cycle. The rabbits were provided with standard rabbit pellets and tap water throughout the study.\u003c/p\u003e \u003cp\u003e The sample size for the animal experimentation in this study was determined in accordance with Russell and Burch's bioethical principles of replacement, reduction, and refinement (1959). These principles aim to minimize animal use by using the minimum number of animals necessary to obtain statistically significant results [33].\u003c/p\u003e \u003cp\u003eThe hip area was prepared for surgery by shaving and cleaning with an iodine solution. Aseptic techniques were employed, and sterile instruments were used. A 2% lidocaine\u0026thinsp;+\u0026thinsp;epinephrine 1:100000 solution was diluted to 1% and injected for infiltration. The skin was incised distally, and the muscles were dilated bluntly. A 5 mm drill was used to create bone defects in the metaphysis of the femur to a depth of 10 mm (Fig.\u0026nbsp;2) [34]. Following this procedure, the rabbits were randomly assigned to one of the four experimental groups using simple randomization. The first group received a bone allograft with platelet-rich plasma (PRP) filling in the bone defects (AG\u0026thinsp;+\u0026thinsp;PRP). The second group received bone allograft with recombinant human bone morphogenetic protein-2 (rhBMP-2) filling in the bone defects (AG\u0026thinsp;+\u0026thinsp;rhBMP-2). In the third group, the bone defects were filled with a bone allograft with zoledronic acid (AG\u0026thinsp;+\u0026thinsp;ZA). Finally, the fourth group (control) received only bone allograft filling in the bone defects (AG). All surgical procedures were carried out by a trained operator.\u003c/p\u003e \u003cp\u003eThe surgical incision was closed using absorbable sutures (4\u0026thinsp;\u0026minus;\u0026thinsp;0 Vicryl, Ethicon, Johnson \u0026amp; Johnson, USA). To prevent wound infection following surgery, the rabbits were given intramuscular injections of the antibiotic gentamicin 0.1 ml/kg (MAPICHEM, Switzerland) two times daily for three days postoperatively. Pain relief was provided by administering ketonal 0.04 ml/kg (Sandoz, Slovenia). The healing process was observed daily after surgery based on a predetermined schedule for several days. At 14-, 30-, and 60-days, the rabbits were euthanized with a lethal dose of Zoletil 50 mg/ml, and the distal femur was collected for histological analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological and histomorphometric examination\u003c/h2\u003e \u003cp\u003eThe bone fragment exhibiting a formed defect underwent histopathological examination subsequent to fixation in 10% neutral buffered formalin for 24 hours, followed by decalcification in Biodec R solution for 24 hours. The resultant samples were rinsed in phosphate buffer (pH\u0026thinsp;=\u0026thinsp;7.4) and processed for optimal decalcification. After a bone incision, the tissue was fixed in 10% formalin at 4\u0026deg;C for 24 hours, washed in tap water, dehydrated in graded alcohol concentrations (70%, 90%, 95%, 100%), cleared in xylene, and finally embedded in paraffin blocks. Subsequently, serial longitudinal sections of 5\u0026micro;m thickness were prepared using a rotary microtome parallel to the sagittal plane and stained using hematoxylin and eosin (for determining the general tissue morphology and cellular composition of the bone defect) and Masson's trichrome staining (for identifying the percentage of fibrous tissue, cartilage tissue, and bone tissue) [35]. The microscopic evaluation of the preparations was performed using a Zeiss AxioLab 4.0 microscope at a magnification of x400. AxioVision 7.2 software was utilized for analyzing and capturing the images. The cellular composition of the bone defect, including osteoclasts, osteoblasts, and osteocytes, was determined by enumerating these cells in each section stained with hematoxylin and eosin. The calculation was based on the enumeration of cells per 1000 cells around the defect zone, and the mean values were expressed to two decimal places for each group. The morphometric measurements of fibrous, cartilaginous, and bone tissue were conducted on the area delimited radially by the defect ends and laterally by the original femur and the outer boundary of the allograft and/or newly formed bone as a percentage of the total area of the defect zone. Each bone defect was evaluated by three slices, and the arithmetic mean was determined. The proportion of the closure of the defect area with bone and cartilage tissue was determined by plotting a horizontal line across the outer part of the inner and outer cortical layer of bone at the edges of the defect. Blood vessels were characterized by the presence of erythrocytes in the lumen and endothelial cell lining, and the number of vessels per area of the formed defect was estimated based on 10 fields of view at x200 magnification. Two certified histologists, who were blinded to the group distribution, performed the histological analysis using a histological bone defect healing score (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHistopathological parameters of bone defect healing score\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eHistological score\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eInflammation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePolymorphonuclear leukocytes*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLymphocytes*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMacrophages/Histiocytes*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eСellular composition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOsteoblasts**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOsteocytes **\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOsteoclasts**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBone defect healing rate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBone defect closure area (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eTissue composition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFibrous tissue (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCartilage (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBone (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNeovascularization ***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e* - assessment of cellular infiltrate was carried out on 100 cells by summing the average values of different cell types in the area of the defect zone\u003c/p\u003e \u003cp\u003e** - assessment of the cellular composition was carried out for 1000 cells by summing the average values of different types of cells in the area of the defect zone\u003c/p\u003e \u003cp\u003e*** - assessment of the number of newly formed vessels was carried out on the area of the formed defect calculated for 10 fields of view\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe experimental data were presented as the median and interquartile range (Q1-Q3). The Chi-Squared Test with Yates Continuity Correction and Mann-Whitney test were applied for comparing the two groups, while Pearson's Chi-Squared Test and Kruskal-Wallis Test were used for multiple comparisons. IBM SPSS Statistics 20.0 and STATISTICA 10 were used for statistical analysis of the research results. A p-value less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003eThe postoperative period in 93 animals of all experimental groups proceeded without visible complications (Fig.\u0026nbsp;3). The animals remained active and maintained their appetite.\u003c/p\u003e \u003cp\u003eHistological analysis showed no evidence of inflammatory cell infiltration near of the allograft in any of the cases. On day 14, the number of osteoblasts in the AG\u0026thinsp;+\u0026thinsp;PRP group was significantly higher than in the AG, AG\u0026thinsp;+\u0026thinsp;rhBMP-2, and AG\u0026thinsp;+\u0026thinsp;ZA groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The number of osteoblasts in the AG, AG\u0026thinsp;+\u0026thinsp;rhBMP-2, and AG\u0026thinsp;+\u0026thinsp;ZA groups did not differ significantly from each other (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). On day 30, the number of osteoblasts was significantly higher in the AG\u0026thinsp;+\u0026thinsp;PRP and AG\u0026thinsp;+\u0026thinsp;rhBMP-2 groups than in the AG and AG\u0026thinsp;+\u0026thinsp;ZA groups (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). There was no significant difference in osteoblast numbers between the AG\u0026thinsp;+\u0026thinsp;PRP and AG\u0026thinsp;+\u0026thinsp;rhBMP-2 groups (p\u0026thinsp;=\u0026thinsp;0.819). On day 60, there was no significant difference in osteoblast number among all groups (p\u0026thinsp;=\u0026thinsp;0.730) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHistopathological evaluation of bone defect healing after 14 days.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 group\u003c/p\u003e \u003cp\u003e(AG\u0026thinsp;+\u0026thinsp;PRP)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 group\u003c/p\u003e \u003cp\u003e(AG\u0026thinsp;+\u0026thinsp;rhBMP-2)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 group\u003c/p\u003e \u003cp\u003e(AG\u0026thinsp;+\u0026thinsp;Zol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 group\u003c/p\u003e \u003cp\u003e(AG)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14 days\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOsteoblasts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e412.0 (410.0; 465.8)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e273.0 (267.0; 304.0)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;0.5059\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;0.1975\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e268.0 (262.8; 289.0)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;0.1134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e298.0 (281.5; 310.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOsteocytes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e335.0 (317.8; 366.8)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e250.0 (241.3; 251.8)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;0.6929\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e265.5 (240.8; 287.0)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e217.0 (193.0; 229.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOsteoclasts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.0 (5.0; 11.0)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;0.4196\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;0.1767\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;0.2582\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;0.5591\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.5 (7.3; 13.5)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;0.5605\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;0.2444\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.0 (5.8; 11.3)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;0.5217\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.5 (4.5; 11.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFibrous tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.0 (32.8; 38.0)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.0 (45.8; 51.8)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;0.8192\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0235\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48.5 (42.8; 55.0)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;0.1134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e47.0 (40.8; 49.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCartilage tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.5 (3.8; 9.0)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.5 (12.0; 14.5)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0078\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.0 (16.0; 18.8)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.5 (10.8; 12.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBone tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e58.0 (54.5; 62.8)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0003\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.0 (36.3; 40.0)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;0.1400\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0178\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36.0 (27.5; 38.0)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0009\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41.5 (38.8; 48.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVessels\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39.0 (33.8; 41.3)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.0 (18.0; 27.3)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0306\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.0 (5.0; 10.3)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.0 (14.3; 20.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eNote:\u003c/p\u003e \u003cp\u003en is the number of samples in a group; p is the significance level;\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared between all groups;\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to AG\u0026thinsp;+\u0026thinsp;PRP and AG\u0026thinsp;+\u0026thinsp;rhBMP-2;\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to baseline AG\u0026thinsp;+\u0026thinsp;PRP and AG\u0026thinsp;+\u0026thinsp;ZA;\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to the corresponding values in control samples AG\u0026thinsp;+\u0026thinsp;PRP and AG;\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to the corresponding values in control samples AG\u0026thinsp;+\u0026thinsp;rhBMP-2 and AG\u0026thinsp;+\u0026thinsp;ZA;\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to baseline AG\u0026thinsp;+\u0026thinsp;rhBMP-2 and AG;\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to the corresponding values in control samples AG\u0026thinsp;+\u0026thinsp;ZA and AG.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHistopathological evaluation of bone defect healing after 30 days.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 group\u003c/p\u003e \u003cp\u003e(AG\u0026thinsp;+\u0026thinsp;PRP)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 group\u003c/p\u003e \u003cp\u003e(AG\u0026thinsp;+\u0026thinsp;rhBMP-2)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 group\u003c/p\u003e \u003cp\u003e(AG\u0026thinsp;+\u0026thinsp;Zol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 group\u003c/p\u003e \u003cp\u003e(AG)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003e30 days\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOsteoblasts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e410.5 (401.5; 446.8)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;0.8192\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0129\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e417.0 (337.3; 457.5)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0047\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;0.2124\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e276.5 (233.3; 354.0)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0104\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e364.5 (316.5; 398.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOsteocytes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e438.5 (428.5; 455.3)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0014\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e425.5 (396.3; 427.8)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;0.2987\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e255.0 (230.0; 371.0)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e408.5 (389.5; 421.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOsteoclasts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.5 (5.8; 7.0)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;0.2278\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;0.8653\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.5 (17.5; 24.0)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.0 (2.0; 11.0)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;0.5465\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.5 (2.8; 11.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFibrous tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.5 (6.0; 8.0)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;0.9008\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0018\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.0 (2.0; 11.0)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0306\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.0 (35.0; 60.0)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.0 (8.5; 14.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCartilage tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.5 (6.8; 9.3)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0026\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0007\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;0.7063\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.5 (1.0; 7.8)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0199\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.0 (11.0; 13.5)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;0.1134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.0 (5.5; 13.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBone tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84.5 (83.8; 85.3)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;0.1559\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0006\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89.5 (85.0; 93.3)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0018\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.0 (29.0; 53.8)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80.5 (76.0; 82.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVessels\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.0 (25.0; 41.3)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0003\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0041\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.0 (14.8; 21.8)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0019\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;0.0673\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.0 (8.0; 11.0)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.5 (19.0; 23.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eNote:\u003c/p\u003e \u003cp\u003en is the number of samples in a group; p is the significance level;\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared between all groups;\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to AG\u0026thinsp;+\u0026thinsp;PRP and AG\u0026thinsp;+\u0026thinsp;rhBMP-2;\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to baseline AG\u0026thinsp;+\u0026thinsp;PRP and AG\u0026thinsp;+\u0026thinsp;ZA;\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to the corresponding values in control samples AG\u0026thinsp;+\u0026thinsp;PRP and AG;\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to the corresponding values in control samples AG\u0026thinsp;+\u0026thinsp;rhBMP-2 and AG\u0026thinsp;+\u0026thinsp;ZA;\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to baseline AG\u0026thinsp;+\u0026thinsp;rhBMP-2 and AG;\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to the corresponding values in control samples AG\u0026thinsp;+\u0026thinsp;ZA and AG.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHistopathological evaluation of bone defect healing after 60 days.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 group\u003c/p\u003e \u003cp\u003e(AG\u0026thinsp;+\u0026thinsp;PRP)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 group\u003c/p\u003e \u003cp\u003e(AG\u0026thinsp;+\u0026thinsp;BMP-2)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 group\u003c/p\u003e \u003cp\u003e(AG\u0026thinsp;+\u0026thinsp;Zol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 group\u003c/p\u003e \u003cp\u003e(AG)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003e60 days\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOsteoblasts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e405.0 (350.8; 421.3)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;0.7302\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;0.8191\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;0.6511\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;0.4286\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e383.0 (341.0; 427.3)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;0.3286\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;0.3714\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e396.0 (370.0; 424.0)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;0.9989\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e402.5 (357.0; 441.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOsteocytes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e422.0 (411.0; 435.0)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;0.8679\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;0.6511\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e412.5 (412.0; 453.0)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;0.6511\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e301.0 (212.0; 314.3)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e422.5 (413.8; 431.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOsteoclasts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.5 (4.8; 7.0)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;0.7063\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;0.9399\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.0 (14.3; 21.8)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.0 (3.5; 8.3)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;0.7630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.0 (3.8; 7.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFibrous tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.5 (4.8; 8.3)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0142\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;0.5465\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.0 (8.8; 41.0)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0004\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0258\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57.0 (47.0; 64.0)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.5 (4.8; 8.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCartilage tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.5 (10.0; 13.3)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;0.3017\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;0.0964\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;0.8802\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;0.4509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.0 (2.5; 10.0)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;0.2279\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;0.1345\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.5 (9.5; 12.5)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;0.4509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.5 (9.8; 11.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBone tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e81.5 (80.8; 84.3)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;0.7711\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;0.8081\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e81.0 (49.3; 89.3)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0009\u003c/b\u003e\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;0.9337\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.5 (26.8; 49.0)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;\u003cb\u003e0.0000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e81.0 (80.8; 82.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVessels\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.5 (10.3; 15.8)\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;=\u0026thinsp;0.4180\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;=\u0026thinsp;0.6475\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;=\u0026thinsp;0.4660\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;=\u0026thinsp;0.5316\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.5 (9.0; 17.0)\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;=\u0026thinsp;0.2617\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;=\u0026thinsp;0.6776\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.5 (8.0; 15.0)\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;=\u0026thinsp;0.7063\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.0 (11.0; 15.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eNote:\u003c/p\u003e \u003cp\u003en is the number of samples in a group; p is the significance level;\u003c/p\u003e \u003cp\u003ep1\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared between all groups;\u003c/p\u003e \u003cp\u003ep2\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to AG\u0026thinsp;+\u0026thinsp;PRP and AG\u0026thinsp;+\u0026thinsp;rhBMP-2;\u003c/p\u003e \u003cp\u003ep3\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to baseline AG\u0026thinsp;+\u0026thinsp;PRP and AG\u0026thinsp;+\u0026thinsp;ZA;\u003c/p\u003e \u003cp\u003ep4\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to the corresponding values in control samples AG\u0026thinsp;+\u0026thinsp;PRP and AG;\u003c/p\u003e \u003cp\u003ep5\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to the corresponding values in control samples AG\u0026thinsp;+\u0026thinsp;rhBMP-2 and AG\u0026thinsp;+\u0026thinsp;ZA;\u003c/p\u003e \u003cp\u003ep6\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to baseline AG\u0026thinsp;+\u0026thinsp;rhBMP-2 and AG;\u003c/p\u003e \u003cp\u003ep7\u0026thinsp;\u0026lt;\u0026thinsp;0.05 - statistically significant difference compared to the corresponding values in control samples AG\u0026thinsp;+\u0026thinsp;ZA and AG.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOn day 14, the number of osteocytes in the AG\u0026thinsp;+\u0026thinsp;PRP group was significantly higher than in the AG, AG\u0026thinsp;+\u0026thinsp;rhBMP-2, and AG\u0026thinsp;+\u0026thinsp;ZA groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The numbers of osteocytes in the AG\u0026thinsp;+\u0026thinsp;rhBMP-2 and AG\u0026thinsp;+\u0026thinsp;ZA groups did not differ significantly from each other (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), but both were significantly higher compared to the osteocyte numbers in the AG group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). On day 30, the osteocyte numbers in the AG, AG\u0026thinsp;+\u0026thinsp;PRP, and AG\u0026thinsp;+\u0026thinsp;rhBMP-2 groups were significantly higher than in the AG\u0026thinsp;+\u0026thinsp;ZA group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). There was no significant difference between AG and AG\u0026thinsp;+\u0026thinsp;rhBMP-2 (p\u0026thinsp;=\u0026thinsp;0.29), although both were significantly lower than in the AG\u0026thinsp;+\u0026thinsp;PRP group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). On day 60, osteocyte cell numbers in the AG\u0026thinsp;+\u0026thinsp;PRP, AG\u0026thinsp;+\u0026thinsp;rhBMP-2, and AG groups were significantly higher than in the AG\u0026thinsp;+\u0026thinsp;ZA group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, there was no significant difference among the AG, AG\u0026thinsp;+\u0026thinsp;PRP, and AG\u0026thinsp;+\u0026thinsp;rhBMP-2 groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eOn day 14, there was no significant difference in osteoclast numbers among the AG, AG\u0026thinsp;+\u0026thinsp;PRP, AG\u0026thinsp;+\u0026thinsp;rhBMP-2, and AG\u0026thinsp;+\u0026thinsp;ZA groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). On days 30 and 60, the osteoclast numbers were significantly higher in the AG\u0026thinsp;+\u0026thinsp;rhBMP-2 group than in the AG, AG\u0026thinsp;+\u0026thinsp;PRP, and AG\u0026thinsp;+\u0026thinsp;ZA groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Moreover, there was no significant difference among the AG, AG\u0026thinsp;+\u0026thinsp;PRP, and AG\u0026thinsp;+\u0026thinsp;ZA groups for osteoclast numbers during this period (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eAt the 14-day interval, new bone formation was observed at the site of the bone defect in three groups: AG\u0026thinsp;+\u0026thinsp;PRP, AG\u0026thinsp;+\u0026thinsp;rhBMP-2, and AG (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The AG\u0026thinsp;+\u0026thinsp;PRP group exhibited significantly greater closure of the bone defect area compared to the other groups, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The newly formed bone adjacent to the graft particles was composed of bone and lacunae containing osteocytes and numerous vascular channels, which were more abundant in the AG\u0026thinsp;+\u0026thinsp;PRP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Histologically, the newly formed bone trabecular meshwork was connected to the allograft bone. The bone beams of the newly formed tissue were mostly thin and heterogeneous, with focal bridge-like areas and single contacts, mainly at the poles of the bone beams. In contrast, the AG\u0026thinsp;+\u0026thinsp;Zol group displayed a predominance of fibrous tissue covering the defect area with minimal resorbed allograft fragments (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The fibrous tissue contained single thin-walled vessels and scant infiltrate, with single bone trabeculae found along the edges of the bone plate in a chaotic pattern.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn day 30, the reparative process in the cortical layer of the bone among groups AG\u0026thinsp;+\u0026thinsp;PRP, AG\u0026thinsp;+\u0026thinsp;rhBMP-2, and AG was characterized by a progressive increase in mature bone tissue with minimal fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In particular, the AG\u0026thinsp;+\u0026thinsp;PRP and AG\u0026thinsp;+\u0026thinsp;rhBMP-2 groups exhibited a notable prevalence of increased bone defect closure and new bone formation compared to the AG and AG\u0026thinsp;+\u0026thinsp;Zol groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The bone tissue in the affected area was observed as randomly located bone beams and strands, which formed lamellar structures. Additionally, the bone beams had a high degree of mineralization and demonstrated active longitudinal growth. In terms of newly formed vessels, the AG\u0026thinsp;+\u0026thinsp;PRP group exhibited a higher prevalence compared to the AG\u0026thinsp;+\u0026thinsp;rhBMP-2, AG\u0026thinsp;+\u0026thinsp;Zol, and AG groups (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). At this point in time, the AG\u0026thinsp;+\u0026thinsp;Zol group still demonstrated a predominance of coarse fibrous connective tissue within the defect zone, as was observed on day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003eg and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt day 60, in groups AG\u0026thinsp;+\u0026thinsp;PRP and AG, complete trabecular bone tissue was observed at the defect site with normal development of bone trabeculae, which were predominantly composed of spindle-shaped osteocytes (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003e). However, in the AG\u0026thinsp;+\u0026thinsp;rhBMP-2 group, the thickness of the newly formed bone tissue was less than that of the cortical plate outside the defect zone, and a U-shaped depression was formed in the defect area. The newly formed bone tissue was located within the cortical plate and did not spread into the intramedullary space. Furthermore, multinucleated giant cells (osteoclasts) were detected in small lacunae and spaces between allograft fragments and bone beams, which were resorbing the bone tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003ej). The intervals between the bone tissues were filled with fibrous tissue without any signs of inflammatory cell infiltration.\u003c/p\u003e \u003cp\u003eIn contrast, the defect area in the AG\u0026thinsp;+\u0026thinsp;Zol group was primarily covered with fibrous\u003c/p\u003e \u003cp\u003etissue and bone formation were limited (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Thin, randomly located newly formed bone trabeculae extended from the edge of the bone plate into the intramedullary space (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003ek and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003ek). The boundary between the bone plate and the newly formed bone was evident. The newly formed bone tissue was primarily composed of randomly located, thin bone trabeculae extending into the intramedullary space. The surface of the newly formed bone beams and the edge of the cortical plate defect were surrounded by a fibrous layer without reactive infiltration.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study examined the use of heat-treated Marburg bone in combination with PRP, rhBMP-2, and zoledronic acid to fill bone defects in an experimental rabbit femur model.\u003c/p\u003e \u003cp\u003eOur research findings reveal that the application of a PRP-perforated allograft combination resulted in superior bone regeneration after 14 days in comparison to the use of rhBMP-2, zoledronic acid or bone allograft alone to fill the defect. Furthermore, after 30 days, this combination demonstrated better results compared to the usage of zoledronic acid in combination with allograft and filling the defect with allograft alone. This fact was confirmed by the analysis of histological and histomorphometric data of osteogenesis and angiogenesis in the groups. The results obtained are consistent with clinical and experimental studies demonstrating the positive effect of PRP on bone regeneration at an early stage due to the release of several growth factors, such as PDGF, TGF-β and VEGF [22\u0026ndash;24]. Furthermore, growth factors contained in PRP stimulate angiogenesis and proliferation of osteoprogenitor cells only in the very early period after transplantation [36,37]. In our study, the use of PRP resulted in faster bone formation compared to the other groups in the early stages.\u003c/p\u003e \u003cp\u003eConcerning the acceleration of bone formation when using a bone allograft with rhBMP-2, it is well-known that allografts function as carriers of rhBMP2 [20,38,39]. Previous studies, as well as our results, have shown that the incorporation of rhBMP2 into the allograft significantly accelerates bone formation from an early stage up to 4 weeks after transplantation [40,41]. The combined use of a bone allograft with 5 \u0026micro;g of rhBMP2 induced bone formation was higher than that with the allograft alone at day 30. On the contrary, at 60 days, there was a thinning of the cortical plate of the femur at the site of the defect and the presence of many giant cells. This phenomenon is possibly related to the fact that BMPs, in particular rhBMP-2, not only accelerate bone formation but also cause premature bone resorption mediated by osteoclasts through RANKL-RANK signaling, a side effect that is often overlooked [39, 43]. Further study is underway to investigate if bone resorption and formation occur simultaneously at different dosages and when combined with bisphosphonates.\u003c/p\u003e \u003cp\u003ePre-treatment of bone grafts in a bisphosphonate (BP) solution may offer a potential preventive measure against bone graft resorption. The direct influence of BPs on osteoclasts is well established, and this effect on bone formation is noteworthy. The administration of BPs can alter the function of osteoclasts by inducing the secretion of an osteoclastic inhibitory factor by osteoblasts. The regulation of osteoclastic activity is primarily controlled by osteoblastic cells during bone remodeling. BPs enhance the proliferation and differentiation of osteoblasts and reduce apoptotic cell death [25,44,45]. The dosage and application technique of BPs are critical factors that determine the interaction between BPs and bone tissue. Research has revealed that BPs exhibit a biphasic effect on bone cells, with low concentrations stimulating cell proliferation and tissue formation, while high concentrations restrict these processes [26, 46\u0026ndash;49]. This study used zoledronic acid 0.05 mg/ml in combination with a bone allograft. We found a decrease in resorption, as expected, and a decrease in the amount of new bone in the bone defect at all follow-up periods compared with other groups. In this group, the bone defect was filled mainly with fibrous tissue at all stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003e). During the remodeling phase, our data support the idea that bisphosphonates may reduce bone resorption and inhibit osteogenic cell activity. Although in previous studies [26,27], the authors showed that the use of zoledronic acid 0.05 mg/ml promotes osteogenesis, in this study, the use of zoledronic acid 0.05 mg/ml led to the inhibition of both resorption and bone formation. The application of a thermally treated bone allograft in combination with zoledronic acid has potential applications in joint revision surgery, specifically in scenarios necessitating impaction with bone tissue and prevention risk of endoprosthesis loosening.\u003c/p\u003e \u003cp\u003eThe strength of this study is the comparative characterization of the use of a bone allograft prepared according to the Marburg system in combination with PRP, rhBMP-2, and zoledronic acid both in the early stages and in the late stages of bone defect healing. This made it possible to reveal the stimulation of osteoregeneration in the early stages with the use of PRP and rhBMP-2, as well as the inhibition of bone allograft resorption with the use of zoledronic acid. The use of a standardized rabbit femoral defect model and histological and morphometric analysis is also a strength of the study.\u003c/p\u003e \u003cp\u003eLimitations are the absence of an empty defect in the model, which could demonstrate the initial efficiency of the regenerative potential of the bone without any bone grafts, as well as the need to draw blood from the heart before surgery in the group with PRP, which could affect the recovery process after surgical intervention.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, the present study showed that PRP and rhBMP-2, in combination with a Marburg bone allograft, can markedly promote bone formation in bone defects at early stages. Although the mechanisms that lead to the stimulation of bone regeneration remain unclear, enhancing the osteoinductivity of bone substitutes may provide a highly relevant approach to clinical bone reconstruction in the near future. In this study, we found a strong effect of bisphosphonates on graft resorption as well as inhibition of new bone ingrowth into grafts. However, due to the impossibility of direct extrapolation of the results to human conditions, further studies are ongoing in controlled clinical trials.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research has been funded by the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan (Grant №AP09260954).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, D.S., Y.K. and E.T.; methodology, D.S. and E.T.; software, E.T.; validation, B.T., D.R. and Y.K.; formal analysis, D.S.; investigation, E.T. and Y.K.; resources, B.T.; data curation, E.T.; writing\u0026mdash;original draft preparation, E.T. and Y.K.; writing\u0026mdash;review and editing, S.D. and D.R.; visualization, E.T.; supervision, S.D.; project administration, S.D.; funding acquisition, Y.K.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study protocol was approved by the\u0026nbsp;University Animal Care Committee (UACC) of\u0026nbsp;Karaganda Medical University (protocol № 27 27.09.2020).\u0026nbsp;The standard principles of animal experiment ethics were followed in all the animal experiments, and they adhered to the protection guidelines of DIRECTIVE 2010/63/EU for animals used in scientific research. The study was conducted while adhering to the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWinkler T, Sass FA, Duda GN, et al. A review of biomaterials in bone defect healing, remaining shortcomings and future opportunities for bone tissue engineering: The unsolved challenge.\u0026nbsp;Bone Joint Res. 2018;7(3):232-243. doi: 10.1302/2046-3758.73.BJR-2017-0337.R1\u003c/li\u003e\n \u003cli\u003eMolina CS, Stinner DJ, Obremskey WT. Treatment of traumatic segmental long-bone defects: a critical analysis review.\u0026nbsp;JBJS Reviews. 2014;2(4). doi: 10.2106/JBJS.RVW.M.00062\u003c/li\u003e\n \u003cli\u003eRoberts TT, Rosenbaum AJ. 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The Effect of Bisphosphonates on Fracture Healing Time and Changes in Bone Mass Density: A Meta-Analysis.\u0026nbsp;Front Endocrinol (Lausanne). 2021 Aug 30;12:688269. doi: 10.3389/fendo.2021.688269.\u003c/li\u003e\n \u003cli\u003eThe Code of the Republic of Kazakhstan on the Health of the People and the Healthcare System of July 7, 2020 №360. Chapter 24 Donation and Transplantation. Available from: https://adilet.zan.kz/rus/docs/K2000000360.\u003c/li\u003e\n \u003cli\u003eOrder of the Minister of Health and Social Development of the Republic of Kazakhstan dated 04.05. 2019 on the Approval of the Rules for the Formation and Maintenance of Registers of Tissue Recipients (part of the tissue) and (or) Organs (part of the organs), as well as Tissue Donors (part of the tissue) and (or) Organs (part of organs), Hematopoietic Stem Cells. Available from: https://adilet.zan.kz/rus/docs/V1500011477.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eTuleubaev B, Saginova D, Saginov A, et al. 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Regenerative Efficacy of Supercritical Carbon Dioxide-Derived Bone Graft Putty in Rabbit Bone Defect Model.\u0026nbsp;Biomedicines. 2022 Nov 3;10(11):2802. doi: 10.3390/biomedicines10112802.\u003c/li\u003e\n \u003cli\u003eBurkitt HG, Young B, Wheater JW. Wheater\u0026apos;s Functional Histology: A Text and Colour Atlas. 3rd edition. New York: Churchill Livingstone; 2015.\u003c/li\u003e\n \u003cli\u003eJung RE, Schmoekel HG, Zwahlen R, Kokovic V, Hammerle CH, Weber FE. Platelet-rich plasma and fibrin as delivery systems for recombinant human bone morphogenetic protein-2.\u0026nbsp;Clin Oral Implants Res. 2005;16(6):676-682. doi: 10.1111/j.1600-0501.2005.01162.x.\u003c/li\u003e\n \u003cli\u003eMarx RE. Platelet-rich plasma (PRP): What is PRP and what is not PRP?. Implant Dent. 2001;10(4):225-228. doi: 10.1097/00008505-200110000-00002.\u003c/li\u003e\n \u003cli\u003eGillman CE, Jayasuriya AC. FDA-approved bone grafts and bone graft substitute devices in bone regeneration. Mater Sci Eng C Mater Biol Appl. 2021 Nov;130:112466. doi: 10.1016/j.msec.2021.112466.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eCheng TL, Leblanc E, Kalinina A, Cantrill LC, Valtchev P, Dehghani F, Little DG, Schindeler A. A bioactive coating enhances bone allografts in rat models of bone formation and critical defect repair.\u0026nbsp;J Orthop Res. 2019 Nov;37(11):2278-2286. doi: 10.1002/jor.24409. PMID: 31283054.\u003c/li\u003e\n \u003cli\u003eAlam MI, Asahina I, Ohmamiuda K, Takahashi K, Yokota S, Enomoto S. Evaluation of ceramics composed of different hydroxyapatite to tricalcium phosphate ratios as carriers for rhBMP-2.\u0026nbsp;Biomaterials. 2001;22(14):1643-1651. doi: 10.1016/s0142-9612(00)00404-8.\u003c/li\u003e\n \u003cli\u003eLee YM, Nam SH, Seol YJ, Kim TI, Lee SJ, Ku Y, Rhyu IC, Chung CP, Han SB, Choi SM. Enhanced bone augmentation by controlled release of recombinant human bone morphogenetic protein-2 from bioabsorbable membranes.\u0026nbsp;J Periodontol. 2003;74(6):865-872. doi: 10.1902/jop.2003.74.6.865.\u003c/li\u003e\n \u003cli\u003eKaneko H, Arakawa T, Mano H, et al. Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts. Bone. 2000;27(4):479-486. doi: 10.1016/s8756-3282(00)00360-5.\u003c/li\u003e\n \u003cli\u003eYamamoto Y, Udagawa N, Matsuura S, et al. Osteoblasts provide a suitable microenvironment for the action of receptor activator of nuclear factor-\u0026kappa;B ligand. Endocrinology. 2006;147(7):3366-3374. doi: 10.1210/en.2005-1581.\u003c/li\u003e\n \u003cli\u003eDunford JE, Rogers MJ, Ebetino FH, Phipps RJ, Coxon FP. Inhibition of protein prenylation by bisphosphonates causes sustained activation of Rac, Cdc42 and Rho GTPases.\u0026nbsp;J Bone Miner Res. 2006;21(5):684-694. doi: 10.1359/jbmr.060201.\u003c/li\u003e\n \u003cli\u003ePatntirapong S, Singhatanadgit W, Chanruangvanit C, Lavanrattanakul K, Satravaha Y. Zoledronic acid suppresses mineralization through direct cytotoxicity and osteoblast differentiation inhibition.\u0026nbsp;J Oral Pathol Med. 2012;41(9):713-720. doi: 10.1111/j.1600-0714.2012.01154.x.\u003c/li\u003e\n \u003cli\u003eHuja SS, Fernandez SA, Phillips C, Li Y. Zoledronic acid decreases bone formation without causing osteocyte death in mice.\u0026nbsp;Arch Oral Biol. 2009;54(9):851-856. doi: 10.1016/j.archoralbio.2009.05.008.\u003c/li\u003e\n \u003cli\u003eHuja SS, Fernandez SA, Phillips C, Li Y. Zoledronic acid decreases bone formation without causing osteocyte death in mice.\u0026nbsp;Arch Oral Biol. 2009 Oct;54(10):851-6. doi: 10.1016/j.archoralbio.2009.06.002. PMID: 19615831.\u003c/li\u003e\n \u003cli\u003evon Knoch F, Jaquiery C, Kowalsky M, Schaeren S, Alabre C, Martin I, Rubash HE, Shanbhag AS. Effects of bisphosphonates on proliferation and osteoblast differentiation of human bone marrow stromal cells.\u0026nbsp;Biomaterials. 2005 Nov;26(32):6941-9. doi: 10.1016/j.biomaterials.2005.04.056. PMID: 15990113.\u003c/li\u003e\n \u003cli\u003eGao Y, Liu X, Gu Y, Song D, Ding M, Liao L, Wang J, Ni J, He G. The Effect of Bisphosphonates on Fracture Healing Time and Changes in Bone Mass Density: A Meta-Analysis. Front Endocrinol (Lausanne). 2021 Aug 30;12:688269. doi: 10.3389/fendo.2021.688269. PMID: 34526966; PMCID: PMC8435630.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bone regeneration, bone graft, platelet-rich plasma, BMP-2, zoledronic acid","lastPublishedDoi":"10.21203/rs.3.rs-2813335/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2813335/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe establishment of a reliable technique for promoting bone formation without resorting to autografts remains an unresolved challenge. Currently, platelet-rich plasma (PRP), bone morphogenetic protein (BMP), and bisphosphonates are under intense scrutiny for their potential clinical utility in such instances. Despite their widespread investigation, the effect of these agents on bone formation is still a matter of debate, and the observed variations in their efficacy are influenced by multiple factors, including the biomaterials employed. The aim of this research was to assess the impact of bone allograft combined with platelet rich plasma, recombinant human bone morphogenetic protein-2 (rhBMP-2), and zoledronic acid on bone formation in rabbit femur defects.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMaterials and methods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA total of 96 rabbits were used, and femoral bone defects with a diameter of 5mm and depth of 10mm were created. The bone allografts used were prepared from femoral heads according to the Marburg system. The rabbits were divided into four groups: (1) bone allograft with PRP (AG\u0026thinsp;+\u0026thinsp;PRP), (2) bone allograft with rhBMP-2 5\u0026micro;g (AG\u0026thinsp;+\u0026thinsp;BMP-2), (3) bone allograft with zoledronic acid 5\u0026micro;g (AG\u0026thinsp;+\u0026thinsp;ZA), and (4) bone allograft only (AG) as the control group. Histopathological and histomorphometric analyses were performed to evaluate bone defect healing after 14, 30, and 60 days.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe findings from the histomorphometric analysis showed that the new bone formation inside the bone allograft was significantly greater in the AG\u0026thinsp;+\u0026thinsp;PRP group compared to AG and AG\u0026thinsp;+\u0026thinsp;Zol groups after 14- and 30 days (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000). The use of bone allograft with rhBMP-2 induced higher bone formation compared to AG group on days 14 and 30 (p\u0026thinsp;\u0026lt;\u0026thinsp;0,000), but excessive osteoclast activity was observed on day 60. The local co-administration of ZOL with heat-treated allograft inhibits allograft resorption as well as new bone formation in the bone defect at all periods.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn conclusion, the study demonstrated that PRP and rhBMP-2, combined with a Marburg bone allograft, can significantly promote bone formation in the early stage of bone defect healing.\u003c/p\u003e","manuscriptTitle":"Comparative evaluation of heat-treated bone allograft combined with platelet rich plasma, recombinant human bone morphogenetic protein 2 and zoledronic acid: An experimental study on the histopathological and histomorphometric aspects of bone healing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-20 17:42:05","doi":"10.21203/rs.3.rs-2813335/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b6c1ed45-fe90-49f8-bdee-445d96fdc1d1","owner":[],"postedDate":"April 20th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-03T05:44:19+00:00","versionOfRecord":[],"versionCreatedAt":"2023-04-20 17:42:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2813335","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2813335","identity":"rs-2813335","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}