Effect of Concentrated Growth Factor (CGF) on the Promotion of Osteogenesis in Bone Marrow Stromal Cells (BMSC) in vivo

The therapeutic method traditionally used in bone defect reconstruction is autologous bone grafting. The most common problems affecting this type of repair approach are bone absorption and donor trauma. The approach taken in this study overcomes these problems. Bone marrow stromal cells (BMSCs) provided the crucial seed cells. Fibrin biological scaffolds were formed by combining the BMSCs with concentrated growth factor (CGF). BMSCs were isolated from Wistar rat femurs; CGF was prepared from rat heart blood. Five repair groups were created for comparative purposes: (A) CGF + BMSCs; (B) CGF; (C) collagen + BMSCs; (D) collagen; (E) blank. After three months, the rats were sacrificed, and histopathology and three-dimensional CT images produced. Bone regeneration was significantly higher in the (A) CGF + BMSC group; osteogenesis was lower in the (B) CGF and (C) collagen + BMSC groups, at very similar levels; the (D) collagen and (E) blank groups scored the lowest results. Our research suggests that combining CGF with BMSCs leads to the formation of fibrin scaffolds that have a powerful effect on osteogenesis as well as a subsidiary angiogenic effect. SEM images of the CGF scaffolds cultured with BMSCs confirmed good CGF biocompatibility. The superior osteoinductive activity of the CGF + BMSC combination makes it an excellent biomaterial for bone regeneration.


Materials and Methods
Isolation and culture of Bone Marrow Stromal Cells. All animal operations were performed strictly in accordance with NIH guidelines and ethical principles for the care and use of laboratory animals approved by the Animal Research Committee of Shanghai Ninth People's Hospital affiliated to Shanghai Jiao Tong University, School of Medicine. All experimental animals were managed humanely. The animals were housed in 12-hour light/dark cycles, in an air-conditioned environment (22 ± 2 °C) and given free access to food pellets and water. Eight-week-old Wistar rats were sacrificed and bilateral femurs were harvested under aseptic conditions. Both ends of the femurs were cut off and the bone marrow was flushed out using a 25-gauge needle with DMEM (Hyclone, Logan City, UT) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 U/ml penicillin and 100 μg/ml streptomycin. The primary BMSCs were cultured in a humidified 37 °C and 5% CO 2 incubator and the medium was renewed every three days. The cells were passed with 0.25% trypsin (Hyclone) till they reached 90% confluence. BMSCs cultured to the third passages were used in this study.
Flow cytometry identification of BMSCs. BMSCs were identified by flow cytometry analysis of cell surface markers. BMSCs were digested using 0.25% trypsin to a concentration of 1 × 10 6 cells/ml. Cell suspensions to which antibodies had been added served as experimental groups: CD9 (BD), CD29 (BD), CD31 (BD), CD34 (BD), CD45 (Biolegend) and CD90 (Biolegend); those without antibodies were controls. After being incubated in a dark place at 4 °C for one hour with phycoerythrin (PE) or fluorescein isothiocyanate (FITC) and then washed with PBS, the labeled BMSCs were resuspended for analysis using a flow cytometer (Epics Altra, USA).
Fabrication and characterization of concentrated growth factor extracts and fibrin scaffolds. For the in vivo experiments, CGF was obtained from the heart blood of healthy male Wistar rats (age 8 weeks). Donor rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.4 ml/100 g); blood was taken by cardiac puncture directly into a sterile tube without anticoagulants and was immediately centrifuged in a special centrifuge device (Medifuge TM ; Silfradent srl, Sofia, Italy) for approximately 13 minutes. CGF, the fibrin buffy coat used in this in vivo study, was separated using scissors (Fig. 1). The three-dimensional (3D) structures of CGF and BMSCs cultured with CGF for 7 days were observed by scanning electron microscopy (SEM, JEOL, Japan).
In vivo reconstruction of calvarial defects in rats. Thirty male Wistar rats were divided randomly into five calvarial defect groups. The rats were anaesthetized by intraperitoneal injection of 10% chloral hydrate (0.4 ml/100 g), a 1.5 cm sagittal incision was made in the scalp, and then the calvarium was exposed by blunt dissection. Two bilateral critical-sized defects without perichondrium were created (Fig. 2) using a 5.5-mm diameter trephine burr (Medesy, Italy). In the experimental group, we used BMSCs that had been initially cultured with CGF for 7 days in vitro and then transferred in vivo. The collagen used as control scaffolds in the study was type I rat tail tendon collagen (Shengyou, China). Non-filled calvarial defects served as blank controls. Finally, sixty critical-sized calvarial defects in thirty Wistar rats were randomly filled as follows: (A) CGF + BMSCs; (B) CGF; (C) collagen + BMSCs; (D) collagen; (E) blank.
Microcomputed tomography (Micro-CT) measurement. The samples were scanned with a micro-CT system (uCT-80, Scanco, Switzerland) using 10 μm spot size and 55 KVp maximum voltage parameters to determine the amount of newly formed bone. 3D images were generated. Histological and immunohistological observation. The specimens were decalcified in 10% EDTA for two weeks after being examined by micro-CT, dehydrated in 75% to 100% ascending alcohol concentrations, and then embedded in paraffin. Histological sections were prepared for Hematoxylin and Eosin (H&E), Masson staining and immunohistological analysis. The primary antibodies CD34 (1:1000 dilution; Servicebio) and the secondary antibody (1:1; DAKO) were successively subjected to immunohistological assay. The samples were then observed and photographed under a high quality microscope.
Material characterization. The SEM images of CGF scaffolds cultured with BMSCs for 7 days are presented in Fig. 4. The images show that the samples have regular fibrinogen structures and contain white blood cells, red blood cells and blood platelets. BMSCs can be clearly seen growing well on the surface of the CGF.
Histological and immunohistological analysis of bone regeneration. An osteogenic histological assessment of the five groups was carried out 6 and 12 weeks after the rat calvarial defect operation. Bone sections that had undergone H&E and Masson staining were examined under a microscope. Histological results revealed different bone regeneration results in the different groups. In the (D) collagen and (E) blank control groups, almost no new bone was found in the defect site 6 weeks postoperatively and the fibrous connective tissue over the empty defects was sparse. This pattern of bone formation was also observed after 12 weeks; a large bone defect still remained in these two groups.
In the (B) CGF and (C) collagen + BMSCs groups, at 6 weeks postoperatively, fibrous connective tissue was observed over the defects and some new bone had formed along the boundary of the bone defect. In contrast with the (D) collagen and (E) blank groups, Masson staining showed matrix collagen fibrils were widely distributed in the (B) CGF and (C) collagen + BMSC groups. The new bone grew gradually from the bone defect boundary to the central area. Twelve weeks after operation, newly formed bone covered almost half the bone defect area. The difference between the (B) CGF and (C) collagen + BMSC groups was that neovascularization was observed in histological sections of the (B) CGF group.
In the (A) CGF + BMSC group 6 weeks postoperatively, newly formed bone was found both in the center and the boundary of the defect site, with wide distribution of the collagen fibrils of the matrix (Fig. 7). Micro-CT images confirmed the islands of newly-formed bone in contrast with the other groups. Distinct neovascularization around the new bone was observed in histological analysis and confirmed by CD34 immunohistology (Fig. 8). The regenerated bone gradually matured. 12 weeks postoperatively, the defect was almost filled by new bone. The newly formed bone had the same histological structure as normal bone.

Discussion
Autogenous bone grafting or artificial materials can be used to reconstruct bone defects, but reducing bone absorption and enhancing new bone formation has remained problematic; the risk of bioincompatibility, infection and immunological rejection are high both for allogenic bone grafting and artificial materials. Tissue  engineering technology may make bone defect reconstruction feasible. In some clinical studies, autologous materials acting as a biological scaffold were used to ensure graft stabilization; satisfactory long-term results were obtained 16 . For example, Hibi et al. applied PRP and autologous mesenchymal stem cells to repair the alveolar cleft of a 9-year-old female patient and regenerated bone was observed by CT after three months with 79.1% remaining after nine months 17 .
In our experiment, the properties of bone marrow-derived mesenchymal stem cells were characterized using cell surface markers. The detection of surface molecule expression revealed that BMSCs were negative for CD45, CD34, CD31 and CD9 but positive for mesenchymal associated markers such as CD90 and CD29. The BMSCs played a key role in the bone regeneration 6 .
In the present study, we compared the effects of different groups -CGF or collagen with or without BMSCswhich have not been discussed previously in the literature. CGF, the third-generation platelet concentrate, has a higher GF concentration and better regenerative capacity than PRF (the second-generation platelet concentrate) and PRP (the first-generation platelet concentrate) 18,19 . Our study confirms the positive effects of a combination of bone marrow stromal cells and CGF in an experimental bone defect model. In our study, micro-CT and histological examination found new bone formation not only at the periphery of the bone defect, but also in the central area of the bone hole in the (A) CGF + BMSC group, starting 6 weeks after the operation. Newly formed bone appeared not only at the periphery, but also in the centre of the bone defect. Other groups had a gradual progression of new bone from the bone defect periphery to the center. New bone grew in this direction because the cells that induce bone formation were mainly supplied from the cut edge of the bone defect. The different phenomenon in (A) CGF + BMSC group showed that CGF provides a three-dimensional scaffold for BMSCs to survive and grow continuously and steadily. After twelve weeks, critical-size bone defects were almost closed over  in the (A) CGF + BMSC group, whereas large bone defects remained in the (D) collagen and (E) blank control groups. In the (B) CGF and (C) collagen + BMSC groups, new bone grew slowly. These results demonstrate that the CGF fibrin buffy coat is a biomaterial that can both bear BMSCs and secrete growth factors and thereby promote new bone formation, as reported by Honda et al 20 . By making comparisons between five different groups -CGF + BMSCs, collagen + BMSCs, CGF, collagen and blank group -we were able to identify the most powerful osteogenesis effect while excluding BMSC interference. In addition, compared to the other groups, starting six weeks postoperatively, new bone mass significantly increased in the (A) CGF + BMSC and (C) collagen + BMSCs groups, and this tendency continued for 12 weeks, confirming the importance of bone marrow-derived stem cells.
Not only does CGF provide a three-dimensional fibrin scaffold structure for BMSCs, it releases multiple growth factors which regulate cell proliferation and differentiation via specific receptors 21 . Vascular endothelial growth factor (VEGF) stimulates angiogenesis, an important step in the bone regeneration process because the supply of blood favors osteogenesis 22 . It has been shown that angiogenesis occurs before osteogenesis in the healing of bone defects, and that angiogenesis and osteogenesis have a mutually reinforcing effect on bone regeneration 23 . Given that angiogenic factors play an important role both in healing and in regeneration 24 , VEGF may induce mobilization, recruitment, proliferation and differentiation of endothelial progenitor cells (EPC), as well as the recruitment and survival of osteoblasts 25,26 . Our histological and immunohistochemical analysis showed that CGF promotes neovascularization better than groups without CGF. Neovascularization may enhance impaired angiogenic capacity and facilitate bone healing. Based on these results, we believe that CGF may induce osteogenic differentiation of BMSCs as well as angiogenesis.
Combination CGF/BMSC therapy has the full panoply of osteogenesis tissue engineering advantagesosteogenic seed cells: BMSCs, cell-bearing scaffold: CGF fibrin network, osteoinductive and osteoconductive cytokines: concentrated growth factors -that favour effective bone growth and regeneration. Preparing CGF and cells cultivated with CGF is quick and easy. Most importantly, CGF is autologous, spontaneously degraded, and absolutely safe. These in vivo results indicate that BMSC-stimulating CGF is an ideal biological material for bone regeneration.

Conclusions
This study demonstrates that CGF not only promotes the superior osteoinductive activity of BMSCs to enhance bone formation, but also outperforms collagen in stimulating angiogenesis. We conclude that CGF is an excellent cell growth factor biomaterial. Combined with BMSCs, it has a strongly positive effect on both osteogenesis and angiogenesis, making it a very promising material for bone regeneration.