| Large bone defects often arise from trauma, skeletal diseases, congenital malformations, and tumor resections. Traditional approaches for bone reconstruction include autologous bone grafting and use of alloplastic implants, but their applications remain limited by the low availability of bone grafts, donor site morbidity, risk of infection, and immunoreactions. In the field of bone tissue engineering, numerous bone scaffolds have been developed; however, their applications have not proceeded to clinical practice because the current strategies remain limited in fully meeting the golden rules of bone tissue engineering. It is critical to develop a biomaterial that can chemically and structurally mimic the native extracellular matrix of the bone. Calcium phosphate cement (CPC) biocomposite has been widely used as a scaffolding biomaterial in bone tissue engineering. Due to its noncytotoxic and osteoconductive properties, CPC is an important candidate as a filling material in orthopedic applications. CPC can be combined with antibiotics, drugs, and growth factors and can stimulate certain biological responses at the local site. Studies on animal models revealed that CPC produced good tissue response. CPC is a promising biomaterial in the application of bone cement for bone defect repair.The combination of bioactive molecules and bone scaffolds seems to be a good solution for bone tissue defects. One, of the tissue engineering strategies to obtain osteoinductivity and enhance the repair of critical-sized bone defects is to utilize bone morphogenetic protein-2 (BMP-2) therapies; however, its short half-life limits the local delivery and does not always exhibit efficacy in bone defect repair in vivo. Hence, it is obviously important to devise more sophisticated and proper approaches to deliver this bioactive molecule in a controlled and sustained manner and simultaneously minimize its side effects. Most of the studies have focused on the approaches or strategies for designing and fabricating the scaffolds as well as the properties such as biocompatibility, biodegradability, and osteogenic differentiation capability of the scaffolds; however, the mechanism behind the supportive function of the scaffolds remains poorly known. Urist was the first to iaolated BMP in 1982. BMP is the only growth factor to induce the ectopic bone formation and enhance the bone repair individually. BMP-2 have the strongest ability to induce differentiation of bone marrow mesenchymal stem cells into osteoblasts. The amount of BMP-2 contained in the natural bone is limited. Researchers have solved this problem by genetic engineering and the recombinant human bone morphogenetic protein-2 (rhBMP-2) was prepared. At present, the recombinant human bone morphogenetic protein-2 (rhBMP-2) application defects include:complicated preparation technology, multiple biological effects, low biological activity, short half-life, lack of affinity to the bone, et al. The study found that in the natural human BMP-2 mature peptide of 114 amino acids, the core domain is composed of 5-20 amino acids, which really play the role of bone structure. P24 peptide derived from BMP-2 knuckles clusters, which can greatly exert the biological role of BMP-2. P24 and BMP-2 is not only the peptide chain length, unique advantage performance aspects:1) were synthesized on a large scale; 2) the linear structure, the active site more fully exposed, high biological activity, good stability; 3) specific interaction and no side effects; 4) low immunogenicity; 5) without osteoclast activation; 6) easy surface modification. P24 polypeptides have been shown to regulate the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs), and to induce ectopic bone formation in vivo. In addition, P24 peptides contain aspartate and phosphorylated serine, which can form an acidic environment, promote the deposition of calcium and phosphorus, accelerate the formation and mineralization of the core.A good controlled release effect can be achieved by the combination of the osteogenic factor and the suitable carrier material. There are two kinds of methods of chemical crosslinking and physical adsorption, which are active factor and biological material carrier. Chemical crosslinking method in scaffolds were prepared by adding the active factor in the process, this method can make the active factor in the scaffolds uniformly and achieve the purpose of sustained release, but because in the preparation process, there will be more involved in organic solvent, active factors were directly exposed to organic solvents, its biological activity is usually physical adsorption is obviously weakened; will have good scaffolds for a certain period of time in a solution containing the active factor in the activity factor reached to drug adsorption materials, the method is simple and feasible, to maximize the retention of the biological activity factor, but its distribution in the scaffold may not be uniform, and the faster release speed, affecting its normal biological function of play. In the field of bone tissue engineering, microspheres have great potential application as a three-dimensional (3D) matrix to support cellular expansion and cell differentiation after implantation into the damaged tissues, such as bone defects. Spherical particulates can be used as carriers of tissue cells to increase the capacity for manipulation of efficient cell adhesion, migration, or proliferation, which ultimately promotes the repair of bone defects. The chemical composition of the spherical particulates, also consisting of bioactive ceramics, such as calcium phosphates, can significantly affect their in vivo bone-forming ability. Moreover, the 3D shape and morphology of the microspheres are also important to effectively load and contain growth factors or tissue cells. Some recent studies have reported the development of porous or evacuated calcium phosphate microspheres as an effective drug-delivery system and bone defect fillers.Objective:The aim of the present study was to develop new calcium phosphate cement (CPC) microspheres loaded with bone morphogenetic protein-2 (BMP2)-derived peptide P24 (P24/CPC) and to evaluate the effect of the P24/CPC microspheres on osteogenesis and bone regeneration in vitro and in vivo.Methods:The calcium phosphate cement (CPC) used in this experiment consisted of equimolar tetracalcium phosphate (TECP) and dicalcium phosphate anhydrous (DCPA). The peptide 24 (P24) was derived from BMP-2 (Nâ†'C:KIPKA SSVPT ELSAI STLYL SGGC). P24 was hydrated in 10 mL of 0.1 M HC1, and CPC powder was added with continuous stirring until uniform distribution. Microspheres were prepared with hybrid of P24 and CPC by using of the dripping procedure, then microspheres were lyophilized at-50 ℃ and 20 Pa using the sphere mold to obtain the P24/CPC microspheres. CPC was used as control and different amounts of P24 (2% or 4% of the weight of CPC:2% P24/CPC or 4% P24/CPC microspheres) were used in in vitro and in vivo tests. For the in vitro experiment, the P24 release profiles from P24/CPC microspheres were determined in vitro by high performance liquid chromatography system (HPLC, Shimadzu 10Avp, Japan). BMSCs were seeded onto CPC,2% P24/CPC, and 4% P24/CPC microspheres at a concentration of 2 × 104 cells/cm2 and cultured in DMEM supplemented with 15% fetal bovine serum and 1% penicillin-streptomycin liquid (100 U/ml). Then, we assessed the cell shape and proliferation of rat BMSCs on P24/CPC by SEM scaning, CCK-8 test and fluorescence (DAPI staining). Furthermore, we detected the the mRNA levels of osteogenic-specific genes(OCN and Runx2). In addition, we analyzed the alkaline phosphatase (ALP) activity of BMSCs cultured on the scaffolds after osteogenic induction. For the animal experiment, firstly, the ectopic bone formation experiment was performed.18 healthy SD female rats (weighing an average of 150 g), supplied by the Animal Research Center of Guangdong Province, were equally divided into three groups (A, B, and C). Rats in A, B, and C groups, respectively, were implanted with CPC,2% P24/CPC, and 4% P24/CPC microspheres on the muscle pouches on either side of the back. Computed tomography (CT) scanning, hematoxylin and eosin (H&E) or Masson’s trichrome staining were preformed at 4, and 8 weeks after implantation. Finally, we surgically created the rabbit femur cavity defect model and inplanted the CPC,2%P24/CPC and 4%P24/CPC microspheres into the femur cavity defect of the rabbit, respectively. The cavities without any filling material were used as the negative control. At 4,8, and 12 weeks after implantation, the femora were removed and subjected to CT analysis and histological studies to investigate the efficacy of P24/CPC in inducing new bone formation and regeneration. The data obtained in this study were all measurement data. All data were expressed in the. form of x±SD, and the data were analyzed by SPSS20.0 software. We used the factorial analysis or repeated measures analysis of variance for statistical analysis, and chose the corrected or uncorrected F values and P values according to the results of homogeneity test of variance (Levene’s test). Single factor analysis of the multi factor data between the two groups were compared using t test, and single factor analysis of the multi factor data between the multiple groups were compared using univariate analysis of variance (one-way ANOVA and LSD multiple comparisons). P< 0.05 was considered statistically significant.Results:The in vitro studies showed that the bioactivity of P24 was preserved in the P24/CPC microspheres and the P24 release duration lasted up to 39 days. The P24/CPC microspheres with different proportions of P24 (2% and 4%) induced high proliferation of bone marrow stromal cells (BMSCs) and well supported cell adhesion on the sphere surfaces. Furthermore, the cells exhibited higher alkaline phosphatase activity and higher expression of osteogenic-specific genes (OCN and Runx2) in 4% P24/CPC than in the CPC and control groups (P< 0.05). Therefore, these findings indicated the enhanced osteoblastic differentiation of BMSCs on P24/CPC microspheres. In vivo osteoinductive studies revealed that the degrees of ectopic osteogenesis in the dorsal muscle pocket of rat were significantly higher in the P24/CPC microspheres than in the CPC microspheres. Finally, the CPC or P24/CPC microspheres were implanted into rabbit femoral condyle defects and P24/CPC serial groups were observed for 4,8, and 12 weeks. Computed tomography (CT) and histological and histomorphological studies proved that P24/CPC microspheres were biocompatible, bioabsorbable, and osteoinductive.Conclusion:These results confirmed that BMP2-derived peptide P24 can have a beneficial effect on the bone-generating properties of CPC microspheres. P24/CPC microspheres were biocompatible, bioabsorbable, and osteoinductive. In conclusion, this study demonstrated that P24/CPC microspheres can enhance osteogenesis in vitro and promote bone regeneration in vivo, suggesting that P24/CPC microspheres are strong potential bone graft substitutes in bone tissue engineering. |
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