Study On Calcium Phosphate Cement Loaded With Rhizoma Drynaria

Calcium phosphate cement (CPC) is the ideal bone filler due to its various advantages, such as in vivo self-setting and the perfect fit with the implantation bed, in particular, the hydration product is similar to the main inorganic component of natural bone. It is needed to load with various drugs or bioactive substances, released gradually at bone defects, in order to prevent the infection, treat bone disease in clinic or deal with bone defect filling and drug therapy. The low-temperature setting of CPC allows the incorporation of a variety of drugs and biologically active molecules. Many reports confirmed that CPC was a good carrier for local drug delivery and could avoid the side-effects of systemically administered drugs, reduces the risks from resistant bacteria and keep the local drug concentration.There are many kinds of Chinese herbal medicines confirmed to be effective for bone regeneration, among which rhizoma drynaria (GSB) has been proven to be effective in the promotion of bone healing. In this study, the physicochemical properties of CPC containing drynaria and in vitro drug release kinetics were characterized. Furthermore, the biological activities in vitro and in vivo were also investigated in this study. The aim of this study was to investigate the effect of CPC containing drynaria on bone defects reconstruction and its clinical feasibility.The Biocement D composition was adopted in the present study. The solid phase of CPC consists of 58 wt% a-tricalcium phosphate (a-TCP),25 wt% dicalcium phosphate dehydrate (DCPD),8.5 wt% hydroxyapatite (HA) and 8.5 wt% calcium carbonate (CaCO3); phosphate buffered solution (PBS, pH 7.4) was used as the liquid phase. The CPC powder was mixed with PBS at a liquid/powder ratio of 0.3 (mL/g) to form a cement paste. Two methods were used in the preparation of drug-loaded CPC. One was by means of synthesizing drug-loaded DCPD, the extraction of drynaria with various concentrations was added into DCPD paste to form drug-loaded CPCs which were marked as CPC-GSB. CPCs containing different concentrations of drynaria were marked as CPC-5 wt% GSB. CPC-10 wt% GSB and CPC-15 wt% GSB. respectively. Another method was by means of mixing CPC powder with drynaria directly and CPCs prepared in this way were marked as CPC-GSB. CPCs containing different concentrations of drynaria were marked as CPC+5 wt% GSB, CPC+10 wt% GSB and CPC+15 wt% GSB, respectively.The effects of drynaria on the physichemical properties and drug release behaviors of CPC were characterized by Gilmore needle tests. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR). compressive strength tests, scanning electron microscope (SEM) and UV-Vis spectrophotometry (UV-Vis). The biological evaluation was studied by co-culturing the disk-shaped CPCs with osteoblasts in vitro. Cell morphology, around the CPC disks, was observed by inverted microscope, while cell adhesion was observed by SEM. The influence of drynaria on proliferation of osteoblasts was investigated by Alamar Blue assay. The differentiation of osteoblasts cultered with drug-loaded CPC was studied by determining the activity of alkaline phosphatase (ALP). Cavities of 2 mm in diameter and 8 mm in depth at the femur of four New Zealand white rabbits were prepared for the implantion of cylindrical CPCs. All the specimens were carried out by general observation. X-ray observation and histological observation at 2,4 and 8 weeks after the operation, respectively.It was found that the initial and final setting time of both CPC-GSB and CPC+GSB increased by the addition of drynaria. The setting time of CPC-5 wt% GSB could meet the clinical requirement. The results of XRD showed that the addition of drynaria by both methods had no influence on the phase composion of CPC but inhibited the subsequent conversion ofα-tricalcium phosphate to HA. CPC converted to hydroxyapatite(HA) gradually during the hydration. Amouts of platy crystals could be observed after 24 hours, and the structure of CPCs containing drynaria was more compact compared to that of the control. The addition of drynaria increased the compressive strengths of CPC-GSB and CPC+GSB in dose-dependent manner. Furthermore the compressive strength of CPC-GSB was higher than those of CPC+GSB correspondingly. The drug release profiles of both kinds of CPC exhibited an initial fast release stage followed by a slow release stage, which were fit to the diffusion-controlled Higuchi model. In the process of drug release, CPC converted to HA gradually with a small amout of a-TCP remaining which was closely related to the concentration of drynaria. Amounts of acicular crystals in CPC-GSB and irregular laminate structure in CPC+GSB could be observed after released for 15 davs. which were more compact than those of the control proup. Compared with CPC before drug release, the compressive strength of CPC-GSB and CPC+GSB increased in dose-dependent manner. The compressive strengths of CPC-GSB were higher than those of CPC+GSB correspondingly.The results of cell culture indicated that the effects of CPC-GSB on the proliferation of osteoblasts were time and dose-dependent. There were no significant differences between CPC-GSB and CPC at day 3, while CPC loaded with 5wt% and 10wt% drynaria significantly promoted osteoblast proliferation after cultured for 5 days. However, the proliferation of cells cultured with CPC-GSB was decreased at day 7. CPC-5 wt% GSB significantly promoted osteoblast differentiation after cultured for 7 days. Osteoblasts with a normal morphology adhered on the surface of drynaria-loaded CPC, indicating an excellent biocompatibility. X ray observation indicated that the gap between CPC and the natural bones became smaller and smaller until finally they were connected with each other. Histological observation showed that newly formed bones accompanied by a number of active osteoblasts could be seen around the bone defects. The new bones were getting mature and the new-born trabecular bones grew from the edge into the depth of the materials with time. After 8 weeks, the number of mature bones increased significantly, the new bones and the natural bones linked with each other closely. In the mean time, fewer new-born trabecular bones were formed in the control group. The results showed that the addition of drynaria in CPCs accelerated the repare of bone defects.In this study drynaria-loaded CPCs with three concentations were prepared, among which CPC-5 wt% GSB was demonstrated to have excellent properties and promoted cell proliferation and differentiation. Accordingly, CPC-GSB has double effect of bone filling and drug therapy.