Experimental Studies Of Injectable Nano-hydroxyapatite/Collagen/Calcium Sulfate Hemihydrates(nHAC/CSH) Composite Cement

ObjectiveBony defects caused by infection, tumor, trauma, surgery, joint fusion, and fracture nonunion have increased as the population ages. Facing the problems of the irregular bony defects and the increasing popularity of minimally invasive techniques, especially in the application of craniofacial regions, the search for injectable biodegradable scaffold materials with satisfactory compatibility and rapid ossify performance became one of the major hotspots in bone tissue engineering.Injectable bone cements can be classified into four classes:calcium phosphates, acrylic bone cements, calcium sulfates, and composites. Among these materials, calcium sulfate has a long clinical history for use as bone graft substitute in its form known as plaster or gypsum for its self-setting ability "in situ" after filling the defect, the lack of inflammatory response, and the promotion of bone healing. It has been utilized in periodontal disease, endodontic lesions, alveolar bone loss and maxillary sinus augmentation. However, calcium sulfate is inferior to autografts in bone defect repair. However, calcium sulfate cement has some drawbacks that significantly limit its clinical applications, such as lacking the ability to form a chemical bond with bone tissue at the early stage of therapy because of its poor bioactivity. The ideal bone scaffold should promote early mineralization and support new bone formation while at the same time allowing for replacement by new bone. Based on the biomineralization principles, nano-hydroxyapatite/collagen (nHAC) composite, inspired from investigation on natural bone, had been developed by mineralizing typeⅠcollagen in our lab, which has attracted great attention. In this material collagen molecules and nano-hydroxyapatite assembled into mineralized fibril, with high similarity of natural bone both in composition and hierarchical structure. Extensively studies indicated that the nHAC material is bioactive and biodegradable. In order to optimize bioactivity and the performance of bone regeneration of CSH, the previous study suggested that hydroxyapatite (HA) could be incorporated into CSH, creating a composite grout that could be used for bone regeneration as an alternative or adjunct to autogenous bone.In the present study, a novel injectable bone cement was designed as a composite of nHAC and CSH, A series of such injectable composite cements with different ratio of nHAC and CSH were prepared for adjustable self-setting time. Their mechanical properties, in vitro bioactivity and degradability, in vitro and in vivo biocompatibility of the composite cements were determined in this study, which is aimed to have complementary advantages for the non-loading bearing bone defect repair in orthopedics application, such as bone tumor resections and osteoporosis.Methods1. In order to evaluate the biocompatibility and bioactivity of the composite cements, a series of such injectable composite cements with different ratio of nHAC and CSH were prepared for adjustable self-setting time. Their mechanical properties, in vitro bioactivity and degradability, in vitro and in vivo biocompatibility of the composite cements were also determined in this study. Through this research, the suitable proportion of nHAC should be screened for clinical practice.2. Quantitative and qualitative biocompatibility assays with bone marrow stromal cells cultured with materials in vitro were performed for evaluating the biocompatibility. Under the induction of condition culture medium, marrow mesenchyma stem cells were induced to marrow source osteoblast and express the performance of ossification. In vitro, the marrow source osteoblast was detected by MTT assay and Flow cytometric analysis. BMSCs grew on the surface of two samples were detected under scanning electron microscope. Type I collagen and integrinβ1 mRNA relative expression level were also detected to test the mechanism of cell adhesion.3. The the hematolysis experiment, the pyrogen experiment and the intracutaneous response experiment were used to appraise biocompatibility of the composite cement. A critical box-shaped defect model in the mandible of the rabbit was used to evaluate the bone-remodeling ability.4,8,12 weeks after operation, rabbit mandibular in box-shaped defect regions were extracted. X-ray study was used to observe the situation of each group of animal's bone defect. BMD analysis the of the defect regions were also used to evaluate the level of ossification. Using the decalcify and not the decalcify tissue slice and the dyeing technology to observe the degradation, the shape change and the bone formation of the defect region and also evaluate the ability of bone regeneration of the composite cement.Results1. XRD was used for testing the composition of cement with a serious of different ratio of nHAC and CSH. The main composition of the final setting cement of nHAC/CSH is nHAC and CSD. The diffraction of the nHAC and CSH powders prepared in this paper were the same as that reported previously. The composite cement was synthesized successfully. The results demonstrated that setting behavior, injectability, and mechanical properties of the bone cement were depend on the composition ratio of nHAC and CSH. nHAC has a retarding effect on the setting time of composite cement, a higher setting time was obtained with the increase of the amount of nHAC. The mechanical results indicated that compressive strength and compressive modulus of composite the cement decreased with the increase of the content ratio of nHAC. The results from the EDX analysis indicated that the bioactivity of the nHAC/CSH composite cements has been improved significantly compared to that of the pure CSH cement. It can be seen that there was no significant difference of the degradation rate between the nHAC/CSH composite cements and the pure CSH cement, except that of 30% nHAC/CSH composite cement at 14th day and 21st day. The nHAC/CSH composite cement can be adjusted by modifying the ratio of nHAC and CSH for various clinical applications.10% nHAC was carefully screened for clinical practice.2. Under the induction of condition culture medium, marrow mesenchyma stem cells were induced to marrow source osteoblast. In vitro, favourable biocompatibility was detected by MTT assay and Flow cytometric analysis. BMSCs grew on the surface of two samples were found to be adhered firmly on nHAC/CSH surface compared with pure CSH under scanning electron microscope. Type I collagen and integrinβ1 mRNA relative expression level also suggested nHAC/CSH composite cement have a higher ability to promote cell adhesion than the pure CSH. Results in vitro indicated that the nHAC/CSH significantly improved bioactivity compared with that of CSH, especially in promoting cell adhesion. 3. In vivo experiment indicated that this composite cement has not caused the obvious inflammation, does not have hemolytic, not sends the thermal property, the non-skin irritating quality, has the satisfactory biological security, conforms to the national biology medical material related standard. The in vivo results also show that nHAC/CSH composite cement exhibit favorable bioactivity of bone regeneration than that of CSH, it also could cause an earlier accelerator and better osseointegration for bone repair.Conclusions1. The setting time and injectability and mechanical properties of the composite cement could be controlled by adjusting the content ratio of nHAC and CSH. nHAC/CSH exhibit better bioactivity compared to pure CSH.10% nHAC was carefully screened for clinical practice.2. The in vitro cell culture test showed that the composite cement have no significant cytotoxicity could benefit cell proliferation and adhesion compared with pure CSH cement.3. Furthermore, the in vivo results mean that nHAC/CSH composite have better biocompatibility and a higher bone remodeling activity than that of CSH, especially at the early stage of remodeling. Hence, nHAC/CSH composite cement can be further researched as injectable scaffold for bone regeneration.