A novel antibacterial bone graft substitute was developed to repair bone defects and to inhibit related infections simultaneously. This bone composite was prepared by introducing vancomycin (VCM) to nanohydroxyapatite/collagen/calcium sulphate hemihydrate (nHAC/CSH). XRD, SEM, and CCK-8 tests were used to characterize the structure and morphology and to investigate the adhesion and proliferation of murine osteoblastic MC3T3-E1 cell on VCM/nHAC/CSH composite. The effectiveness in restoring infectious bone defects was evaluated
Bone defect-related infections especially chronic osteomyelitis are quite common in open fracture and trauma in clinical treatment, which continues to be very difficult to treat and brings challenges to clinicians. It is difficult to make effective bone repair and inhibit infection at the same time. Vancomycin hydrochloride (VCM) is an antibiotic drug that is specifically used against
Therefore, it is critical to select an appropriate scaffold for constructing drug delivery system, in which the scaffold should be biocompatible, osteogenic, operable, biodegradable, and antibacterial. Primarily, the scaffold should possess suitable ingredients and structure for cell attachment, proliferation, and osteogenic differentiation. Li et al. did some related studies [
PMMA is often incorporated with antibiotics such as gentamicin as one of the most widely used bone materials in clinical applications. However, the inherent biological inertia of PMMA leads to poor osseointegration between bone tissue and cement interface, apart from other shortcomings such as nonabsorbability, impermeability to antibiotic, monomer toxicity, and high polymerization temperature [
Calcium sulfate hemihydrate (CSH) itself has a long clinical history as a bone graft substitute, known for its bioresorption, satisfactory handling properties, or self-setting ability
To optimize the performance of bone regeneration of CSH, some previous studies in our group suggested that nanohydroxyapatite/collagen (nHAC) could be incorporated, which was prepared from mineralizing type I collagen with excellent osteoconductive properties, and was thought to be a new scaffold material for its high similarity of natural bone both in composition and in hierarchical nanostructure in bone tissue engineering [
The aim of the present study was to investigate nHAC/CSH as a carrier material for vancomycin in the treatment of chronic osteomyelitis to repair bone defects and inhibit related infections simultaneously. An
The powders of nHAC and CSH were prepared as previously described. In brief, nHAC were obtained by precipitation of Ca2+ and
The composition of materials was characterized by X-ray diffraction (XRD, D/max-2500X) using monochromated CuK
Composite samples were sputter-coated with gold film for scanning electron microscopy (SEM, Quanta 200 FEG, Netherlands) examinations at the voltage of 20 kV.
In order to evaluate the biocompatibility of cement, cell counting kit-8 (CCK-8) method was used to quantitatively evaluate the proliferation of murine osteoblastic MC3T3-E1 (a clonal osteogenic cell line derived from newborn mouse calvarias, which is often used in bone tissue engineering research). Cell growth and adhesion behaviors on the scaffolds surfaces were examined by SEM observation. MC3T3-E1 cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 50
The animal experiments were carried on by the approval of the Ethics Committee of the General Hospital of People’s Liberation Army. The
After surgery, rabbit femoris in defect regions were extracted and fixed in 4% paraformaldehyde and then decalcified in EDTA and dehydrated in ethanol before they were embedded in paraffin. Sections were prepared and stained with hematoxylin and eosin and Masson’s trichrome. Histomorphometry was carried out using a light microscope (BX51, Olympus) under 40x magnification. For the evaluation of fibrosis, picrosirius red staining was performed using 0.1% picrosirius red solution. Micro-computed tomography was used to observe the bone reconstitution.
All the data were statistically analyzed using SPSS 13.0 software and expressed as the standard deviation of the mean. The
The conversion of CSD phase to
SEM micrographs of (a) nHAC, (b) CSH crystal, and (c) CSD crystal and XRD patterns of (d)(A) CSD, (d)(B) CSH, (d)(C) nHAC, (d)(D) compound of VCM/nHAC/CSH, and (d)(E) set cement of VCM/nHAC/CSH.
Also the characteristic peaks of nHAC located at 25.91°, 31.82°, 32.20°, 32.93°, and 34.10° and correlated with crystal planes of 002, 211, 112, 300, and 202 (Figure
In our previous study, it was demonstrated that final setting time was about 15~20 min. The porosity of the scaffold was 38.8% and the compressive mechanical strength was about 4.8 MPa, which was more than the lower limit of natural cancellous bone (1 MPa) [
In order to evaluate the cell toxicity of VCM, CCK-8 method was used to measure the proliferation of MC3T3-E1 cells on the scaffold surfaces. Figure
(a) Cells proliferation on the nHAC/CSH and VCM/nHAC/CSH scaffolds evaluated by CCK-8 assay and SEM micrographs of cells on (b) VCM/nHAC/CSH and (c) nHAC/CSH.
In the previous study, the materials were shown to be
Picrosirius red staining is one of the best techniques of collagen histochemistry. Sirius red enhances the birefringence in oriented collagens as it attaches to collagens in parallel; then we can see the red-orange colored light from sirius red stained collagens in polarized light microscopy. As the enhancement of birefringence is limited in fibrous tissues, this method can be used to identify fibrous collagen. In this study, the tissues were fixed in a solution of 10% neutral buffered formalin, embedded in paraffin, sectioned at a thickness of 5
Histological cross sections (4
The results also were confirmed by the micro-computed tomography graphs of implanted materials on the antibacterial bone defect as shown in Figure
Micro-CT graphs taken 12 weeks after focal debridement. (a) Cross section position (red line), (b) normal bone, (c) nHAC/CSH group, and (d) VCM/nHAC/CSH group.
All results above suggested that the inflammation may actually inhibit the growth of bone tissue in nHAC/CSH group, and the treatment of inflammation along with bone repair was effective in VCM/nHAC/CSH group.
The best treatment of contaminated or infected bone defects, such as chronic osteomyelitis, is to control infection and repair bone defect at the same time. This requires an osteoinductive bone graft composite with ideal release antibiotic capabilities, mechanical properties, and other related properties. In this study, the nHAC/CSH was used as a carrier of vancomycin (VCM) for the treatment of osteomyelitis, and the VCM/nHAC/CSH composite has ideal self-setting, antibacterial, porous, degradable, good mechanical properties, and better osteogenic activity. So the materials can act not only as void filler facilitating tissue regeneration but also as carrier for inhibiting infection in the healing process.
The bioactivity of the scaffold was determined by the components such as CSH and nHAC, of which CSH as the main ingredient has been used in bone augmentation for many years in virtue of its self-setting ability
Since the 1980s, vancomycin has become the first choice for treating refractory hip infections because of its high efficacy against gentamicin-resistant bacteria, its rare bacterial resistance, and its low incidence of side effect [
In
In this study, we first used the VCM/nHAC/CSH bone substitute as a degradable local antibiotic delivery system for the treatment of chronic osteomyelitis. The implants were successful as vancomycin carriers in inhibiting infection in rabbits osteomyelitis mode during the course of this study. Moreover, the implants performed excellent biocompatibility. Our results suggested that the implants can be considered a new system for local vancomycin delivery and the effectiveness of VCM/nHAC/CSH bone substitute in the treatment of
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work is in part supported by the Natural Science Foundation for Young Scientists of Shanxi Province (no. 2014021039-6), the Qualified Personnel Foundation of Taiyuan University of Technology (QPFT) (no. tyut-rc201270a), the Youth Foundation of Taiyuan University of Technology (nos. 1205-04020102, 2013Z020, and 2014TD066), the National High-Tech Program (no. 2011AA030105), the Technical Services Project of Taiyuan University of Technology (no. 143230043-J), the National Science Foundation of China (nos. 50830102, 51303119, 21161003, and 20701010), MOST of China (no. 2011DFA31430), Grants of Science and Technology of Guangdong Province (no. 2010B031500005), and the Natural Science Foundation of Jiangsu Province (BK20130309).