Objectives: Bone grafts with both antibacterial and osteoinductive abilities are greatly needed for treatment of infected bone defects. In this study we developed such a bone graft with a sequential release system: burst release of the powerful antimicrobial agent, hydroxypropyltrimethylammonium chloride chitosan (HACC), followed by sustained release of the osteoinductive agent, BMP-2. Our hypothesis was that this would kill infection-associated bacteria and eventually induce osteogenesis to repair an infected bone defect. Methods: The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time–kill curve of HACC against methicillin-resistant Staphylococcus aureus (MRSA) were evaluated. The influence of HACC at concentrations of 1000, 200 or 40μg/mL on the proliferation of pre-osteoblasts was evaluated by measuring cell viability. The influence of 40μg/mL HACC on the BMP-2-induced differentiation of pre-osteoblasts was evaluated by assessing activity of alkaline phosphatase (ALP) , osteocalcin (OCN) expression and mineralisation. BMP-2 was first internally incorporated into biomimetic calcium phosphate granules to form BMP-2-BioCaP. HACC solution was then adsorbed onto its surface to obtain a BMP-2-BioCaP/HACC complex. Confocal laser scanning microscopy (CLSM) was used to observe the structure of BMP-2-BioCaP/HACC using FITC-BSA as a substitute for BMP-2 and staining HACC solution with rhodamine B. The release kinetics of FITC-BSA and rhodamine B were determined by quantitative spectrophotometric analysis. To evaluate osteoinductivity of the complex and to optimise the HACC concentration in vivo, the following groups (n=6) were randomly implanted into subcutaneous pockets in rats: (i) BMP-2-BioCaP/HACC complex (BMP-2-BioCaP/HACC-20, BMP-2-BioCaP/HACC-4 and BMP-2-BioCaP/HACC-0.8 according to content of 20, 4 or 0.8μg HACC); (ii) BMP-2-BioCaP; (iii)BioCaP/HACC; (iv) BioCaP. Five weeks after implantation, samples were retrieved for histological and histomorphometric analysis. Results: The MIC and MBC of HACC for MRSA were 40μg/mL. BioCaP/HACC completely eliminated MRSA within 24 hours. HACC at levels of 1000μg/mL and 200μg/mL (but not 40μg/mL) significantly suppressed cell viability. HACC at a level of 40μg/mL did not significantly influence BMP-2-induced ALP, OCN or mineralisation. Microscopy showed that BMP-2 homogenously distributed throughout BMP-2-BioCaP granules within BMP-2-BioCaP/HACC complex, forming a thin layer of HACC along the surface. The in vitro release profile showed rapid release of HACC, which was completely exhausted after 3 days; BMP-2 was gradually and slowly released, with only about 20% depletion after 30 days. Bone formation was observed only in the BMP-2-containing groups in the in vivo pro-fibrotic environment (subcutaneous sites). In comparison with the positive control group (BMP-2-BioCaP), BMP-2-BioCaP/HACC-4 and BMP-2-BioCaP/HACC-0.8 complexes resulted in similar levels of new bone formation, while BMP-2-BioCaP/HACC-20 was associated with significantly less new bone formation. Conclusions: BMP-2-BioCaP/HACC-4 complex can rapidly eliminate antibiotic-resistant bacteria and efficiently promote new bone formation both in vitro and in vivo – making this a promising material for repairing infected bone defects. HACC is a strong antibiotic that can be readily combined with other bone substitutes, such as Bio-OssTM/® to provide in situ antibacterial activity for bone regeneration associated with infection.

Overview: Part 1 - Incision and mobilization of the mucoperiosteal flap - Cleaning the bony defect - Screw insertion for support, membrane application and fixation - Suture closure Part 2 - Opening of the mucosa above the region of augmented bone - Preparation of tunnel and bone bed for an open screw-type endosteal implant (Straumann® implant) - Use of single button sutures for tension-free wound closure Contents: This female patient presented with bone loss due to a radicular cyst at tooth 27 and peri-implantitis at tooth 26. Four months after extraction of the respective tooth and implant, guided bone regeneration (GBR) was carried out using supporting screws and an e-PTFE membrane for augmentation of the posterior tooth region of the maxilla. Four months after GBR the insertion of an implant took place.