Craniofacial Bone Regeneration Using a Novel Nano-Structured Calcium Phosphate Carrier
There has been significant research in the development of polymer and ceramic cement based scaffolds for bone tissue engineering. However, most of these systems are not amenable for in situ incorporation of cells, growth factors and/or biological systems.
The objective of this study to determine the efficacy of novel nano-structured Calcium Phosphate (CaP) based cements containing nano-sized CaP nanoparticles (NanoCaPs), as carriers, with or without BMP-2 combined with an organic phase to enhance bone regeneration in an experimentally induced critical sized bone defect.
A novel aspect of the cement is the generation of a moldable putty like structure that would set within minutes containing pore formers as well as nanosized particulates as delivery agents of proteins, growth factors and plasmids under physiological conditions.
METHODS
Novel calcium phosphate cements were prepared containing nano-sized calcium phosphate (NanoCaP) particulates that serve as carriers of growth factors and proteins. The NanoCaP nanoparticulate solutions were pre-fabricated by mixing Ca precursors containing BMP-2 with phosphate precursors to form the nano-sized particles containing BMP-2 for in vivo use.
Twenty-four skeletally mature adult New Zealand White rabbits were used. Following induction to general anesthesia, a critical sized defect was created in each rabbit by removing 15mm of full thickness calvarial bone. In the osteotomy space the following groups were implanted: Group 1: Commercially available cement (Norian). Group 2: CaP cement+BMP-2. Group 3: CaP cement alone. Group 4: Control, empty. The periosteum was approximated and closed with interrupted 4-0 Vicryl (Ethicon, Somerville, NJ) suture. The skin was closed in a running fashion with 4-0 Vicryl suture. All animals received post-operative analgesics, three doses of antibiotic prophylaxis for infection and routine postoperative care. Eight weeks following surgery, all the rabbits were euthanatized. The skulls were removed, stripped of soft tissues, and prepared for analyses.
Animals were assessed at regular intervals at 2, 4, 6 and 8 weeks postoperatively with radiographs. At 8 weeks computed tomography (CT) analysis using a Scanco Medical AG μCT 40 system followed by histological assessment was carried out. The percentage areas of the calvarial critical sized bone defect occupied by newly formed bone were calculated.
Non-parametric tests were used and the significant level was considered at p<0.05.
RESULTS
The CaP cements containing NanoCaPs were fabricated under neutral pH show the formation of nano-structured hydroxyapatite (HA). The cements also show excellent cell attachment and cellular migration. Microstructural changes occuring in the cement resulting in the formation of nano-sized HA is an added indication of the likely faster resorption kinetics of the cement when implanted. Our results of the radiographical, micro-CT and histological assessment of the new regenerative bone showed that the addition of BMP-2 to the CaP-cement yielded higher bone regeneration compared to other groups. Subsequently the CaP-cement group also yielded higher regeneration potential than the control of organic matrix alone.
CONCLUSIONS
Based on our results the incorporation of BMP-2 bound to NanoCaP carriers in the CaP-cements does yield bone healing enhancement compared to controls. Our current on-going research delves into the optimization of resorbable cements that are amenable to the addition of growth factors.
REFERENCES
1. D. Olton et al., Nanostructured calcium phosphates (NanoCaPs) for non-viral gene delivery: influence of the synthesis parameters on transfection efficiency Biomaterials 28, 1267 (Feb, 2007).
2. P. Kumta, C. Sfeir, D.H. Lee, D. Olton and D. Choi, Nanostructured calcium phosphates for biomedical applications: novel synthesis and characterization, Acta Biomater 1 (2005) (1), pp. 65–83