An Experimental Study On Reconstruction Of Rabbit Cranial Critical-sized Bone Defect With N-HA/PA66 Combined With MSCs And BMP2

Many clinical conditions such as trauma, tumor resection and congenital deformities, can lead to bone defects in craniomaxillofacial region and the bone defects restoration pose great challenge to maxillofacial surgeons. More and more methods have been introduced for reconstruction of bone defects and these methods include autogenous bone graft, allogenous bone graft, exogenous bone graft, as well as distraction osteogenesis, an endogenous bone tissue engineering technique. Despite of above methods, bone tissue engineering has recently become one of important techniques to repair bone defects.Bone tissue engineering includes three key components, bone scaffold, bone forming cells and osteogenic growth factors. Nano-hydroxyapatite/ Polyamide66 (n--HA/PA66) is a newly synthesized bone scaffold among dozens of scaffolds used in bone tissue engineering and the material has better biological and physical characteristics when compared with artificial materials commonly used. Nano-HA/PA66 has been successfully used for reconstruction of bone defects in vertebrae and long bones. However, reparation of bone defects in craniomaxillofacial region with this scaffold remains far less investigated, especially the combination use of this scaffold with bone forming cells, such as bone marrow mesenchymal stem cells (MSCs) and osteogenic growth factors such as bone morphogenetic protein (BMP). Therefore, further studies are quite necessary to elucidate the effects of these materials on reconstruction of bone defects in craniomaxillofacial region.Objective: To explore the biological characteristics of MSCs cultured on n-HA/PA66 and to study the effects of n-HA/PA66 combined with MSCs and BMP2 on reconstruction of rabbit cranial critical-sized bone defects.Methods: (1) MSCs of passage 3 were cultured on scaffold n-HA/PA66 and underwent osteogenic differentiation. Cell proliferation and alkaline phosphonase (ALP) activity were detected to determine the biological characteristics of MSCs. Scanning electron microscope examination was also performed to observe cellular attachment of MSCs on n-HA/PA66. (2) Forty-five New Zealand rabbits were used in this study and n-HA/PA66 combined with MSCs and BMP2 was used to reconstruct critical-sized bone defects in rabbit cranium. The animals were randomly divided into five groups: group A (n-HA/PA66), group B (n-HA/PA66 +BMP2), group C (n-HA/PA66+MSCs), group D (n-HA/PA66+BMP2+ MSCs), and group E (control without scaffold). Animals were sacrificed at 4 and 12 weeks after grafting surgery and specimens were examined by histological and SEM observation.Results: (1) MSCs were cultured on n-HA/PA66 and examined for cell proliferation and ALP activity. The results indicated that the scaffold used did not affect cell proliferation and ALP activity, while osteogenic induction could decrease cell proliferation and ALP activity. SEM observation indicated that good attachment of MSCs on n-HA/PA66 was achieved.(2) At 4 weeks after reconstruction of bone defects with n-HA/PA66, new bone was formed in all four groups at the border of original cranium and scaffold, surface of scaffold and inside of scaffold. However, new bone was less and much connective tissue was observed in the bone grafting area. For comparison, scaffold-bone contact rate (SBCR) at the border and new bone volume (NBV) at the implant area were highest in group D (p<0.01) when compared with other groups, followed by group B and C, and lowest in group A (p<0.01). Surface new bone volume (SNBV) at the inner side of scaffold was also highest in group D (p<0.01), followed by group B and C, and lowest in group A (p<0.01). SNBV was higher in group B than group C. At 12 weeks after bone reconstruction with n-HA/PA66, much more bone was formed at the grafting area when compared with 4 weeks and new bone had been mature trabecular bone. For comparison, SBCR,NBV,SNBV were all highest in group D and lowest in group A (p<0.01), while the above indices were much higher in group B than group C.Under SEM observation, much new trabecular bone was found at the border in all of four groups. However, much more new bone was found in group D and the new bone contacted directly with scaffold and original cranium. New bone was less in group A and C and some connective tissue could be found at the border. While new bone formation in group B was less than group D but much more than group A and C.Conclusion: n-HA/PA66 does not affect prliferation, differentiation and attachment of MSCs and therefore it is a good scaffold for bone tissue engineering. Combination of n-HA/PA66 with MSCs and BMP2 can effectively promote bone formation during reconstruction of bone defects and simultaneous application of MSCs and BMP2 is better than their single use.