Abstract: BACKGROUND: Bone tissue loss caused by tumors and trauma can have an adverse effect on postoperative rehabilitation. Therefore, scaffold materials are usually implanted during treatment. However, the existing implant materials are relatively simple and lack antibacterial properties. Early implantation may lead to iatrogenic autoinfection and have an adverse effect on osteogenesis.
OBJECTIVE: To construct a KR-12-a5 polypeptide-nanohydroxyapatite-small intestinal submucosa composite scaffold and evaluate its feasibility as a material for promoting bone defect repair.
METHODS: The small intestinal submucosa scaffold and the small intestinal submucosa scaffold containing 25, 50, and 100 mg/mL nanohydroxyapatite (referred to as nHA-SIS scaffold) were prepared by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide cross-linking method. The appropriate scaffold was screened for subsequent experiments by mechanical property testing. The antibacterial properties of KR-12-a5 polypeptide solution against Staphylococcus aureus, Streptococcus gordonii, and Fusobacterium nucleatum were detected. The nHA-SIS scaffolds were immersed in 250, 500, and 1 000 μg/mL KR-12-a5 peptide solutions for 24 hours, and then freeze-dried to obtain peptide-loaded nanohydroxyapatite-porcine small intestinal submucosa composite scaffolds (denoted as P-nHA-SIS scaffolds). The sustained-release properties of the three groups of scaffolds were characterized. The nHA-SIS scaffolds and the three groups of P-nHA-SIS scaffolds were co-cultured with Staphylococcus aureus, Streptococcus gordonii, and Fusobacterium nucleatum for 24 hours or 48 hours. The scaffolds with strong antibacterial ability were screened by live and dead bacteria staining and scanning electron microscopy for subsequent experiments. The degradation properties and water absorption rates of the uncross-linked small intestinal submucosa scaffolds, cross-linked small intestinal submucosa scaffolds, nHA-SIS scaffolds, and P-nHA-SIS scaffolds were characterized. The extracts of cross-linked small intestinal submucosal scaffolds, nHA-SIS scaffolds, and P-nHA-SIS scaffolds were co-cultured with MC3T3-E1 cells. CCK-8 assay and live-dead cell staining were performed. The effects of the extracts of the three scaffolds on the migration of MC3T3-E1 cells were detected by Transwell chamber assay. 
RESULTS AND CONCLUSION: (1) The elastic modulus and compressive strength of 25, 50, and 100 mg/mL nHA-SIS scaffolds were higher than those of small intestinal submucosal scaffolds (P < 0.05), among which the elastic modulus and compressive strength of 25 mg/mL nHA-SIS scaffolds were the highest, and this group of scaffolds were selected for subsequent experiments to load peptides. (2) KR-12-a5 peptide had strong antibacterial activity against common bacteria in bone defects (Staphylococcus aureus, Streptococcus gordonii, and Fusobacterium nucleatum). The three groups of P-nHA-SIS scaffolds all had sustained release properties. With the increase of peptide mass concentration, the antibacterial property of P-nHA-SIS scaffold was enhanced. Among them, the P-nHA-SIS scaffold loaded with 500 μg/mL peptide had achieved a satisfactory antibacterial effect, and this group of scaffolds would be selected in the future. (3) The degradation rate of the three groups of cross-linked scaffolds was lower than that of the uncross-linked scaffolds, and the water absorption rate was greater than that of the uncross-linked scaffolds. P-nHA-SIS scaffolds could promote the proliferation and migration of MC3T3-E1 cells without affecting the activity of MC3T3-E1 cells. (4) The results show that P-nHA-SIS scaffolds have strong antibacterial properties and the ability to promote the proliferation and migration of MC3T3-E1 cells, and are expected to be used in bone defect repair.

Key words: antimicrobial peptide, small intestinal submucosal scaffold, nanohydroxyapatite, bone tissue engineering, three-dimensional scaffold, surface modification, scaffold modification, tissue engineered bone material