Influence of bone formation by composite scaffolds with different proportions of hydroxyapatite and collagen

Composite scaffolds with different proportions of hydroxyapatite (HA) and collagen (COL) produced different bone induction results.

To examine the composite scaffolds with optimal proportion of HA and COL to achieve earlier bone induction and maximum bone formation.

Composite scaffolds with the HA/COL weight ratio of 7:3, 3:7, 5:5 and 9:1 were prepared, as HA powder was added to collagen solution at 130℃ for 48 h. Then, the composites with different proportions of HA/COL were implanted into the extraction socket of right upper central incisor of C57BL/6 J mice. The bone formation of the extraction socket was observed by Hematoxylin-eosin (HE) and Masson-trichrome (Masson) staining at 1 and 2 weeks after operation. Five weeks later, the bone formation of extraction socket was observed by micro computed tomography (micro-CT). After MC3T3-E1 cells were co-cultured with materials of different proportions for 3 days, the number of cells attached on the surface of the materials and entering the materials were counted, and the expression of osteogenic related genes (Runx2, Ocn. Osx and Alp) was detected by reverse transcription polymerase chain reaction (RT-PCR). The composite scaffolds with different proportion of HA/COL with and without mouse bone marrow mesenchymal stem cells (BMMSCs) were implanted into the back of adult mice and cultured subcutaneously for 30 days, and observed histologically by HE and Masson staining.

After one week implantation with the composite HA/COL scaffolds with the weight ratio of 7:3, 3:7, 5:5 and 9:1, there was no new bone formation in the extraction socket in mouse. However, two weeks later, new bone was firstly observed in the tooth socket with the composite HA/COL scaffolds of 7:3. 5 weeks later, micro-CT scanning showed that the total amount of newly formed bone, trabecular width and bone mineral density of the HA/COL scaffolds of 7:3 were higher than the other HA/COL scaffolds (P < 0.05). After MC3T3-E1 cells were co-cultured with different composite HA/COL scaffolds for 3 days. The number of cells on the surface and inside of the HA/COL scaffolds of 7:3 was more than the other materials, and the difference was statistically significant (P < 0.05). The expression levels of Ocn and Osx of MC3T3-E1 cells were also the highest in the HA/COL scaffolds of 7:3 (P < 0.01). Bone formation was observed in the composite HA/COL scaffold of 7:3 with BMMSCs subcutaneously in mouse for 30 days, while only osteoid formation was observed in the same scaffold without BMMSCs. but bone formation was not detected in the other proportions of the HA/COL scaffolds.

Compared with other proportions of HA/COL, the composite HA/COL scaffolds of 7:3 has stronger ability to promote bone formation, recruit osteoblasts to attach and enter into the scaffolds, and promote the osteogenesis of BMMSCs.