[A research on ectopic osteogenesis and vascularization of tissue engineered bone promoted by 1,25-(OH)2D3]

To study the ectopic osteogenesis and vascularization of the tissue engineered bone promoted by an artificial bone composite that consists of coral hydroxyapatite (CHA), 1,25-(OH)2D3, human marrow stromal osteoblast (hMSO), and human umbilical vein endothelial cell (hUVEC).

After the isolation and the culture in vitro, hMSO and hUVEC were obtained. Then, hMSO (5 x 10(5)/ml) and hUVEC (2.5 x 10(5)/ml) were seeded at a ratio of 2 : 1 onto the CHA scaffolds coated with 1,25-(OH)2D3 (the experimental group) or onto the CHA scaffolds without 1,25-(OH)2D3 (the control group). The scaffolds were cultured in vitro for 3 days, and then the scaffolds were implanted into the pockets that had been made on the backs of 18 nude mice. Then, 6 of the mice were implanted with one experimental engineered bone bilaterally; another 6 mice were implanted with one control engineered bone bilaterally; the remaining 6 mice were implanted with one experimental engineered bone and one control engineered bone on each side. At 4, 8 and 12 weeks after operation, the retrieved scaffolds and cells were examined by the nake eye and histology as well as by the scanning electron microscopy. The quantitative assessment of the newly-formed bone and the quantitative analysis of the newly-formed blood vessels were performed.

The evaluations by the histology revealed that at 4 weeks the original bone tissues grew into the scaffolds in all the groups, but significantly more newly-formed bone tissues and newly-formed blood vessels were found in the experimental group. At 12 weeks the newly-formed bone tissues were found in all the groups, but there was a typical bone unit found in the experimental group. There was a significantly smaller amount of capillary vessels in the control group than in the experimental group at all the time points. The evaluations by the scanning electron microscopy revealed that at 4 weeks in the experimental group there were great amounts of extracelluar matrix that embedded the cells, and plenty of capillary vessels were found on the surface of the implanted bone materials and some of them grew into the materials; however, in the control group there was a smaller amount of capillary vessels although much extracelluar matrix was still found there. At 8 weeks sarciniform osteoids were found on some of the implanted materials, with much extracelluar matrix and many newly-formed capillary vessels in the experimental group; however, in the control group there were fewer capillary vessels and lower degrees of the bone maturity. The quantitative assessment of the newly-formed bone showed that the new-formed bones were 3.1 +/- 0.52 in the experimental group but 2.30 +/- 0.59 in the control group at 8 weeks (P < 0.05), and 4.63 +/- 0.55 vs. 3.53 +/- 0.62 at 12 weeks. There was a significant difference at these two time points between the two groups (P < 0.05). The quantitative analysis of the newly-formed blood vessels showed that the vascular areas were 28.74% +/- 7.81% in the experimental group but 19.52% +/- 4.57% in the control group at 4 weeks (P < 0.05), and 24.66% +/- 7.38% vs. 17.84% +/- 5.22% at 12 weeks. There was a significant difference at these two time points between the two groups (P < 0.05).

1,25-(OH)2D3 as an active factor can increase the interaction between hMSO and hUVEC, and thus promote the ectopic osteogenesis and vascularization in the tissue engineered bone.