The Biocompatibility Of Nacre Artificial Bone And Application In The Spinal Fusion

Objective1. To evaluate the biocompatibility of nacre.2. To observe and analysis the effect of nacre composite artificial bone on biological behavior of human osteoblast in vitro.3. To study the osteogenic effect and biodegradation of nacre artificial bone in a canine posterolateral lumbar fusion modele.Methods1. The iliac bone of human were treated with collagenase-trypsin, a lot of osteoblasts were released out. Then the osteoblasts were purified and cultured. Subsequently, the 3rd generation cultured human osteoblasts were inoculated with the extract of nacre and rubber. The appearance of osteoblast was observed under the phase contrast microscope. Microstructure of the osteoblast was observed under transmission electron microscope. Additionally, to determine whether these materials affect cell proliferation, the numbers of BrdU-labeled cells were examined at these two groups. MTT assay was tested to observe the viability of human osteoblasts.2. Human osteoblasts were inoculated with the extract of the nacre polylactide artificial bone. Neutral red assay was tested to observe the viability of human osteoblasts, and alkaline phosphatase (ALP) activity and protein content were detected by spectrophotometric methods respectively.3. Osteoblasts were incubated with nacre composite artificial bone. Theappearance of human osteoblasts was observed under the phase contrast microscope, adhesion of the cell on the scaffold was observed under Scanning Electron microscopy. Microstructure of the osteoblast was observed under transmission electron microscope.The flow cytomety were used to observe the effect on the proliferation of osteoblasts.4. Human osteoblasts isolated from iliac bone were incubated with nacre composite artificial bone and polylactic acid. The appearance of human osteoblasts was observed under the phase contrast microscope.The activity of alkaline phosphatase and the formation of mineralized nodules were identified by histomorphometric analysis.5. Twelve adult canines underwent intertransverse process L4-51umbar fusion with nacre artificial bone, and decortication of the transverse processes of L2 and L3 was performed as control.Results1. Under the phase contrast microscope, the appearance of the osteoblasts in the nacre groups was spindle or triangle like, and black nodes were seen in confluent cell layer 48 hours after cultured. The observation of electron microscopy showed that the nucleus of the osteoblasts in the nacre groups was big and round, and there were a large number of mitochondrion and rough endoplasmic reticulum. Stereological analyses of the numbers of BrdU-labeled cells revealed that. Nacre has no harmful effect to proliferation of human osteoblast.2. Neutral red assay shown the nacre artificial bone had no harmful effect to viability of human osteoblasts. The OD values for tests (protein content and ALP activity) were similar with the negative control.3. Under the Scanning Electron microscopy, the artificial bone skeleton was composed of crystals and irregular pores and micropores, and the morphology of an adhered differentiation cell after incubation was a flat cell with extending pseudopodia. The observation of transmissionelectron microscopy showed that the nucleus of the osteoblasts were big and round, and there were a large number of mitochondrion and rough endoplasmic reticulum within the oseoblasts.4. There was significant increase of ALP positive cells in the experimental group, and a large number of mineralized nodules were identified 21 days after subculture in the experimental group and only a few in the control group.5. At 18 weeks after surgery, continuous of trabecular bone between the transverse processes were observed at the plain radiographs and CT scans confirmed the presence of complete bone growth into the nacre blocks from the transverse processes, and it was considered fused. The histological findings demonstrated that the specimen had c...