Purification Of Low Molecular Weight Porcine Amelogenin And Their Effects On The Dental Mesenchymal Cells

Amelogenin, being secreted by the inner enamel epithelium (ameloblasts), as the major componentsof enamel matrix proteins, play specific functional roles in matrix mediated enamel biomineralization and dental root development. In 1999, the Food and Drug Administration (FDA) approved Emdogain for use as an adjunct to a minimally invasive surgical technique. The FDA approval indicated that Emdogain could be utilized in conjunction with scaling and root planing for the treatment of periodontal intrabony defects. In recent years, it was reported that Emdogain promoted the proliferation of many kinds of dental mesenchymal cells. Emdogain increased the osteogenic effect, mineralized nodule formation and alkaline phosphatase (ALP) capacity of rat bone marrow cells, meanwhile increased up to two-fold both their number and the amount of gingival fibroblasts matrix produced. Emdogain enhanced the formation of both reparative dentine and dentine bridges during wound healing of amputated rat molar pulp. It is likely that EMD stimulated angiogenesis directly by stimulating endothelial cells and indirectly by stimulating the production of angiogenic factors vascular endothelial cell (VEGF) by periodontal ligament cells (PDLCs). Importantly, the data are consistent with the concept that EMD enhanced bidirectional communication between human microvascular endothelial cells (HMVECs) and PDLCs during angiogenesis associated with healing. Enamel matrix derivative limits the release of pro-inflammatory cytokines induced by lipopolysaccharide or peptidoglycan in human blood, suggesting that it also has anti-inflammatory properties.In recent years, a number of studies focused on the enamel matrix proteins (EMPs), however, the extraction of amelogenin and its further function are rarely reported, where reachers did not obtain the high purity and active low molecular weight porcine amelogenin. So we developed current extraction technology, low molecular weight porcine amelogenin (LMWPA) proteins were extracted from pig tooth germ enamel powder by Liquid Chromatography method. SDS-PAGE was used to determined the protein molecular. We used Edman degradation technology to analyze the N-terminal region, and then, we used electronmicroscope to detect the form of LMWPA. After which, we observed the effects of LMWPA on cell proliferation and ALP activity activity of HDPCs, HPLCs and RDPCs. Meanwhile, the biomineralization of the protein as also been studied. Finally, tooth papilla of first molar germs of postnatal 4 days SD rat were combined with LMWPA gel. The reconstructions were implanted in renal capsules of adult SD rat respectively. After 2 weeks the implanted tissues were harvested. The formation of hard tissue and differentiation of dental epithelia were analyzed histologically. The detailed methods and results of each experiment are:1.Extraction of LMWPA, measuring of molecular weight and N-terminal region amino acid determination.Tooth germs of permanent molars were surgically extracted with a hammer and chisel from the upper and lower jaws of 6-month-old pigs, within minutes of each animal's termination. The enamel organ epithelia and dental pulp tissue were removed and the hard tissue was quickly frozen in liquid nitrogen. As much dental enamel as possible was removed by scraping with a curette. The alkaline enamel extract was applied to a Q Sepharose Fast Flow column(ion exchange column), collected the chromatographic peaks and centrifuge them, then chromatographic peaks were applied to a Sephacryl S-100HP Column (size exclusion column). The protein we got showed typical molecular weight distribution of amelogenin through SDS-PAGE. The collected weight distribution concentrated in 5.0KD. The amino acid analyses and Edman degradations were performed at the Academy of Military Medical Sciences. N-terminal region of the protein was Met,Pro,Leu,Pro,Pro,His,Pro,Gly,His and Pro, and thus can be concluded it was amelogenin.2.The ultrastructure of LMWPA.Transmission electron microscope(TEM) studied 100μg/ml LMWPA, we observed that the protein which was purified by liquid chromatography technology from enamel matrix is uniform, like sphere structure, 50–100nm in diameter irregular. Scanning electron microscope(SEM) studied 1000μg/ml LMWPA, the figures seen on the ultra-structure is clear that fibrils grouped into rod structures, which are like the tree extend throughout the whole crystal, 100 nm in diameter.3.Bioactivity of LMWPA. MTT was used to observe the effects of LMWPA on cell proliferation activity of HDPCs , HPLCs and RDPCs in vitro. The results showed that 50～100μg/ml LMWPA significantly promoted the proliferation of HDPCs and HPLCs. As for RDPCs, in the first 3 days, 25,50,100μg/ml LMWPA significantly promoted the cells proliferation, compared to control group, the reaults had significant difference(p<0.05). The results showed that the LMWPA which we extracted has good bioactivity in certain concentration.4.The effects of LMWPA on ALP activity of HDPCs, HPLCs and RDPCs in vitro.The influence on the activity of ALP of HDPCs, HPLCs and RDPCs were evaluated by enzyme dynamics methods, in order to analyze the possible mechanism of promoting the mineralize ability of these cells by LMWPA. 25μg/ml LMWPA could increased the ALP activity of HDPCs and HPLCs in the first 5 days, later on this effects weakened, compared to control group, there was no significant difference(p>0.05). 50μg/ml and 100μg/ml LMWPA could up-regulate the ALP activity of HDPCs and HPLCs from first day to fifth day, this effects became greater, later on this effects weakened,≥150μg/ml LMWPA down-regulated the ALP activity of HDPCs and HPLCs. As for RDPCs, from third day to fifth day, 50μg/ml and 100μg/ml LMWPA increase the activity of ALP of the cells, compared to control group, the reaults had significant difference(p<0.05), however, five days later, the effects of 50μg/ml and 100μg/ml LMWPA weakened, other groups did not have any significant difference(P>0.05). This study demonstrated that LMWPA had good ability to promote cells mineralization in certain concentration.5.The influence on the forming mineralize tuber of RDPCs by LMWPA was evaluted by Von-Kossa. 50μg/ml LMWPA dissolved in 2% fetal bovine serum(FBS)DMEM, RDPCs were cultured in the solution. The results showed that LMWPA at this concentration was able to accelerate the forming mineralize tuber of the cells. This was not only owe to the ability of enhancing the mineralize ability of the cells, but also owed to the ability of promoting the proliferation of the cells.6.The study of LMWPA PGA gel combined with newly born rat papilla implantation in vivo.50μg/ml LMWPA and 6% PGA gel were made in vitro, which combined with the newly born 4 day rat first molar. We used the bioprotein to encapsulate reconstructions and implanted them in renal capsules of 6 weeks rat. After 2 weeks the implanted tissues were harvested. The formation of hard tissue was analyzed by HE and immunohistochemistry. The control group was the 6% PGA gel which did not contain LMWPA. After 2 weeks of transplantation, thin dentin structures could be seen in transplants, large amount bone like tissue formed.Immunohistochemistry results showed that Col-Ⅰwas positive in dentin in experimental group, while the control group was negative. These results demonstrated that 50μg/ml LMWPA combined with PGA gel could induce dental mesenchymal cells development and promote dentin formation.