The Effects Of Osteoblasts On The Three Dimensional Constructions Of Type I Collagen And Self-Assembled Of Collagen

Collagen, which is synthesized and secreted by osteoblast, can be assembled to theembryonic form of bone by bone cells. Then step by step the inorganic salts (such ashydroxylapatite) are deposited in the embryonic bone. Ultimately, the bone is forming.The designing and manufacturing processes of bone-tissue-engineering materials, whichwere used as bone substitude, will achieve new breakthrough, if the above-mentionedprocesses could reappear in vitro. This new idea about mimic manufacture deserve to bestudyIn this paper, the interactions between type I collagen and osteoblast were studiedfor demostrating the possibility of reappearing the bone developing process in vitro. Tryto study whether cell could build the tissue just using basic materials.In this work, type I collagen which was extracted from the tail of SD rat dissolvesin acidic solution. After co-culturing type I collagen and osteoblast, the pattern, theproliferation and the Alkline phosphatase(ALP) activity of osteoblast were determined.the results were used to evaluate the ability of type I collagen affecting on osteoblast. Atthe same time, the three dimensional constructions of collagen, which were used toevaluate the ability of osteoblast affecting on type I collagen, were determined bystaining techniques, scanning electron microscope (SEM) and Micro-ComputerizedTomography (Micro-CT).The main works and conclusions are included as follows:①extracting, purifying and identifying of type I collagen: collagen was extracted andpurifyed by Schor method, identifyed by Fourier infrared spectroscopic analysis,sodium dodecyl sulfate polyacrylamide gel electropheresis(SDS-PAGE), HEstaining and Masson staining. The results showed that the extractive object has thesame molecular structure and molecular weight with Rat tail tendon collagen Type Ifrom Shengyou Biotechnology Co Ltd. The result of HE staining and Massonstaining also provided evidence to demostrate the object is type I collagen.②cell culture and identification: osteoblast were obtained from neonatal Wistar rats bytissue blocks culture method. After cultured for a few days in vitro, the cellspresented columnar or cubical shape. The result of alizarin red staining showed thatthe cell had characteristics of osteoblast, which means that the cells could be usedin the following experiments. ③the effection on osteoblast’ shape, proliferation and physiological function caused bydifferent three dimensional construction of type I collagen at differentconcentrations: the three dimensional construction of8different concentrations(including0.5mg/ml,1.0mg/ml,1.5mg/ml,2.0mg/ml,2.5mg/ml,3.0mg/ml,5.0mg/ml,8.0mg/ml) were characterized by SEM. The SEM results showed thatthere were difference among the three dimensional constructions of collagen atdifferent concentrations. Although it did not changed in a linear fashion, the overalltrend showed that the diameter of inner pores of collagen would be reduced and thenumber of pores per unit area would be increased with the increase of collagenconcentration. the cell shapes, proliferation and ALP activity were detected at8groups of collagen at different concentrations by DAPI staining, MTT and ALP kit.Osteoblasts were seeding into collagen and co-cultured, then cell proliferation wasdetermined by MTT assay at2,4,6,8th day. And a growth curve was drawn, whichshowed that the proliferation power and growth curves were different at differentcollagen concentrations. Cells in8concentration groups all possessed proliferationpower. The power at lower collagen concentrations was notablely stronger than athigher collagen concentrations. ALP activity was detected at2,4,6th day by ALPkit. The result showed that All8groups appeared high ALP activity. ALP activity atlower collagen concentrations was notablely stronger than at higher collagenconcentrations. All the physiological function contacted with the results of SEMwould reveal the relationship between osteoblast physiological function (includingcell proliferation, ALP activity) and the three dimensional constructions of collagenat different concentrations. Osteoblast performance better physiological function onthe surface of smaller inner pores of collagen and less number of pores per unit areaat lower collagen concentrations than the surface of larger inner pores of collagenand more number of pores per unit area at higher collagen concentrations.④The change on the three dimensional constructions of collagen caused by osteoblast:Osteoblasts were seeding into collagen and co-cultured2,4,6days. Then usingDAPI and HE staining, Micro-CT and SEM to detect the change on the threedimensional constructions of collagen cuased by osteoblast. Control group, whichjust contained collagen at the same concentration with the experimental group, wassetted-up to evaluate the effect of osteoblast. The results showed that: withinosteoblasts, the inner pores of collagen observably shrinked, at the same time amass of deposited matter appeared on the surface which implied that the three dimensional constructions of collagen had a remarkable change. The network ofcollagen appeared to be ordered array nearby osteoblast, which did not happened incontrol group.