Experimental Study On Human Umbilical Cord Mesenchymal Stem Cells In Combination With NHA/PA66 Material For Bone Tissue Engineering

The bone defect caused by trauma,infection,tumor and so on,are one of the common diseases.Damaged bone will spontaneously heal provided the defect is not over a critical limit,but when the defect is critical-sized,it will be filled by fibrous tissue because the osteoblast cells can not pass the gap between two broken bones.The clinic treatment of wide-boud bone defects is still hard to tackle at present.Autogenous bones,allogenic/allogenous bones,and artificial bone substitutes are now usually used as obturation materials for bone defects,which have their respective strong and weak points.Bone tissue engineering is a subject which applying engineering and medicine principles to repair and reconstruct bone defects.Recently,researchers are mainly focusing on constructing bioactive compounds with seed cells and biological scaffolds.According to the basic principle of bone tissue engineering, we would isolate and culture the human umbilical cord mesenchymal stem cells (hUCMSCs) and then induce cells ossification, Latterly we seed osteoblastic-induced hUCMSCs in nano-hydroxyapatite crystals and pnolyamide 66 (nHA/PA66) material to construct cells-scaffold composites, and then observe its ossification circumstances after raising this compound in nude mouse's body. Establish large animal model of bone tissue engineering and further satisfy treatment bone defection and damage of clinical demand in order to provide theories foundation.Chapter 1 Experimental study of the isolation?cultivation?proliferation and biological characteristics of human umbilical cord mesenchymal stem cellsObjective To investigate methods for the isolation, cultivation, proliferation and biological characteristics of human umbilical cord mesenchymal stem cells(hUCMSCs) in order to provide seed cells for bone tissue engineering.Methods The full-term umbilical cords from healthy neonate were collected and washed in D-Hank's balanced salt solution.The mesenchymal tissue was then diced into cubes of approximately 1mm3.We isolated and cultured MSCs derived from human umbilical cord in vitro by utilizing collagenase digestion method.We cultivated origin cells by the DMEM/F12 which contain the fetus cow blood-serum physical volume a score to 0.1. While needing a cell growth to 80-90% fusion,1:3 spread generation culture. We observed the characteristics of the cells by the inverted microscope, and measured the P3?P5 and P7 cells growth curve by MTT method. Flow cytometry examined the surface antigen of the P3 cell.Results In 24 hours a few adherent cells could be observed after cultivation. The primary hUCMSCs indicated spindle-shape or multangular shape after the second-3rd day while it could be observed cell division. Fibroblast-like cells manifold progressively and proliferate rapidly after the 5th-6th day.Cells fused more than 80%?90% after the 10th?12th day. The shapes of cells are homogeneous long spindle-shape after the second passage, and then it could be observed circinate-like in fusion time.Flow cytometry shows that 97%,95%,96% and 97% of the 3rd passage cells expressed CD105,CD29,CD44 and CD 13 respectively, while only 0.8%,1.8% and 1.2% of the cells expressed CD45,CD31 and HLA-DR respectively.The growth curve showed that proliferation ability of the 3rd,5th and 7th passage cells was powerful.Conclusion The method of isolation and culture of hUCMSCs was simple and feasible. The characteristics of cells obtained from human umbilical cords were similar to the mesenchymal stem cells and could be accorded as a kind of seed cells for tissue engineering.Chapter 2 Osteogenic differentiation of human umbilical cord mesenchymal stem cellsObjective To explore the osteogenic differentiation potential of hUCMSCs cultured with a specific medium in vitro and make preparations for the further research about bone tissue construction in vivo.Methods (1)Human umbilical cord mesenchymal stem cells were isolated and cultured according to chapter I. (2) The 3rd passage hUCMSCs were cultured in the high sugar DMEM which contained 10% fetus cow blood-serum, dexamethasone, antiscorbic acid,vitamin C and?-sodium glycerophosphate to be induces into osteoblast. Be observed the metamorphosis of cells by the inverted microscope and HE staining.The Alkaline Phophatase(ALP) staining was verified and the levels of ALP were measured.The formation of calcium nodules were confirmed by Alizarin red S staining. (3) The expression of osteopontin?osteocalcin and alkaline phosphates mRNA were examined by RT-PCR in 7 and 14 days. (4) The secretion of collagen type I were verified by the immunocytochemistry staining and Western blotting in 7 days.Results (1)After osteogenic induction,the cells became larger and polygonal, and could assemble themselves together.(2)The ALP staining appeared positive after osteogenic induction for 14 days.The levels of Alkaline Phophatase gradually became higher in 14 days.The Alizarin red S staining appeared positive,which verified the formation of calcium nodules,when osteoinducted 14 days. (3)The expression of OPN?OCN and ALP mRNA showed positive at 151bp?155bp and 182bp after osteogenic induction for 7 and 14 days. There were more expression for 14 days.(4)The secretion of collagen type I were positive with immunocytochemistry staining and the Western blotting after osteoinduction for 7 days.The Western blotting verified the expression of collagen type I in the experiment group were obviously higher than that of the control group(P<0.05)Conclusion The hUCMSCs could be osteoinduced to osteoblastic and stably expressed osteoblastic phenotype in vitro. The hUCMSCs could be used to construct bone tissue.Chapter 3 The study on biocompatibility of nano-hydroxyapatite crystals and polyamide 66 (nHA/PA66) material composite with human umbilical cord mesenchymal stem cells in vitroObjective To study the biocompatibility of nHA/PA66 material composite with hUCMSCs and explore the possibility of nHA/PA66 material as scaffold used in the carrier for hUCMSCs.To provide theoretical foundation for further experiments in bone tissue engineering.Methods (1) hUCMSCs were isolated and cultured according to chapter?.(2)The 3rd passage osteoblastic-induced hUCMSCs were seeded in nHA/PA66 material in vitro to construct cells-scaffold composites. We observed the compound situation of cells and the scaffold by the inverted microscope;tested the influence that the scaffold has upon cells multiplication differentiation by MTT.(3) The staining of Alkaline Phophatase(ALP) and collagen type I were verified. The levels of ALP were measured by MTT. (4) The adhesion ratio of nHA/PA66 scaffold composite with the 3rd passage osteoblastic-induced hUCMSCs were detected;The RGR in this scaffold were measured by MTT and counted the cytotoxicity of this material in 2?4?8d.(5) The expression of osteopontin?osteocalcin and alkaline phosphates mRNA were examined by RT-PCR in 7 days.(6) The growth and proliferation of hUCMSCs in this scaffold were observed by scanning electron microscope.Results (1)The 3rd passage osteoblastic-induced hUCMSCs had a good quality of growth, multiplication and differentiation on the surface of nHA/PA66 scaffold.The cellular activity and function were not affected by the materials,and no statistical difference were found between two groups(P>0.05).(2)After compound cultivation,the secretion of ALP and collagen type I were positive with immunocytochemistry staining. The levels of Alkaline Phophatase gradually became higher in 14 days. (3) The adhesion ratio of the 3rd passage osteoblastic-induced hUCMSCs were 69.1±2%?78.4±2%?82%±2% and 91%±2% in the nHA/PA66 scaffold at 4?8?12?24h. (4) The cytotoxicity of this scaffold to cells ranged from zero to one degree and the scaffold were all considered innocuity.(5)The expression of OPN?OCN and ALP mRNA in the experiment group showed positive at 151bp?155bp and 182bp after compound cultivation for 7 days. The RT-PCR verified the expression of OPN?OCN and ALP mRNA in the experiment group were obviously higher than that of the control group(P<0.05).(6)The osteoblastic-induced hUCMSCs adhered and grew on the surface of nHA/PA66 scaffold after one day, The cells extened and proliferation in the nHA/PA66 scaffold were observed by scanning electron microscope after 7 days.Conclusion The nHA/PA66 material could be a good scaffold for hUCMSCs in bone tissue engineering. It was an innocuity?better histocompatibility material and hadn't a negative impact on the growth?proliferation and osteoinduction of cells.It could satisfy the demand of bone tissue engineering.Chapter 4 The study on ectopic bone formation of nHA/PA66 material composite with human umbilical cord mesenchymal stem cellsObjective To explore the ability of ectopic bone formation of nHA/PA66 material composite with osteoblastic-induced hUCMSCs in nude mouse back and provide theories foundation for further large animal experiments of bone tissue engineering.Methods (1) The 3rd passage osteoblastic-induced hUCMSCs were seeded in nHA/PA66 material in vitro to construct cells-scaffold composites according to chapter?.(2)The osteoblastic-induced hUCMSCs, nHA/PA66 material, nHA/PA66+ osteoblastic-induced hUCMSCs were implanted into the muscular pouch of nude mouse back respectively.All of implant were performed examination of HE staining and immunocytochemical staining of osteocalcin protein to observed the expression after operation 4?8?12 weeks.(3) The composites and products were observed by the scanning electron microscope.Results Neither osteoblastic-induced hUCMSCs nor nHA/PA66 material were sclerotized in the muscular pouch of nude mouse back.The nHA/PA66+ osteoblastic-induced hUCMSCs were maturely sclerotized in large amount,the material partly absorbed.Conclusion The nHA/PA66 material composite with osteoblastic-induced hUCMSCs have a good ability of ectopic bone formation.