| The substituted materials for bone tissue already couldn't be satisfied clinical needing of the defection and damage which caused by the trauma, surgical operation and cutting off for bone tumor. People was developing a kind of biologic substitute which could repair, maintain and improve the function of the hurt bone organization, and was absorbed by the economy. Using it to solve a huge bone defection and damage is the point of bone tissue engineering nowadays research. People gradually understand the triangle mode of cell breeding which set up to take scaffold material as framework, compound stem cell, and assist with growth factor is more ideally choice of tissue engineering. According to the basic principle of bone tissue engineering, we designed a new enhanced tricalcium phosphate - collagen-variety growth factor (PRP) compound scaffold which compound platelet-rich plasma with enhanced tricalcium phosphate at first, then reacted with coagulant. Latterly we seed the beagle's bone marrow stem cells in this scaffold, and then observe its ossification circumstances after raising this compound in beagle's body. Investigate the ideally engineering rudiment product for setting up tissue engineering bone, 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 The isolation,cultivation,purification and proliferation about the dog marrow mesenchymal stem cellObjective To investigate the experiment foundation for method of isolation and culture of bone marrow mesenchymal stem cells, evaluated their growth and proliferation, and expected it for seed cell of bone tissue engineering.Methods By puncture and sample beagle's bone marrow in axenic condition and the method of density gradient centrifugation to isolate the bone marrow mesenchymal stem cells. We cultivated origin cells by the low sugar DMEM 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 P10 cells growth curve by MTT method. Flow cytometry examined the surface antigen of the P5 cell.Results In 24 hours a few adherent cells could be observed after cultivation. The primary BMSCs indicated long or short spindle-shape or multangular shape at 48 hours later while it could be observed cell division. Fibroblast-like cells manifold progressively after the 3rd day. Logarithm growth period appeared in the 5th day. Cells fused more than 90% in the 8～10th day. Passage cells with 1:3 ratio began to adhere within 2 hours after sub-culture, proliferate rapidly, most fibroblast-like. Passage cells confluence after the 6～7th day. The shapes of cells are homogeneous long spindle-shape after the 3rd passage, and then it could be observed circinate-like in fusion time. Broad brimmed-like cells appeared after the 10th passage. Flow cytometry shows that 95% and 96% of the 5th passage cells expressed CD44 and CD29 respectively, while only 5% of the cells expressed CD34. The growth curve showed that proliferation ability of the 3rd passage and 5th passage cells was more powerful than the 10th passage cells; the 3rd passage cell had the most powerful ability; but the proliferation of the 10th passage cell was weaker then before.Conclusion The method of isolation and culture of BMSCs is simple and feasible. BMSCs can be cultured in a long period and grow stably, proliferate rapidly, may be accorded as a kind of seed cells for tissue engineering.Chapter 2 Osteoblastic reaction of the dog marrow mesenchymal stem cell was influent by PRP in vitroObjective To explore osteoblastic of the dog bone marrow mesenchymal stem cells cultured with a specific medium in vitro; to explore the best PRP gel concentration for the dog bone marrow mesenchymal stem cells and the influence of osteoblastic reaction of it while joined with PRP gel; and to observe and analysis the proliferation of the dog bone marrow mesenchymal stem cells was influent by PRP gel. Methods (1) Beagle's bone marrow mesenchymal stem cells were isolated and cultured according to chapter I. (2) Haemospasia 27 beagles' dog in vein of the posterior limb, then prepare PRP by two-stage centrifugation. Count the plasma number of PRP and whole blood by manual work, then prepared gel by confused PRP with 10 percent calcium chloride solution containing ox-thrombin 100u/ml as ratio 1:1. (3) Measured the influence of proliferation of the 3rd passage bone marrow mesenchymal stem cells were interfaced by different concentration PRP gel and explore the best concentration-effect group. (4) The 3rd passage cells were collected and derived for 4 groups. Low glucose DMEM containing fetus blood-serum of 0.01 volume fraction was group I ; high glucose DMEM containing fetus blood-serum of 0.01 volume fraction, dexamethasone 1×10-7mol/L, beta-sodium glycerophosphate 10mmol/ L, antiscorbic acid 50mg/ L was group II; low glucose DMEM containing the best effective concentration PRP gel was group III and high glucose DMEM containing the best effective concentration PRP gel, dexamethasone 1×10-7mol/L, beta-sodium glycerophosphate 10mmol/ L, antiscorbic acid 50mg/ L was group IV. Be observed the metamorphosis of all groups by inverted microscope and HE stain. The levels of alkaline phosphates were measured by alkaline phosphates kit; the formation of calcium nodules were confirmed by Alizarin red S staining; the expression of osteocalcin protein was examined by immunocytochemical stain and the expression of osteocalcin,alkaline phosphates and collagen type I mRNA were examined by RT-PCR for all groups.Results (1) It has 143.5±33.1×109/L plasma in whole blood and 1239±174.07×l09/L plasma in PRP. (2) All different concentration PRP gel can promote the proliferation of canine bone marrow mesenchymal stem cells at first stage, but different concentration group appeared different changes along with time. Gradually decreased could be observed in high concentration groups, but Contrarily the low concentration PRP groups characterize better and the 6.25% concentration group has the best powerful. (3) The cells of group II,IV became triangle,polygon and roundness shape, and could assemble themselves together by inverted microscope and HE stain. It was distinctly different with the group I,III, the group III became more proliferation than I . The levels of alkaline phosphates of all groups became crescent along with time. The alkaline phosphates level of group IV was strongest. The Alizarin red S staining appeared positive, which verified the formation of calcium nodules, when osteoinducted 14 days in group IK IV. The secretion of osteocalcin was positive with immunocytochemistry staining after osteoinduction for 7 and 14 days in group II,IV. The expression of osteocalcin,ALP and collagen I protein mRNA showed positive at 185bp,292bp and 145bp after osteogenic induction for 7 and 14 days. There was more expression of osteocalcin,ALP and collagen I protein mRNA in group II,IV than group I,III. There was no different of the expression between with group I and III, and the group IV has the strongest expression.Conclusion PRP gel can promote the proliferation of canine bone marrow mesenchymal stem cells; the intensity of proliferation was correlated with concentration. The proliferation were inhibited in high concentration groups were observed. The BMSCs could be osteoinduced to osteoblastic and stably expressed osteoblastic phenotype in vitro. PRP gel could clearly boost up the synergistic effect of osteoblastic for revulsant, but could not osteoinduced BMSCs to osteoblastic behave by use it alone.Chapter 3 The preparation and histocompatibility research of intensified beta-tricalcium phosphate/platelet-rich plasma gel multi-scaffold and constructed tissue engineering bone in vitroObjective To explore the preparative method of intensified beta-tricalcium phosphate/platelet-rich plasma gel multi-scaffold, make the histocompatibility and biomechanical analysis of it. To explore the possibility of this scaffold used in the carrier for BMSCs.Methods (1) PRP was prepared according to chapter II. (2) The pillar-like intensified beta-tricalcium phosphate were cleaned the dust in surface by brush and be flushed by normal saline, then be prepared for high temperature antisepsis after drying. (3) Measured the biomechanics by general purpose material test instrument. (4) The intensified beta-tricalcium phosphate would be dipped in PRP, and then constructed multi-scaffold by confused with 10 percent calcium chloride solution containing ox-thrombin 100u/ml as ratio 1:1. (5) Beagle's bone marrow mesenchymal stem cells were isolated and cultured according to chapter I. The 3th passage cells were collected and seeded in alone intensified beta-tricalcium phosphate and intensified beta-tricalcium phosphate/platelet-rich plasma gel multi-scaffold. (6) The adhesion ratio of the 3rd passage was detected by cell counted in 4,8,12,24h. (7) The RGR in scaffold were measured by using MTT and counted the cytotoxicity of this material in 2,4,8d. (8) The growth,proliferation and production of BMSCs is this scaffold were observed by scanning electron microscope in two weeks.Results (1) The intensified beta-tricalcium phosphate was a white,no peculiar smell and toughness material. It has a spongy-like structure, crude surface. PRP gel were full of holes of intensified beta-tricalcium phosphate after coagulate reaction. (2) The biomechanical analysis vertical compress showed the average max-load of the intensified beta-tricalcium phosphate were 942.49N, the average max-intensity were 13.634MPa. (3) The adhesion ratio of the 3rd passage were 52±2%,81±1%,92±2% and 98±1% in the intensified beta-tricalcium phosphate/platelet-rich plasma gel multi-scaffold at 4,8,12,24h. (4) The RGR in the intensified beta-tricalcium phosphate/platelet-rich plasma gel multi-scaffold were all exceeded than 1.0 and the cytotoxicity of this material was considered innocuity. (5) The BMSCs could be adhered the surface of the intensified beta-tricalcium phosphate/platelet-rich plasma gel multi-scaffold and growth more hearty than the alone intensified beta-tricalcium phosphate by scanning electron microscope in 2 weeks. The cells extended in the intensified beta-tricalcium phosphate/platelet-rich plasma gel multi-scaffold were observed and much more extracellular matrices were found in it. But little extracellular matrices were found in controlled.Conclusion The intensified beta-tricalcium phosphate/platelet-rich plasma gel multi-scaffold could be used for cells carrier in bone tissue engineering. It was an innocuity,better histocompatibility material and could be doubled as osteo-conductibility and osteo-inductivity for satisfying the demand of bone tissue engineering.Chapter 4 The experiment research of the construction of tissue engineering bone in vivo from dog bone marrow mesenchymal stem cells seeded in intensifiedβ-TCP/PRP gel multiple-scaffold.Objective To investigate the construction of tissue engineering bone and the ability repairing bone defection with dog bone marrow mesenchymal stem cells in intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold Implanted dog's tibia upper segment after directed differentiation of stem cells into osteoblasts in vitro.Methods (1) Dog BMSCs was seeded in and then cultured or osteoinduced with intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold in vitro for 3 weeks according to chapter 3. (2) The cells compound with alone beta-tricalcium phosphate(group I ) or intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold (group II) were randomly implanted in dog's left or right circular tibial upper segment and were compared with autogenetic Ilium(group III). The changes of implants surface,the form of osteotylus,the reparation of bone defection and tissue reaction around the implant were observed. (3) All of implants were performed X-ray examination and observed the changes after operation 4,8,12 weeks. (4) All of implant were performed examination of HE stain and immunocytochemical stain of osteocalcin protein to observed the expression after operation 4,8,12 weeks. (5 ) The expression of OCN,ALP and Col1 A1 mRNA were detected by RT-PCR after operation 4,8,12 weeks. (6) Appertain tissue of the tibia upper segment of beagles' were excised, and then were examined in general purpose material test instrument which named INSTRON8032.Results ( 1 ) The wound growth all right, inflammation and toxicity reaction in body or local part were not observed in all groups. (2) The X-ray showed that the group of II,III were all became Osseo-concrescence in 12 weeks, but group III were in Contrarily. Material-osseous interface were clearly, and shadow appeared about the materials. (3) The ossifications of new cartilages were observed in all groups by HE stain and immunocytochemical stain of osteocalcin protein. The expression of the cell/intensified beta-tricalcium phosphate/ platelet-rich plasma gel multiple-scaffold compounds were better positively than the cells/ intensified beta-tricalcium phosphate compounds. (4) The expression of osteocalcin,ALP and collagen I protein mRNA were showed positive at all groups after operation for 4,8 and 12 weeks. The group II,IIIexpressed all protein mRNA more positive than group I at 8 and 12 weeks. And there was no different between the expression of group II,III. (5) The group III showed no different between group II, but was better intensity than group I in the examination of vertical compress.Conclusion The intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold was an ideally material for bone tissue engineering which was satisfied in histocompatibility,the speed of degradation,the efficiency of biomechanics and ossification. Canine BMSCs seeded in the intensified beta-tricalcium phosphate/platelet-rich plasma gel multiple-scaffold could construct tissue engineering bone by osteoinductation in vivo. |
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