Adipose-derived Mesenchymal Stem Cells And Xenogeneic Bone Carrier Construct Tissue Engineering Bone

BackgroundOn the clinical organ and organization's damage is puzzle multitudinous clinicians and patient's disease, the traditional method uses much from the body or the foreign body organ histological transplantation reconstructs and the repair. Because the traditional method is restricted donor origin as well as the immunity repel, dissemination disease and so on the dangerous factor influence, the people are very early start to seek implant the substitute to meet the clinical needs. The tissue engineering is the swift developing new discipline about the near 20 years, to will give organ and tissue repairing and the substitution treatment a epoch-making significance. The tissue engineering appearance, has brought the new hope for orthopedist and the bone damage patient. The tissue engineering is composed approximately by four essential factors: seed cells acquisition; growth and differentiation of seed cells; cell and carrier compound shift in vivo; the transplant comformed with recipient site, the organ and tissue is formed. The seed cells and the carrier material are two most basic essential factors which the bone tissue project need. seeking new suitable support material became a hot spot of bone tissue engineering research.Now, processing the natural bone to make the carries still at to start the stage, but overseas similar product had being come out, and obtains the very good effect in the clinical practice. Although the mechanics performance and biological compatibility of homogeneous bone are good, but the material origin are few, also it exist law and ethics aspect and so on all sorts of questions. The xenogeneous bone originates widely, the price is inexpensive. And materials passed through suitable processing be able to reserve its bone induction, and provides bone support enable it to have the bone conduction function.In the bone tissue engineering, osteoblast's gain and culture are the foundation and important component element. There are rich mesenchymal cell in the fat tissue, including adipose tissue-derived mesenchymal stem cells. Adipose tissue-derived mesenchymal stem cells in the human fatty tissue can differentiate to fat, cartilage, bone, muscle and nerve. It is one kind of ideal seed cells.ObjectiveIn order to construct the tissue engineering bone, we have prepared the pig source xenogeneous bone carrier, and contrast with the homogeneous bone carrier. We examinated their physics and chemistry performance and toxicity. We confirmed the feasibility of adipose tissue-derived mesenchymal stem cells being the seed cell of bone tissue engineering. And we attempted to culture the adipose tissue-derived mesenchymal stem cells with pig source xenogeneous bone carrier in vitro to construct tissue engineering bone.Materials and methodsUltra low temperature freezing 6 monthes iliac bone of adult swines and fresh health adult human cadavers, ultrasonic cleaning, H₂O₂ and alcohol soaking, freeze drying and radiation treatment were processed to xenogeneic bone carrier and allogeneic bone carrier. And then contrast their factor of porosity, Young's modulus, the contents of protein, Ca and P.We manufactured the leaching solution of xenogeneous bone support material and the human homogeneous bone support material, and used them to culture adipose tissue-derived mesenchymal stem cells. Inverted microscope observation cell, appraisal cell. PI method in flow cytometry examination cell life cycle. Hypodermic implants 2 materials. 4 weeks, 8 week, 12 week, 16 week after operation drew the materials from rabbit and made paraffin section and HE dyeing to observe and observated by scan electron microscope.adipose tissue-derived mesenchymal stem cells. were isolated by differential adhesion and primary culture. And contrasted their CD44 masculine. The F2 adipose tissue-derived mesenchymal stem cells. were divided into three groups:â‘ bone induction group: used oseogenesis medium;â‘¡adipogenic induction group: used adipogenic medium;â‘¢control group: used basal medium. Bone induction group and control group were detected alkaline phosphatase. Adipogenic induction group and control group were detected oil red O staining.Cultured F2 adipose tissue-derived mesenchymal stem cells with xenogeneous bone carries or human homogeneous bone carrier. Taked the material after 7d to make processing to scan electron microscope observation. Continual 7 days evaluated cytoactive by MTT method and draw up the cell multiplication curve. In 4d, 8d, 12d determined cell ALP activity.ResultsXenogeneic bone carrier and allogeneic bone carrier both had intrinsical bone trabecula, trabecular spaces and Bone cavity system. Both of them had unabridged natural three dimensional network structure. The 3D supporting frame of them were complete. The xenogeneic bone carrier had more spaces than allogeneic bone carrier. The size of both carrier were approximation, about 400p.m. The interval porosity of xenogeneic bone carrier (57.20±1.37%) was higher than the allogeneic bone carrier (53.21±1.63%). And the protein of xenogeneic bone carrier (23.36±0.48%) was not so many as it of allogeneic bone carrier (26.50±0.23%) . The contents of Ca and P of xenogeneic bone carrier were 1.7×10⁵μg/g and 1.0×10⁵μg/g. The contents of Ca and P of allogeneic bone carrier were 1.8×10⁵μg/g and 1.0×10⁵μg/g. The Young's modulus of xenogeneic bone carrier and allogeneic bone carrier were 1089.89±915.65MPa and 550.34±435.80MPa.The pig source xenogeneous bone carrierl leaching solution group cells assume the shuttle, have thick ecphyma, and interconnected in ecphyma, the cell nucleuses are big, circular, the proportion of nucleus and kytoplasm is big. The pig source xenogeneous bone carrier leaching solution group, homogeneous bone carrier leaching solution group as well as the blank control group's cells grow good, the multiplication is exuberant, the shape does not have the obvious difference. PI method in flow cytometry examinated cell life cycle to find 3 group of cell G1 time, the G2 time cell percentage is close. Hypodermic implants experimental animals arefine.4 weeks after operation, around 2 kinds of materials had obvious connective tissue. The histology slice observation, we discovered has connective tissue proliferation, and few inflammatory cell infiltration. And some osteoblast around the material arrangement regulation, around the material had a kind of ossein and the chondroid structure appearance. After 8 weeks, non-inflammation found in material. The material had some part of degeneration. After 12 weeks, tissue around the material had non-inflammation respond. The material degraded much. 16 weeks after operation, majority of material degenerated. The pig source xenogeneous bone carrier degraded more rapidly than homogeneous bone carrier. the surplus material were few.Scan electron microscope can observate obvious connective tissue around two kinds of materials 4 weeks after operation. The connective tissue proliferated, the material surface is smooth, the material with and connective tissue had obvious boundary. 8 weeks after operation, material surface became roughness, the part of carrier degraded, the material and around tissue hadn't clear boundary, had few connective tissue entering in the material. After 12 weeks, the material surface were rougher, degraded many, massive connective tissue entered in the material. the material were incomplete, and massive blades could be seen. 16 weeks later, the material surface were rougher than 12 weeks, and could not see the integrity bone tissue. connective tissue around material extended massive organizations to the material. pig source xenogeneous bonecarrier degraded more rapidly than homogeneous bone carrier.After 2 hours, the ordinary culture cell has few cells pasted the wall. After 48 hours, cells of differential adhesion and ordinary culture fast pasted the wall completely. The cell multiplication is rapid. About 4 days, cells tended to the single-layer convergence and the cell boundary is unclear. the major part cells assume spindle-shaped or polygon, and sometimes had many ecphyma. Differential adhesion and ordinary culture cells after generation, in 24 hours pasted the wall completely, 3d the majority achieved of fusions. the cells assumed spindle-shaped or polygon, ecphyma decreased. Generation after 7 generations, the cells still maintained good multiplication ability, the cellular form is very even, the shape changed not obviously.Adipose tissue-derived mesenchymal stem cells of differential adhesion had higher CD44 masculine(86.12±1.55%) than adipose tissue-derived mesenchymal stem cells of primary culture(74.03±1.06%). Alkaline phosphatase of bone induction group was high than alkaline phosphatase of control group. Oil red O Staining of adipogenic induction group were positive. Oil red O staining of control group were negative.The adipose tissue-derived mesenchymal stem cells after compound culture with carrier, the cell multiplication number presents the tendency which increasing along with the culture time lasting. Scans electron microscope observeed obviously arrives adipose tissue-derived mesenchymal stem cells survival on 2 materials and attached with the material, the cells assumes spindle-shaped or polygon, stretches out catwhisker to material. The cell number increases gradually along with the culture time lasting. The alkalinity phosphatase activity of adipose tissue-derived mesenchymal stem cells cultured with pig source xenogeneous bone carrier or human homogeneous bone carrier change tendency is basically consistent with multiplication situation of adipose tissue-derived mesenchymal stem cells, presents increasing along with the culture time lasting.ConclusionsThe adipose tissue-derived mesenchymal stem cells are one kind of fine bone tissue engineering seed cells. The pig source xenogeneous bone carrier in physics and chemistry performance and material toxicity aspects are approximation with homogeneous bone carrier, can definitely substitute for the human homogeneous bone carrier to be used in the bone tissue engineering. Because the results of adipose tissue-derived mesenchymal stem cells cultured with 2 materials are not really ideal, therefore has the necessity in the material preparation and the processing beautification, the seed cell induction splits up, the support material and the seed cell three dimensional compound culture makes improvement.