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The Construct of Tissue-Engineering Adipose with Progenitors of Human Adipose Mesenchymal Stem Cells

Author: LiChunMing
Tutor: LiuYi
School: Lanzhou University
Course: Surgery
Keywords: Human adipose -derived mesenchymal stem cells Differentiation Fat cells Osteoblasts Scaffolds Scanning electron microscopy Tissue Engineering Fat PLGA Filler Frozen storage
CLC: R318
Type: Master's thesis
Year: 2007
Downloads: 178
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Abstract

Soft tissue defects of the application of the most widely used filling material is solid silica gel and transplantation of autologous fat particles, the latter of which is due to the effect, a rich source of rejection, but the absorption rate of 60-70%, and how to overcome its shortcomings, adipose tissue vitality in many a year of breast augmentation surgery and soft tissue augmentation filler is the best choice. Development of tissue engineering will pose new hope. Tissue engineering is the application of cell biology and engineering principles, research and development can be repaired, a science of biological substitutes to maintain or improve the form and function of the damaged tissue. Adipose tissue engineering is the use of tissue engineering technologies will have on the activity of fat cells were grown in biodegradable stent, to produce a receptor histocompatibility good, non-immunogenic, vibrant, long durability, plasticity strong artificial fat. This ideal fat with self-healing capabilities for growing patients to provide the ability to grow, thereby enhancing the long-term efficacy of the transplanted fat. Development of tissue engineering has brought new hope for the organization, and at the same time pointing out the direction for the new cosmetic professional development and improvement of the filling material. Tissue engineering techniques include: seed cells to obtain seed cells uniformly planted in the three-dimensional scaffold to form a cell - scaffold composite material, jointly carried out culture, tissue engineering fat of in vitro constructed and animal body of experimental observation of the experimental adipose-derived mesenchymal stem cells PLGA scaffolds after adipogenic planted in the complex - biodegradable scaffolds to construct tissue engineered fat as a filler material transplanted into the subcutaneous tissue of the rabbit back the formation of fat cells. At different points in time, from the general to organize the formation of fat staining and scanning electron microscopy of tissue engineering. Objective: 1. The biological characteristics of mesenchymal stem cells and in vitro adipogenic and osteogenic understand cultured human fat. 2. Their cattle material characteristics of mesenchymal stem cells in human adipose cryopreservation of MSCs ideal preservation method. 3. In this study, separation and purification, amplified human adipose mesenchymal stem cells on the basis of whether the change in their biological characteristics, through cryopreservation, to explore the feasibility of this freezing method. 4. The study human adipose mesenchymal stem cell the biodegradable bone matrix material polylactic acid - glycolic acid (PLGA) on the adhesion, proliferation and differentiation, replantation of in vivo research base. 5. The feasibility of PLGA for fat tissue engineering scaffolds formed as a soft tissue filler material. Method: 1. Vitro culture of human adipose mesenchymal stem cells and passaged count rows into the bone and adipogenic cell proliferation cycle and surface molecules by flow cytometry. 2. Isolated and cultured in vitro amplification of normal adult adipose-derived mesenchymal stem cells in the the cryoprotectant conditions under 5% DMSO-30% FBS-DMEM, the use of two-step cryopreservation March trypan blue dye recoveries (TBR) ADSCs were growth curve, ADSCs surface antigen expression and its multi-differentiation ability to detect, compare cryopreserved ADSCs biological characteristics. 3. -70% Of the human adipose mesenchymal stem cells are cultured in vitro and amplified by a conventional method, digestive cells, and washed centrifugal added 10% dimethylsulfoxide, 30% FBS, 60% MEM cell cryopreservation system, directly into the refrigerator storage. Cryopreservation 4, 8 and 12 weeks in the 37 ° C water bath rewarming, by detecting the cell cycle, cell phenotype and multi-directional adipogenic differentiation potential. The geometric mixing polylactic acid (PLA) and polyglycolic acid (PGA), and the organic solvent by an injection molding method Preparation of PLGA. Mesenchymal stem cells cultured in vitro fat, composite of PLGA by scanning electron microscopy of human adipose mesenchymal stem cell adhesion on the PLGA, proliferation, and cell morphology changes in adipogenic case. 5. Experimental group: Rabbit fat mesenchymal stem cell line in vitro amplification of the 3rd generation of culture, adipogenic reach a certain number of one week, were seeded in PLGA scaffolds continue to induce one week, placed in the the rabbit back of the shoulder blades on both sides subcutaneous , respectively, at 4 and 8 weeks from gross and histological observation of fat formation; the control group: simply transplanted to unvaccinated cells bracket on both sides of the rabbit back scapula subcutaneous detected at the same point in time. Results: 1. Human adipose stem cells after in vitro culture of uniform positive expression of CD44, CD106, CD49d, CD34, CD45 and HLA-DR expression was negative. Cell cycle analysis showed that: G ₀ / G ₁ S and G ₂ / M proportion of 79.1%, 19.7% and 1.3 %. Isolated cells to differentiate into bone and adipogenic differentiation induction system. 2. The cryopreserved ADSCs proliferative capacity and to the osteoblast, adipocyte differentiation no significant impact and before freezing, P> 0.05 3. After the traditional procedural cooling cryopreservation technology digested directly at -70 ℃ storage, no significant changes in the cell biological characteristics, cell adipogenic differentiation potential still exists. 4. Synthesis of PLGA was porous, the porosity of 85%, a pore size of 100 ~ 400um. Human adipose mesenchymal stem cells in PLGA adhesion, proliferation and secretion of extracellular matrix, and can differentiate into adipogenic agent the round adipogenic like cells. 14 days after the composite human adipose mesenchymal stem cells and the extracellular matrix has been completely covered by the matrix material particles. 5. The composite after 4 weeks of the experimental group was implanted in the bracket formation of fat, and visible cell inrush scaffold; scaffold after 8 weeks of continued degradation, fat tissue formation significantly, accompanied by a small amount of vascular ingrowth. Control group stent material gradually disintegrating no adipose tissue formation. Conclusion: 1. Adipose mesenchymal stem cells have special biological characteristics, are able to in vitro osteogenic and adipogenic differentiation. 2. 5% DMSO-30% FBS-DMEM the cryopreservation protection solution and two-step cryopreservation system can be more complete to keep ADSCs biological characteristics, cryopreserved ADSCs ideal solution. 3. Human adipose mesenchymal stem cell suspension directly at -70 ℃ storage does not affect its biological characteristics, is a viable method of cryopreservation. 4. Human adipose-derived mesenchymal stem cells in PLGA adhesion, proliferation, PLGA as mesenchymal stem cells in human adipose carrier and tissue engineering scaffold. 5. PLGA as a scaffold for the formation of fat as a soft tissue filler material for tissue engineering provides a good idea.

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