Construct Engineered Adipose Tissue Using Adipose-derived Stem Cells With Human Extracellular Matrix Scaffold And BFGF-PLA Nanospheres Delivery System

BACKGROUNDAt present, as the development of adipose tissue engineering, a new way has been opened up in the field of repairing soft tissue defects without damage and in the true sense of morphological, structural and functional reconstruction completely. But there are still many factors restricting the development of adipose tissue engineering. No suitable scaffolds and biological cytokines are two important factors among them.Appropriate biological scaffold is one of the important part in tissue engineering, it is not only the carriers of cells and bioactive factors into the body, but also supporting cell proliferation, differentiation and secretory activity of the cytokine in vivo. Ideal adipose tissue engineering scaffolds should have the following characteristics:①suitable three-dimensional structure for cell adhesion, proliferation and differentiation;②has well mechanical properties, not only match with the host’s soft tissue characteristics, but also could resist soft tissue pressure, so it can support seed cells’growth and development;③has good composition with non-toxic, no side effects and no immunogenicity;④has the ability of bio-stimulation, such as promoting cell proliferation and differentiation, promoting angiogenesis, or promoting the migration and differentiation of host stem cells. Nowadays, there were a lot of studies about the application of scaffolds, but each of the scaffolds had its own limitations, there was no ideal scaffold for adipose tissue engineering. People are still keep grouping for ideal scaffolds. The extracellular matrix (ECM) is macromolecule exist above the cells or between cells, mainly polysaccharides and proteins or proteoglycans. ECM contains a large number of cells active ingredient, which plays an important role in regulating cell adhesion, proliferation, migration, differentiation and gene expression regulation. Adipose tissue not only contains a number of cellular components, but also rich in ECM components, including collagen, reticular fibers, elastic fibers, nerve fibers, vascular matrix, and lymph nodes. There is feasible to extract ECM from adipose tissue and construct to cell scaffolds. There are no related reports of domestic.bFGF has the ability of promoting the adipose-derived stem cells(ADSCs) and bone marrow stromal cells angiogenesis, migration and adipogenic differentiation, it is an ideal cell factor in adipose tissue engineering. However, bFGF diffuse easily, is sensitive to heat and acid, degradated by proteases easily, half-life is only3-5min in vivo, which limited its biological effect severely. The sustained release technology is likely to be a solution to the problem. It could entrapped bFGF in polylactide nanospheres though the technology of entrapped microspheres, so that the cytokine could release to the surrounding environment continuously and exert its biological effects. The slow release technique has great research value in adipose tissue engineering. OBJECTIVES1. Adipose-derived stem cells were isolated from adipose tissue by liposuction with the method of enzymatic digestion. The cells were induced for three-line differentiation. Study its characteristics of stem cells and advantages as the ideal seed cells in adipose tissue engineering;2. Constructed bFGF-PLA polylactide nanospheres in vitro, detected its controlled release ability; investigated the effect of bFGF-PLA release nanospheres on proliferation and adipogenic induction of adipose-derived stem cells in vitro. Study the feasibility of bFGF-PLA release nanospheres used in adipose tissue engineering;3. Collected human adipose tissue and isolated extracellular matrix in vitro. The extracellular matrix was freeze-dried, sterilized and crushing to powders, observed its surface characteristics and explored the feasibility of extracellular matrix powders as scaffolds in adipose tissue engineering;4. Labeled ADSCs with Dil fluorescence in vitro. The Dil-labeled ADSCs were seeded onto the human extracellular matrix powers, and analysised the biocompatibility of ADSCs and extracellular matrix powers. Explored the feasibility of ECM be used to adipose tissue engineering experimental study in vivo;5. Set up reasonable control group, the extracellular matrix scaffold combined with bFGF-PLA sustained release microspheres and ADSCs transplanted subcutaneous of nude mice. Analysis the influence of extracellular matrix scaffolds combined with bFGF-PLA sustained release microspheres on ADSCs to construct engineered adipose tissue.MATERIALS AND METHODS1. Isolation of human adipose-derived stem cells from adipose tissue and multilineage differentiation ADSCs were isolated from subcutaneous tissue of healthy donors who had undergone liposuction through the method of collagenase digestion. The third generation of ADSCs were used to next experimental study. Draw cells proliferation curve using the method of MTT. Induced ADSCs to adipogenic, osteogenic and chondrogenic differentiation with the corresponding inductive culture medium, oil O, alizarin red and alcian blue staining correspondingly.2. Construct bFGF-PLA polylactide nanospheres and investigated the effect of bFGF-PLA release nanospheres on proliferation and adipogenic induction of adipose-derived stem cells in vitroConstructed bFGF-PLA sustained release microspheres though the method of ultrasonic emulsification in vitro. Detected the microspheres drug loading rate and encapsulation rate and observed the condition of encapsulation with transmission electron microscope, measured the diameter of microspheres under microscope. Detected the bFGF-PLA release capability using ELISA kit. The culture medium and inductive medium of stem cells were prepared containing0mg/ml、1mg/ml、2mg/ml、3mg/ml、4mg/ml、5mg/ml basic fibroblast growth factor-polyactide release nanospheres respectively. Adipose-derived stem cells were cultured in96well plate and replaced the culture medium containing release nanospheres the second day respectively. Detected the cells proliferation by the method of MTT every day and quantification of oil red O every other day. Investigate the effect of basic fibroblast growth factor-polyactide release nanospheres on proliferation and adipogenic induction of adipose-derived stem cells in vitro.3. Fabrication of human adipose extracellular matrix powders in vitroCollected the human adipose tissue, after removed blood and oil components, the tissue was homogenized, centrifuged, freeze-dried, and crushing to powders by instrument. The structures of human ECM powders were observed withelectron microscopy. Explored the feasibility of extracellular matrix powders as scaffolds in adipose tissue engineering4. Biocompatibility study of ADSCs and extracellular matrix scaffolds in vitroAt first, marked ADSCs with EGFP or Dil fluorescent, detected cells ability of proliferation and adipogenic differentiation. The labeled ADSCs were seeded onto the human extracellular matrix scaffolds, detected adhesion rate and observated condition of cells adhesion and growth on the surface of scaffolds with fluorescence microscope and scanning electron. Detected cells’growth and proliferation situation after adhesion though the method of MTT and draw cell growth curve, and compared with normal ADSCs growth curve.5. Construct engineered adipose tissue using adipose-derived stem cells with human extracellular matrix scaffold and bFGF-PLA nanospheres delivery systemThe extracellular matrix scaffold combined with bFGF-PLA sustained release microspheres and ADSCs transplanted subcutaneous of nude mice. There were three groups in all:the first group were transplanted human extracellular matrix scaffold+cell culture medium, a total of0.5ml; the second group was human extracellular matrix scaffold+ADSCs suspension, a total of0.5ml, containing1×107cells; the third group was human extracellular matrix scaffold+ADSCs+bFGF-PLA suspension, a total of0.5ml, containing1×107cells and the concentration of bFGF-PLA was4mg/ml. The suspensions above were injected subcutaneously into the backs of nude mice using an18-gauge needle. At eight weeks after the injections, the grafts were explanted, weighted, and fixed with10%paraformaldehyde. The grafts were assessed by hematoxylin-eosin and oil red O staining of frozen sections. The conditions of angiogenesis was observed and the number of angiogenesis was counted under microscopy. Six mice were analyzed per experimental group. STATISTICAL ANALYSISThe data obtained was represent by mean+standard deviation (x±s), using SPSS13.0software package for statistical analysis. P<0.05represent significant differences.RESULTS:1. ADSCs was obtained successfully from adipose tissue of liposuction patients, had strong proliferate ability, similar to fibroblasts in morphology. After2～3weeks induction in the adipogenic, osteogenic and chondrogenic medium, stained with oil red O, alizarin red and alcian blue respectively, the results were positive.2. Constructed bFGF-PLA sustained release microspheres in vitro successfully, the surface of microspheres was smooth, the sphere size was uniform; the average diameter of microspheres were (28.98±0.50) nm, the rate of encapsulation was (89.54±1.45)%, the microspheres drug loading rate was{(17.91±0.29) X10-3}%; Detected the bFGF-PLA releasing capability using ELISA kit. The microspheres could release bFGF slowly within21days. At last, the cumulative release rate was79.85%. The bFGF-PLA sustained release microspheres could promote the proliferation and adipogenic differentiation of ADSCs significantly, the best concentration of microspheres was4mg/ml.3. The human extracellular matrix powders were prepared using simple physical treatments without the addition of chemicals or enzymatic factors. The powders were suitable for ADSCs adherent because thry were highly diverse in size and shape. They had both rugged and smooth surfaces with an extensive surface area, which could be favorable for cell adhesion and proliferation.4. It was not affect ADSCs proliferation and adipogenic differentiation with EGFP or Dil labeled. Adipose-derived stem cells can adhere to the scaffold easily, and the adhesion rate was (88.81±4.81)%and (86.48±4.58)%before and after DII labeled. There were no differences between two groups. EGFP or DII labeled adipose-derived stem cells adhered to extracellular matrix scaffold in high rate and growth in good condition.5.8weeks after transplantation, the6mice all survived, there were new growth tumors in three group. The size of the tumors in group1, group2and group3showed increasing trend. The color of the tumor in group1was white, yellow in group, while the color in group3was closer to normal adipose tissue than group2. The average wet weight in three group was (0.032±0.005) g;(0.041±0.006) g;(0.049±0.005) g respectively. There was significant differences between three groups in wet weight, group3had more weight than the other groups. HE sections showed group3formed a large number of mature adipose cells, group2formed mature adipose cells, but less than group3. There was nearly no mature adipose cell in group1, only a small amount of a single or a few fat cells located within scaffolds. The condition in oil red O sections was similar to HE sections. Under fluorescence microscopy, we could see strong red fluorescence where the adipose cells gathered, the fluorescence was not so much in group2, while no fluorescence was seen in group1. The average amount new blood vessels were (3.00+1.28)/HP,(3.28±1.36)/HP,(4.44±1.15)/HP respectively. Two-sample t test, P values were as follows:the P of group1compared to group2was0.514>0.05; the P of group1compared to group3was0.001<0.01; the P of group2compared to group2was0.008<0.01. We could see from the results above that there was no significant difference between the first two groups in the amount new blood vessels, group3had much more new blood vessels than the first two groups.CONCLUSIONS1. ADSCs were isolated from subcutaneous tissue of healthy donors who had undergone liposuction through the method of collagenase digestion. This method of isolation and cultivation of ADSCs was convenient and easier to carry out. The ADSCs isolated had the capabilities of pluripotential differentiation and could passage rapidly, so we could had enough ADSCs for implantation in short time. Thus, ADSCs could be used as ideal seed cells for adipose tissue engineering.2. Constructed bFGF-PLA sustained release microspheres in vitro successfully, the microspheres could release bFGF slowly, so it could affect surrounding cells continuingly. The bFGF-PLA sustained release microspheres could promote the proliferation and adipogenic differentiation of ADSCs significantly, it could be an ideal cytokine used to adipose tissue engineering.3. The human extracellular matrix could be extracted from human adipose tissue. The extracellular matrix had the advantages of easier to obtain, high degree of diversity in shape and particle size, large surface area et al, which could be favorable for cell adhesion and proliferation. So it could be an expected scaffold in adipose tissue engineering.4. It did not affect the abilities of proliferation and adipogenic differentiation of ADSCs with EGFP or DiI labeled. Both the two label method were suitable as the application of ADSCs tracer in vivo. ADSCs and human adipose tissue ECM scaffolds had good biocompatibility in vitro, the adhesion rate was high, cells’ growth capacity would not be affected.5. Human extracellular matrix scaffold with bFGF-PLA sustained release microspheres and ADSCs could build engineered mature adipose tissue in vivo. Human ECM scaffolds and bFGF-PLA sustained release microspheres could promote the level of ADSCs differentiated into adipose cells and vascularization. They could use as an ideal scaffold material and micro-environmental cytokines in adipose tissue engineering.