Construction Of Engineering Vascularized Adipose Tissue Via Adipose-derived Stem Cells Attached Scaffolds Encapsulated In Muscular Fasciae With Axial Pattern Blood Vessels

[Background and Objective]Factors including trauma,infection and tumors result in the defects of skin and soft tissue especially the tumors excision of the facial surface and breasts often to cause defects of the whole organs.At present clinically,autologous transplantation (skin flaps,myocutaneous flaps) and artificial substitutes were used to repair or fill the defects of soft tissue and surface organs.Although with promising prospects and encouraging results,there's limits such as short of autologous tissue,lack of blood supply and possibility of donor site morbidity,which greatly restrict the application of these techniques.Moreover,other methods including autologous dermis transplantation,autologous fat transplantation and collagen injection,filled with artificial material,and so on.However each method carries considerable disadvantages: for example,synthetic materials invariably result in foreign body reactions,and free adipose tissue grafts shrink to an unpredictable extent.Althogh kinds of methods were used for the repair of soft tissue deformities,No methods was proved to be ideal. Emerging tissue engineering strategies represent an innovative potential solution to many clinical challenges.Tissue engineering involves there items:(1) seed cells,whose growth is needed to be under strict control and high proliferation is alsoessential.(2) Three-dimensional scaffold structure,which is needed to be degradable and to maintain stable volume and shape.Moreover,good compatibility with seed cells is also indispensabile;(3) appropriate micro-environment,which can provide adequate blood supply and nutrition,ensure cell proliferation and functions.Previous studies have identified a putative stem cell population within adipose stromal compartment.This cell population,termed adipose-derived stem cells (ADSCs),can be isolated from adipose tissue.ADSCs can differentiate toward the multiple lineages like adipocyte,Cartilage cells,muscle cells and cardiomyocyte. ADSCs are easier to obtain,carry a relatively lower donor site morbidity,and are available in large numbers of stem cells at harvest.Thus,the use of adult ADSCs as the seed cell of to engineer adipose tissue seems to be more inspiring.In general,Scaffolds include synthetic materials and natural materials.Several popular scaffolds like polylactic acid(PLA),polyglycolic acid(PGA) or both poly lactic-co-glycolic acid(PLGA),PTFE,Hydroxyapatite,polyvinyl alcohol,alginate etal were used to perform multiple tissue engineering.Adipose tissue engineering is a new field so only a few scaffolds were tried including the porous biodegradable materials like PLGA,hyaluronic acid and ePTFE.But no consensus was made of which materials are most ideal for adipose tissue engineering.Considering the lack of research about vascularized engineering adipose tissue,so systemic further researches on it may be of great importance.We investigage the adipogenic,osteogenetic and chondrogenetic differentiation in vitro about the ADSCs which were harvested from adult rabbit's groin subcutaneous fat,and then to assess the biocompatibility of three-dimensional porous type Icollagen sponge scaffolds with ADSCs.Othe other hand,BrdU was used for labeling the ADSCs,then to explore the adipogenic efficacy and the survival improvement of the ADSCs attched scaffold by encapsulated in muscular fasciae with axial pattern blood vessel.This paper's objective is to probe an ideal method of construction of a new vascularized tissue-engineered adipose which could be applied clinically for repairing the defects of soft tissue.[Material and Methods]1.Isolation,expansion and differentiation of rabbit ADSCs The inguinal fat pads were harvested by exairesis from the rabbits(The Southern Medical University institutional ethics committee approved all experiments in advance),and then enzyme digestion method were used for primary culture and observe changes in cell morphology and function of cells.Only cultured cells for 3 passage were used in this study in vivo.The ADSCs were detected the molecular expression which are associated with the surface of the stem cells such as CD29,CD34,CD44 and CD49d by using cellular immunofluorescence method.We make these cells to adipogenic,osteogenic and chondrogenic differentiation in vitro, then Oil Red O staining and Alizarin red staining and Alcian staining to identify the success of differentiation in order to identify the success of multi-directional differentiation.2.Changes of biological properties of ADSCs labeled with BrdU marker in vitroThe three passage of ADSCs was incubated with BrdU at different concentrations(0,5,10,15 and 20μmol/L) for incubating time(12,24,48 and 72 h),to identify the optimal BrdU concentration and incubating time for cell labeling. Immunofluorescence and trypanblau strain were performed respectively to calculate the labeling index(positive rate) and the cells' activity for different time after BrdU labeling in vitro.Then we compare the difference of adipogenic potentiality before and after BrdU labeling in vitro.MTT colorimetric analysis was used to detect of the rate of cell proliferation and cytotoxicity of BrdU.3.Biocompatibility of ADSCs attached with typeⅠcollagen sponge scaffold in vitroADSCs after BrdU labelling attached to the typeⅠcollagen sponge scaffold to form compounds respectively,then contrast phase microscope was used to observe cells growth,adhesion or expansion.Whereafter,we observed the cells disposition and proliferation under scanning electron microscope(SEM) in vitro.We determine the adhesion rates of ADSCs before and after marked by BrdU.Then we compare the difference of adipogenic potentiality between single ADSCs and ADSCs combine with collagen in vitro.MTT colorimetric analysis is used to detect of the rate of cell proliferation. 4.Experimental research of vascularized tissue-engineered adipose via theADSCs-attached scaffolds encapsulated in muscular fasciae with axial pattern blood vessels.All animals(n=10) procedures were performed in accordance with the guidelines of the Southern Medical University Animal Care and Use Committee. Own control was carried out at all the rabbits(n=10).TypeⅠcollagen sponge (10mm×10mm×5mm) containing ADSCs(1×10~7 cell population and without adipogenic differentiated conditions,group A) was transplanted into the fascia musculares sac with blood vessel pedicle of left latissimus dorsi myocutaneous flaps, and the ADSCs(the same cell population and with adipogenic differentiated conditions,group B) attached same size collagen sponge was transplanted into the fascia musculares sac with blood vessel pedicle of right latissimus dorsi myocutaneous flaps at the same rabbits.Another same size collagen sponge containing adipogenic differentiated ADSCs(the same cell population) were also implanted into the random fascia musculares sac without blood vessel pedicle of right gluteus maximus(group C,Control).The rabbits were fed routinely after operation. Euthanasia was performed at the animals after 8 weeks of the ADSCs with scaffolds transplantation.No animal from either the study or control group died during the study period.The neogenetic tissue were observed macroscopically.Then they were dissected out and weighed their humid weight by electronic balance (fidelity=0.0001g).The tissue were embedded in paraffin for conventional Oil red O stain,HE stain and immunofluorescence staining in order to perform histological assessment.Neovascularization was assessed by measuring the number of capillaries in 10 fields of HE stain slide.To analyze and assess the effectiveness of the method of muscular fasciae with axial pattern pedicle encapsulated ADSCs for enhancing vascularization tissue engineering adipose and survival of the ADSCs.[Results]1.The original cultured ADSCs looks like to fibroblasts,but it has strong tendency for high proliferation and multiple differentiation.With the effect of adipogenic, osteogenic and chondrogenic differentiation medium,it is proved to be able to differentiate into mature adipocyte,osteoblast and chondrocyte,respectively.The differentiation can be proved by Oil Red O,Alizarin red and Alcian positive staining respectively.This kind of cell(ADSCs) is also proved to be CD29,CD44 and CD49d positive expression.2.We perform immunofluorescence staining program after labeled by the BrdU.The ADSCs' nucleus show green fluor under fluorescence microscope.On the whole,The labeling ratio increased gradually with the incubating time and concentration of BrdU. There are more than 90%ADSCs were labelled when the cells incubating 48h and the concentration of BrdU maintaining 15μmol/L at the same time,and the living cell more than 98%.but the labeling ratio does not increased with prolong of the time and the increasing concentration of BrdU.It is indicated that the optimal time for labeling ADSCs is 48h and the most appropriate concentration is 15μmol/L of BrdU.The morphous,growth,proliferation and adipogenic potentiality of the labeled ADSCs not to be influenced compare to the unlabelled ADSCs.3.ADSC attached to typeⅠcollagen sponge scaffold and normally grew.The adhesion rates different from the mixing means of cells and scaffold.The adhesion rates is 79.5%±2.5%when we drop ADSCs onto the surface of scaffold(n=3),and the adhesion rates is 92.7%±2.2%when we inject ADSCs into the interior of scaffold(n=3).There was significant difference between group A and group B (t=6.965,P=0.002).On the other hand,The adhesion rates is 91.3%±1.5%when the adipogenic-differentiated ADSCs were injected into the interior of scaffold.There has no significant difference between group B and group C(t=0.889,P=0.424).There has no influence on the morphous,growth,proliferation,adipogenic potentiality and no cytotoxicity when ADSCs attached scaffold. 4.Eight weeks after transplantation,the neogenetic tissue formation were observed macroscopically in the operative area of group A,group B and group C.But there has no neogenetic tissue were observed in the operative area of the group D.HE stain and oil red O stain show that the neogenetic tissue are mature adipose tissue,the positive display of immunofluorescence staining make it sure that the tissue was construted with transplanted ADSCs.The mean humid weight of the neogenetic tissue are 0.0230g±0.0016g(n=10),0.0251g±0.0019g(n=10) and 0.0202g±0.0014g(n=10) respectively.There was a significant difference between the group A and group B, group B and group C,group A and group C(all value of P＜0.05).The scaffolds were degraded thoroughly after 8 weeks at the new formed tissue.Histological evaluation of 20 fields of neogenetic adipose tissue showed that capillary density,an index of neovascularization,increased markedly in Group A and Group B.A statistically significant increase in capillary density was observed after 8 weeks in group A and group B as compared to control(group C):28.5±3.7 capillaries in group A versus 31.2±4.5 capillaries in the group B and 19.3±2.6 capillaries in control group(all value of P＜0.05).[Conclusions]1.We successfully isolated and cultured the ADSCs from inguinal subcutaneous fat pads of adult rabbits.And then we established a set of convenient methods about isolation,culture and identification for the ADSCs.The cells have great potentiality of proliferation and adipogenic,osteogenic and chondrogenic differentiation in vitro.ADSCs should be used for pluripotent seed cells harvested from adipose tissue.ADSCs are ideal in many aspects:they can be easily and effectively harvested,handled and multiplied non-invasively and abundantly;their pluripotency and proliferative efficiency are not less than those of bone marrow-derived MSCs;and donor morbidity is lower than for MSCs harvested from other sites.So ADSCs have great prospect of clinical application.2.The labeling index may achieve over 90%when the continually passaged cells of ADSCs were incubated with 10μmol/L BrdU for 48h.So the optimal time for labeling ADSCs is 48h and the most appropriate final concentration for the BrdU is 15μmol/L.BrdU has no obvious cytotoxicity and influence on the growth, proliferation and differentiation to the cells.3.ADSCs can attach to typeⅠcollagen sponge scaffold,normally grew and proliferated intro.There has a high adhesion rates when the ADSCs were injected into the interior of scaffold.There has no influence on the morphous,growth,proliferation and differentiation and no cytotoxicity when ADSCs attached scaffold in vitro.So it has excellent biocompatibility between ADSCs with typeⅠcollagen sponge scaffold.4.In this study,conclusion can be drawn that the adipose-derived stem cells which attached scaffold encapsulated in muscular fasciae with axial pattern blood vessel pedicle can increased neovascularization and enhanced tissue engineering adipogenesis.So the engineering vascularized adipose tissue can be constructed by transplanted autologous ADSCs-attached scaffold encapsulated in muscular fasciae with axial pattern blood vessel pedicle.Autologous implanted adipose-derived stem cells can be differentiated into endothelial.Both the ADSCs and and endotheliocytes which derived the axial pattern blood vessel pedicle participate in vascularization and then increase the neovascularization.