Experimental Study On Injectable Dermal Filler Using Tissue Engineering Technique And Abundant Cells In Human Body

1.Study background and objectiveFacial rejuvenation has always been an investigating topic for esthetic surgery. except for facelift,many kinds of dermal packing materials are applied to remove wrinkles and skin depression,and more and more injecting substance are launched with more safety and effectiveness.At present,they are classed by the source as follows:Autogenic:autogeneic fat,cells and collagens,etc.Allochthonous:collagen from human corpse,collagen obtained from cultured human fibroblast culture.Heterogeneic source:collagen from cow or pig,hyaluronic acid or relative derivatives from rooster comb or bacterial fermentation.Artificial synthesis:silicones,Polymethylmethacrylate(PMMA), hydroxyapatite ceramic,polylactic acid,polyacrylamides.Non-permanent injecting filling materials mainly include hyaluronic acid or relative derivatives,collagen,polylactic acids and autogenic cells,etc.;permanent injecting materials include silicon resins,PMMA,hydroxyapatite ceramic and autogenic fat.But they all have advantages and disadvantages,autogenic fat has a limited survival rate and high absorbance,and it can only be applied people with rich fat; bovine collagen can easily induce sensitivity and its effective time is short;Artecoll can induce granuloma and can only be injected subcutaneously;sodium hyaluronate has a short effective time;simple fibroblast must be injected for multiple times and the product is expensive with an unsatisfied effect.In recent years,tissue engineering developed fast,and great accomplishments have been obtained in fields of skin,cornea,liver,spleen,cartilage,bone,blood vessels,urinary tract,nervous system,etc.The study of tissue engineering provides a new direction for the study of injecting dermal filling materials,among which cytoscaffold material can choose disposable bio-material Plurolic-F127 and disposable injecting filling materials (sodium hyaluronate,collagen,etc.),seed cells can choose cells which can be easily obtained from human bodies,such as cells from skin and fat.By adopting tissue engineering,autogenic cells can be isolated from the marginal tissues of human body,and seed cells can be produced by in vitro amplification,then mix seed cells and disposable cytoscaffold to form cell-scaffold complex,inject this autogenic complex into the dermal layer under wrinkles,and scaffold will be gradually dissolved and absorbed,autogenic cells will form new connective tissues and secret a great number of cell matrix,therefore,it will repair the former dermal rupture, reconstruct and repair skin structure,thus to achieve the goal of removing wrinkles.The current disposable injecting filling materials mainly include:collagen from cow and pig,non animal originated hyaluronic acid and its relative derivates, hydroxyapatite microsphere,polypropylene,N,N- diallyl dimethyl ammonium chloride.Among these materials,because the non animal originated hyaluronic acid will not induce sensitivity and is easily obtained,and itself is a component of cell matrix,so it is applied as the cytoscaffold material in this study.Skin is the biggest organ of human body with rich cell resources,and it is a huge cell bank.Moreover,the skin is the distributed in body surface,and materials can be easily obtained.Surgical operations commonly need to cut open skin,so at the time of tissue or organ restoration operations,a small amount of skin can be cut down,or even the removed skin tissues in the operation can be utilized to obtain a great number of skin originated cells.It is not only safe and convenient to obtain seed cells from skin,but the discarded skin tissues in the operation can be fully utilized,and the valuable cell resources of human body will not be wasted.Skin fibroblast has be further studies when applied as seed cell,Eaglstein WH has built tissue engineered dermas with fibroblast obtained from infant prepuce and the technique has been extensively applied in clinics to promote trauma healing,Chen Bing et al have applied skin fibroblast to build tissue engineered tendon.Autogenically cultured fibroblast(Isolagen)has also been applied to treat depressed wrinkles and scar induced by acne in America and Europe,etc.,and this is the application in the aspect of injecting wrinkles removing with simple cell technique,and 1450 cases have received the therapy.Human fibroblast can be isolated from skin and in vitro amplified,the cells can be injected into the depressed wrinkles after collected to remove wrinkles,and it is very effective for tiny wrinkles,no relative complications have been observed,but the method is ineffective for deeper wrinkles,which may be related with the facts that the cell number survived is small and the cells are not easy to fix after simple fibroblast has been injected.Since 2001,Zuk firstly demonstrated that cell groups with multi-directional differentiation capacity existed in the fat tissues obtained in liposuction,and the concept of adipose derived stem cells(ADSCs)was explicated,after that,ADSCs were gradually cognized and quickly they entered the study field of tissue engineering seed cells.Compared with BMSCs,ADSCs distribute extensively in human body and the applicable cell number is enormous,the needed fat tissue can be obtained from liposuction(cosmetic operation),the operation is simple with little invasion,and it can remodel the figure of the patient.Therefore,the patient can easily accept the operation with no psychologic obstacle.Provided with the above considerations,we believe ADSCs is superior to BMSCs in the aspect of seed cell resources,and it has a wider applicable prospect when fat tissue is chosen as the seed cell resource.It is only a few years since people learn ADSCs.In 1994,Kaplan et al found ectopic ossification in subcutaneous fatty tissue in some patients,and they proposed that precursor fat cell could transform to osteoblast,but they did not confirm the existence of mesenchymal stem cell in fat tissue.Zuk et al successfully isolated cells with multi-directional differentiation potential which could differentiate into bone,cartilage,fat,muscle cells,etc.,and ADSCs were really known to people. Many studies hereafter also confirmed the multi-directional differentiation of ADSCs,including differentiation to cartilage cells,osteoblast,endothelial cell, muscle cell,or even nervous cells.In the study on pick-up rate of this cell,1×108 mononuclear cells could be isolated from 350ml fat tissue,and the amount is very considerable,so autogenic aspiration could obtain enough cells to perform tissue engineering study.So the experiment adopted tissue engineering technique,disposable biomaterial Plurolic-F127 and sodium hyaluronate as the scaffold materials, human fibroblasts as the seed cells of ADSCs to form cell-scaffold complex,and inject subcutaneously into the naked mice to build soft tissue,thus to provide parameters for clinics to investigate a more safe and effective wrinkle removing method.2.Materials and method 1 Human skin fibroblast and isolation of ADSCsSkin of experiment one was obtained from five male skin transplanting patients with an average age of 22 years old.Skin and fat of experiment two were obtained from three male patients,two of them were boys aging 10 and 13 respectively and whole thickness skin on abdomen and subcutaneous fat were collected.One case was a 24 years old male performing skin flap thinning,and residual skin and fat were collected.Isolate the skin and subcutaneous fat following the principle of sterile operation.Digest skin and fat with collagenase,isolate and collect the original skin fibroblast and isolate ADSCs for later experiment,place the finally obtained fibroblast and ADSCs in DMEM culture and passage,and collect the second generation of cells for seed cells.ADSCs were incubated at the concentration of 4×10~5 nucleated cells/cm~2,3×10~7 cells was usually inoculated in a 100ram plastic culture dish,add DMEM+10%FBS with low content of glucose,culture and passage,collect the cells of second generation as the seeds for determination.2 Preparation,implantation and materials of cell-biological scaffold complex30%plurolic-F127 and sodium hyaluronate were chosen as the scaffold materials and mixed with seed cells to prepare cell-biological scaffold complex.The experiment one had two groups:group(1)mix plurolic-F127 with fibroblast;group (2)simple fibroblast group.Inject 0.2 ml sample in backside subcutaneously in naked mice(five male)and form four spots,divide two spots for each group and collect sample 8 weeks later for examination and analysis.The experiment two had four groups:group(1)mix 25×10~6/ml fibroblast with sodium hyaluronate after diluted; group(2)mix 25×10~6/ml ADSCs with sodium hyaluronate after diluted;group(3) mix 12.5×10~6/ml fibroblast and ADSCs with sodium hyaluronate after diluted, respectively;group(4)simple sodium hyaluronate was set as the control group.Inject 0.2 ml sample in backside subcutaneously in naked mice(six male)and form four spots,divide one spot for each group and collect sample 8 weeks later for examination and analysis.3 Evaluate the weight and volume in the aspects of macro-observation, volume,Histological examinationt and immunohistochemistry for the obtained sample;Determination of Y chromosome in built tissue by RT-PCR technique,3.Results1 Macro-observation and analysis:collect sample 8 weeks later,no node or residue was found in the two groups of experiment one,one of the six naked mice of experiment two died,in the left five mice injected subcutaneously,soft and elastic node tissue was formed,and no node or residue was found in the control group.2 Determination of volume:The mean volume of group(1)was(0.020±0.007) ml which took 10%of the injected volume;the mean volume of group(2)was (0.037±0.094)ml which took 18.5%of the injected volume;the mean volume of group(3)was(0.035±0.012)ml which took 17.6%of the injected volume.It was demonstrated in the statistical analysis that there were significant differences between the volume of group one and group two or three,P＜0.05,while no difference was observed with the volume of group(3),P＞0.053 Histological examination:results of experiment two:there was fibrous coating tissue around group(1),(2)and(3)after HE dyeing,and there were a little inflammatory cells infiltrated in the fibrous tissue,irregular arrange of intermediate tissue,complete decomposition of biological materials,no new vessels growing. Masson trichrome stain could dye collagen into green,elastic fiber into brown, muscle fiber into red,and nuclei into blue dark,it demonstrated a small number of collagen fibers around the formed tissues of group(1)and(2),no collagen fibers in the center,while there were a small amount of elastic fiber or muscle fiber around the formed tissue in group(3),and a large number of irregularly arranged collagen fibers in the center of the formed tissue.Oil Red dyeing indicator showed the fat tissue in red,which demonstrated there was a little fat around the formed tissue of group(1), (2)and(3),but no fat tissue in the center.4 Immunohistoehemistry of collagen typeⅠ:the results indicated there was collagen typeⅠin the formed tissue in group(1),(2)and(3).5 Determination of Y chromosome in built tissue by RT-PCR technique:the results demonstrated two bands of 294bp and 494bp in the formed tissue of group(1), (2)and(3)of experiment two and the positive control group,only one band of 500bp was obtained in the skin tissue of naked female mice,which indicated the tissue of the experiment groups was formed by the implanted cell-biological scaffold complex.4.Conclusion1 Tangible tissue can not be built by adopting tissue engineering technique with disposable biological material 30%Plurolic-F127 as the scaffold material and human fibroblast(25×10~6cells/ml)as the seed cells.2 Tissue engineering soft tissue can be built by applying tissue engineering technique with sodium hyaluronate as the scaffold material and human fibroblast as the seed cells.3 Tissue engineering soft tissue can be built by applying tissue engineering technique with sodium hyaluronate as the scaffold material and mixed cells of human fibroblast and ADSCs as the seed cells.4 Good tissue engineering soft tissue can be built by applying tissue engineering technique with sodium hyaluronate as the scaffold material and ADSCs as the seed cells. 5 Injecting dermal filling material can be built by applying tissue engineering technique with sodium hyaluronate as the scaffold material and human concentrated fibroblast or Adipose derived stem cell(ADSCs)as the seed cells,and the ADSCs have more advantages when applied as the seed cells.This provides a new safe and effective method to remove wrinkles in clinics.