| BackgroundAdipose-derived stem cell(ASC)is recognized for its capability of differentiation into fat,bone and cartilage since 2005[1].Clinical trials utilizing ASCs have then entered an exponential increase due to the immunomodulation function,multipotency and simple acquisition of ASCs[2-4].It is estimated that about 1%of the population is suffering from some form of chronic wound at any given time[5].In some clinical trials and preclinical models,ASCs were observed reparative effects demonstrating paracrine effect,alleviation of chronic inflammation and restart the wound homeostasis,thus strongly suggest a critical role in soft tissue injury healing[2,4,6].Since the adult stem cell’s differentiation and migration behavior haven’t fully understood by scientists,it is difficult for clinician’s to take full control and intervene of stem cell behavior after transplantation.To avoid risks of infection,aberrant or ectopic stem cell differentiation,even tumorogenesis,many country have promulgated regulations regarding stem cell and related products therapeutic use[7].In common,stem cell therapies include the utilization of chemicals(eg.collagenase)and animal bio-product(eg.fetal bovine serum)are strictly regulated required submission to administrative associations.Even that adipose-derived stem cell had great preliminary result from clinical trials relating chronic wound and other diseased condition,utilization of cultured and isolated stem cell are still lack of evidence quality and some inconsistent results and complication cases were seen.Therefore,in Jun.2015,the Chinese national health and family-planning commission had enacted the "Stem cell clinical trial regulation".It defined the cultured ASC and stromal vascular fraction(SVF)as "class Ⅲ medical practice" and strictly administered.Nowadays the stem cells and related products which earned most popularity(eg.ASC,SVF)all involve collagenase digestion and ex vivo culture,so such therapies had meet many obstacles in clinical translation like long submission time,high expenses,advanced apparatus and environment.Herein,in this project,we focus on designing a cellular product for chronic wound,eg.diabetic food,which involved no chemical reagent nor animal product.It had better be enriched stem cells,can be transplanted by minimal invasive mean and has less risk factor.Methods and resultsMicronized cellular adipose matrix(MCAM)injectable was prepared through selective extraction of connective tissue fractions in fat tissue only through mechanical minimal manipulation procedures.One gram of fat pads was cut into tiny pieces with surgical scissors(continuous fine mincing for 5 min).After transfer to a tube containing 2.5 ml cooled phosphate-buffered saline(PBS),the morcellated tissue was thoroughly shaken several times and then centrifuged(800 x g,5 min).MCAM was extracted as tissue sediment,and floating fatty tissue was also sampled.Whole-mount imaging highlighted functional features of MCAM(Figure 1B).As noted by scanning electron microscopy,MCAM was lacking in mature adipocytes(i.e.,the large,round wheat germ agglutinin+ green cells seen in floating fat).However,hoechst+ nucleated cells persisted in MCAM at rather high density.In addition,MCAM retained branches and segments of vessels and identifiable capillaries.Fluorescence-activated cell sorting analyses were performed to delineate cellular composition,once SVFs were individually isolated from floating fat and MCAM through collagenase digestion.SVFs of fat and MCAM were characterized through a combination of surface markers:hematopoietic cells(mainly white blood cells;CD45+),vascular endothelial cells(CD45-/CD31+/CD34+),ASCs(CD45-/CD31-/CD34+),and other cells(CD45-/CD31-/CD34-).The SVF of floating fat consisted largely of CD45-nonhematopoietic cells(～75%),with ASCs accounting for half of the nonhematopoietic fraction.The SVF of MCAM contained all four subpopulations(including ASCs)albeit in differing ratios.ASCs accounted for 13%of MCAM-SVF cells,with hematopoietic cells,vascular endothelial cells and other cells constituting 57.9,0.7 and 28.3%,respectively.By adipogenic,chondrogenic and osteogenic differentiation assay,MCAM contained ASCs with similar differentiating capability to those obtained from regular adipose tissue.Subcutis of full-thickness dorsal skin ulcers inflicted in diabetic mice was injected at wound peripheries with MCAM prepared from wild-type mice,using pBS injections as control.Healing of diabetic ulcers was significantly more rapid with MCAM(vs pBS)injection,with 64%smaller wound size on day 4(p = 0.0082)and 65%smaller size on day 7(p = 0.0043).At the close of week 2,closure was essentially completed in MCAM-treated ulcers,whereas wound beds of pBS-treated ulcers remained hyperemic.DiscussionWound healing impairment in diabetic patients is a significant clinical issue affecting millions of patients worldwide.The major underlying pathology is noted to be chronic inflammation and ischemia based on peripheral vascular dysfunction,where tissue-resident stem cells are considered to be depleted.Although numerous products for wound dressing with bioactive ECM or growth factors are available,the clinical effects on diabetic ulcer are very limited.Thus,we sought to characterize and evaluate MCAM,which contains human adipose derived stem cells and vascular endothelial cells in their original niche of ECM,as a potential therapeutic tool for stem cell-depleted pathological conditions[4,9].ASCs in MCAM occupy their original ECM niche,ready to assume original roles.As shown by whole-mount staining and flow-cytometry,vessels and capillaries of MCAM,as well as ASCs and other stromal cells,retained their natural states and positions.Functional aspects of ASCs in MCAM were also well preserved,as partly indicated by differentiation assays.ASCs are thought to be potent sources of trophic factors and to have multilineage differentiation capacity.Nonetheless,in transplantation of dissociated(suspended)ASCs,local retention was found to be poor(many disappearing in 1 week),thus nullifying therapeutic intent or resulting in unexpected stem cell behaviors.As the size of injectable MCAM ranges 100-400μm,the ECM of MCAM may also provide better mechanical support and anchorage for ASCs to avoid their rapid and seemingly detrimental migration.In these cases,ASCs alone or in conjunction with ECM(as in MCAM)may provide sufficient therapeutic effect,as we have shown.According to the International Diabetic Federation Diabetes Atlas 2015,there’re 114 million diabetes patients in China,among which 15%will develop diabetic ulcer in course of disease.Diabetes food requires repetitive long term wound care,and types of dressing were normally used.However the therapeutic effect is usually less than optimal due to the persistent local ischemia.In contrast,mesenchymal stem cell/adipose-derived stem cell have demonstrated better efficacy in clinical trials[10,11].Diabetes is considered a great market for stem cell therapy,and many different types of medical service provide had entered the field while many of the participants do not meet the requirement of Good Manufacture Practice(GMP).MCAM is superior for the undemanding preparing procedure.We believed that after the method is refined,it can be prepared at bedside and directly transferred to patient in the same surgery with liposuction.Furthermore,this ASC enriched product is generated with only "minimal manipulation" so many risks can be avoided and thus bypasses the time-demanding and complicated approval procedures before its clinical application.ConclusionsMicronized cellular adipose matrix is an autologous/allogeneic injectable of bioactive ECM and functional cellular components generated through minimal manipulation of adipose tissue.Smaller-sized and more homogeneous(50-100 μm)particles may be preferable as injectables,but as we already learned,such preparations may jeopardize the viability of cells.Further studies are needed to verify the hypothesized mechanism underlying the efficacy of MCAM and further optimization of preparation methods.Therapeutic effect of MCAM in other stem cell-depleted states,including irradiation damage and fibrous diseases will also need to be tested.Besides,we still need to elucidate the specific functions of adipocytes in clinical fat grafting and to see what benefits of fat grafting will be lost when we use the product MCAM is an autologous/allogeneic injectable of bioactive ECM and functional cellular components generated through minimal manipulation of adipose tissue. |
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