【欧洲整形美容技术】.doc

WOUND HEALINGAdipose Stromal Cells and Platelet-Rich PlasmaTherapies Synergistically IncreaseRevascularization during Wound HealingMatthew W. Blanton, M.D.Ivan Hadad, M.D.Brian H. Johnstone, Ph.D.Julia A. Mund, B.A.Pamela I. RogersBarry L. Eppley, M.D.,D.M.D.Keith L. March, M.D., Ph.D.Indianapolis, Ind.Background: The authors examined the efficacy of adipose stem cells, whensupplied either alone or in platelet-rich fibrin gels, to improve wound healing.Methods: A porcine full-thickness woundmodel was used to compare six topicaltreatments: platelet-poor plasma; platelet-rich plasma; autologous adipose stemcells plus platelet-poor plasma; autologous adipose stem cells plus platelet-richplasma; allogeneic adipose stem cells containing green fluorescent protein plusplatelet-poor plasma; and saline (control). One week after isolation, adiposestem cells were applied to full-thickness wounds on the paraspinal and thoracicregions of three pigs (44 wounds per pig; each treatment was applied to eightseparate wounds). Each wound wasmonitored over 21 days for closure, cosmesis,and histopathology.Results: There was no significant difference in the reepithelialization rate, buttreatments containing adipose stem cells demonstrated increased microvesseldensities (31.75 5.73 vessels/cm2versus 7.93 3.61 vessels/cm2) comparedwith groups without adipose stem cells. Wound cosmesis was improved in theadipose stem cell plus platelet-rich plasma group compared with other treat-ment groups (p 0.05). Vascular endothelial growth factor levels detected inmatrices containing adipose stem cells were approximately 7-fold higher com-pared with platelet-rich plasma or platelet-poor plasma (p 0.05). Localizationof transgenic green fluorescent protein plus adipose stem cells indicated in-corporation near neovasculature.Conclusions: In normal healing wounds, adipose stem cells appear to enhancethe healing process only when provided in a fibrin gel vehicle containing anumber of complementary wound-healing trophic factors. Perivascular adiposestem cell localization suggests a function in enhancing blood supply throughproviding physical and paracrine support to newly forming vessels. (Plast.Reconstr. Surg. 123 (Suppl.): 56S, 2009.)Despite modern advances in wound closuretechniques and devices, and wound main-tenance, there is still a critical need for newmethods of enhancing the healing process toachieve optimal outcomes. One of the promisingyet clinically challenging areas of recent therapeu-tic development involves topical application ofgrowth factors to enhance the normal healingprocess.1Many of these new therapies have in-volved the provision of individual trophic factorswith defined biological activities. They have a re-stricting growth factor release that is dependenton matrix loading, thus potentially limiting theiroverall ability to affect healing. Potentially moredesirable for optimal wound healing would betherapies that augment the normal healing re-From the Department of Surgery, Division of Plastic Surgery,Indiana University School of Medicine; the Krannert Insti-tute of Cardiology; the Indiana Center for Vascular Biologyand Medicine; and Ology M.D. Plastic Surgery.Received for publication November 29, 2007; accepted Au-gust 13, 2008.Presented at the 52nd AnnualMeeting of the Plastic SurgeryResearch Council, in Stanford, California, June 20 through23, 2007.Copyright 2009 by the American Society of Plastic SurgeonsDOI: 10.1097/PRS.0b013e318191be2dDisclosure: None of the authors has any commer-cial associations or financial disclosures that mightpose or create a conflict of interest with informationpresented in this article.www.PRSJ 56Ssponse by continuously supplying a mixture offactors mimicking the natural milieu. Plateletscontain large stores of cytokines and growth fac-tors that are normally released during clot forma-tion at wound sites. Concentrates of platelets fromplasma (platelet-rich plasma) are used for a widevariety of surgical applications, particularly in cos-metic and maxillofacial procedures.2Therapeutic effects of platelet-rich plasma arebelieved to occur through the provision of con-centrated levels of platelet-derived growth factors,such as platelet-derived growth factor (PDGF)-BB,transforming growth factor (TGF)-1, vascular en-dothelial growth factor (VEGF), and epidermalgrowth factor (EGF).2In addition, the fibrin ma-trix that is generated on activationmay potentiallyaid in tissue repair by providing a scaffold fortissue ingrowth.Adipose-derived stem (stromal) cells are anabundant population of pluripotent cells found inthe stroma of adipose tissues that have been shownto differentiate in vitro into various cell lineages,including osteogenic, myogenic, neurogenic, andhemotopoietic.3,4We have also demonstrated thatadipose stem cells are a robust source of bioactivegrowth factors that contribute to recovery fromischemic damage through preservation of skeletalmuscle and restoration of blood flow.3The po-tential for therapeutic translation using adiposestem cells is high given the ease with which theycan be harvested in high yield using simple, min-imally invasive lipoaspiration procedures and re-applied as an autologous therapy with minimalneed for manipulation or expansion.5We hypothesized that an improved tissue de-fect repair or augmentation of underlying supporttissues would be gained by mixing platelet-richplasma (or other matrices) with autologous adi-pose stemcells. This study thus examined the ben-eficial effects of treating full-thickness woundswith adipose stem cells immobilized in fibrin ma-trices derived from platelet concentrates.MATERIALS AND METHODSAutologous Adipose Tissue HarvestFemale Yorkshire pigs (n 3) weighing 40 to45 kg were sedated with amixture of ketamine (10mg/kg) and midazolam (1 mg/kg) followed byendotracheal intubation and maintained under asurgical plane of anesthesia with 1 to 2% isoflu-rane and a 50:50 mixture of nitrous oxide andoxygen (3 to 5 liters/minute) for the adipose har-vest. The dorsal hair was removed with hair clip-pers and the skin was swabbed clean with chlor-hexidine and 70% isopropyl alcohol solution.One 10-cm incision was made into the dorsalhump of each pig to a depth of 3 to 4 cm followedby adipose tissue excision (15.7 4.5 g) with a no.10 blade scalpel. The fat was transferred to sterile50-ml conical tubes (Fisher Scientific, Pittsburgh,Pa.) and transported promptly at room tempera-ture for processing. Wound hemostasis was main-tained with electrocautery followed by reapproxi-mation of the deep dermal tissue with interrupted3-0 Vicryl (Ethicon, Inc., Somerville, N.J.) and arunning 4-0 Vicryl subcutaneous closure. Each in-cision was covered with an occlusive dressing (Te-gaderm; 3M Health Care, St. Paul, Minn.).Preparation of Adipose Stromal CellsThe adipose tissue was minced into smallpieces (approximately 1 cm3) and digested in 50ml of buffer containing 2 mg/ml type I collage-nase (Worthington, Lakewood, N.J.) with inter-mittent shaking in a water bath at 37C for 180minutes.Digestion was neutralized by the additionof culture medium (Dulbeccos Modified EagleMedium, 10% fetal bovine serum, penicillin/streptomycin, amphotericin-B, and gentamicin).This cell suspension was centrifuged (300 g for 7minutes at 25C) followed by removal of the su-pernatant and resuspension of the cell pellet infresh Dulbeccos Modified Eagle Medium culturemedium with 10% fetal bovine serum and peni-cillin/streptomycin, amphotericin-B, and genta-micin. This resuspension was filtered with a100-m nylon cell strainer (GIBCO, Carlsbad,Calif.) followed by another centrifugation (300 gfor 7 minutes at 25C) and supernatant removalwith the resultant cell pellet used for culture. Be-fore the cells were placed in the culture flasks, theywere counted with a hemocytometer and viabilitywas determined using trypan blue exclusion. Onreaching 90 percent confluence, the isolated cellswere trypsinized, counted, and subcultured fortwo passages using a 1:5 plating ratio. At passage2 (7 days), cells were trypsinized, counted, andprepared for the appropriate treatment applica-tions.Preparation of Wound Treatment CombinationsOn the day of surgery, platelets were separatedfrom 55 ml of autologous peripheral blood takenfrom the femoral artery of each pig. Briefly, theblood was mixed with anticoagulant citrate dex-trose solution A (Citra, Braintree, Mass.) andplaced into a Gravitational Platelet Separation de-Volume 123, Number 2S Revascularization and Wound Healing57Svice (GPS II; Biomet Biologics, Inc.,Warsaw, Ind.).The blood was separated by means of a single,12-minute centrifuge spin, reducing the total vol-ume to approximately 6 ml of platelet-rich plasmaand 30 ml of platelet-poor plasma per 55 ml ofseparated whole blood. Three milliliters of eitherplatelet-rich plasma (platelet count, 998 39.8 103/l) or platelet-poor plasma were admixedwith autologous adipose stem cells (cell count,18.3 1.87 105) to yield their respective treat-ment suspensions (platelet-poor plasma only,platelet-rich plasma only, adipose stem cells plusplatelet-poor plasma, adipose stemcells plus plate-let-rich plasma, and green fluorescent proteinadipose stem cells plus platelet-poor plasma) andeach was placed into a FibriJet surgical sealantapplicator (Micromedics, Germany). A 20-gaugeFibriJet dual cannula applicator tip was placed onall applicators. According to the GPS II instruc-tions, a solution of 5000 IU topical thrombin(Jones Pharma, Inc., Bristol, Va.) and 5 ml of 10%calcium chloride was placed in the complemen-tary compartment of the applicators using a 10:1volume ratio of platelet-rich plasma, platelet-poor plasma, and adipose stem cell treatmentcombinations to the thrombin/calcium chloridesolution.Wound Model and Treatment ProcedureSeven days after adipose tissue harvest, thesame three female Yorkshire pigs were weighed,sedated, intubated, prepared, and anesthetized asabove. After thoracic and dorsal hair was removedwith hair clippers and the skin cleaned with chlor-hexidine and 70% isopropyl alcohol solution, atemplate was used to define the wound sites, whichwere organized in four rows on the lateral paraspi-nal and thoracic areas (Fig. 1, above). Beforewounding, each wound site was demarcated witha sterile marker (Fig. 1, center) and the rows andcolumns were labeled with tattoo dye. A no. 15blade was used to excise a 1.5-cm2wound, takingcare not to injure the underlying musculature.The full thickness of skin and underlying subcu-taneous layers was removed and covered withmoist gauze to avoid desiccation. The wounds hadan average depth of approximately 7.9 1.2 mm.Each pig had 22 wounds created on the left andright sides, thus creating a total of 44 wounds perpig and a grand total of 132 wounds in the study(Fig. 1, below).Each wound was treated randomly with one ofthe following treatments: (1) adipose stem cellsand platelet-poor plasma; (2) adipose stem cellsand platelet-rich plasma; (3) platelet-poor plasma;(4) platelet-rich plasma; (5) green fluorescentprotein and adipose stem cells in platelet-poorplasma; and (6) saline. Each treatment was ap-plied to eight separate wounds. The wound wasthen quickly covered with an occlusive dressing(Tegaderm) to avoid runoff and treatment loss.Fig. 1. Wound placement and wounding patterns used. (Above)Schematic shows the relative anatomical location of the bilaterallywounded regions. (Center) The layout of the 1.51.5-cmfull-thick-ness wound sites (n22) that weremarked on the paraspinal andlateral thoracic skin regions of both sides of the pig. (Below)Appear-ance of a pig obtained immediately after creation of the wounds.The average thickness of each wound was 7.80.4mm.Plastic and Reconstructive Surgery February Supplement 200958SEach wounds volume was completely filled withtreatment (approximately 0.8ml). The thorax andabdomen were then covered with a snug-fitting,nonadherent tube gauze dressing followed by amodified cotton vest (Four Flags Over Aspen,Jamesville, Minn.) to further protect the wounds.The subjects were then awakened and allowed torecover appropriately. Of note, all animals re-ceived cephalexin (50 mg/kg) 30 minutes beforewounding and a fentanyl patch (25 g) for 72hours. Antibiotic prophylactic coverage was con-tinued for the duration of the subjects postoper-ative survival (21 days). Animal care was per-formed according to Guidelines for the Care and Useof Laboratory Animals published by the NationalInstitutes of Health, and the protocol was ap-proved by the institutional animal care committee.Wound AssessmentsThe wounds were examined on postwoundingday 3, 7, 14, and 21. The subjects were sedated witha subcutaneous injection of ketamine (10 mg/kg)and midazolam (1 mg/kg) followed by cleansingof the normal skin with saline only, with carefulattention paid to not de bride within the woundbed. Qualitative wound assessments were re-corded and individual digital photographs weretaken. Wounds were measured by ruler to thenearest millimeter, and open surface area was cal-culated and expressed as a percentage of the orig-inal wound area on the day of wounding. Punchbiopsies were performed on postwounding days 3,7, 14, and 21. These were 5 to 6 mm in width,mediolaterally oriented, and included approxi-mately 3 to 4 mm of nonwounded skin on bothsides of the wound. All biopsy samples were fixedin 10% neutral-buffered formalin, embedded inparaffin, sectioned, and then examined histolog-ically. At that time, all wounds, both those that hadundergone biopsy and those that had not under-gone biopsy, were redressed with adhesive andTegaderm dressings. Once a wound had under-gone biopsy, it was not included in the additionalassessments. The pigs were then allowed to re-cover from anesthesia.Macroscopic Evaluation of Wound HealingOn the final wound assessment day (day 21),a modified subjective wound evaluation score wasperformed to assess the quality of the scar. Twoindependent experienced observers scored thewounds on a five-point scale for wound color (pinkto purple/red), smoothness, and wound supple-ness while blinded to the treatment pattern. Theoverall score ranged from 1 (like normal skin) to5 (excessive scarring).6Histologic and Immunohistochemical Analysisof Wound HealingHematoxylin and eosin stains were used tovisualize cell infiltration and dermal tissue archi-tecture by a board-certified veterinary pathologistblinded to treatment modalities. For immunohis-tochemistry, the 5-m sections taken from theday-14 punch biopsy specimens were stained withanti-smooth muscle actin to determine vesseldensity. The sections were deparaffinized andstained according to a standard three-step immu-nohistochemical procedure.7The primary anti-body used was a monoclonal anti-smooth mus-cle actin clone (1A4; Sigma, St. Louis, Mo.). Afterwashing with phosphate-buffered saline contain-ing 0.05%Tween 20, samples were incubated withconjugated anti-mouse immunoglobulin G anti-bodies (Vector Elite Kit PK-6102; Vector Labora-tories, Burlingame, Calif.) followed by develop-ment with diaminobenzidine as substrate andcounterstaining with hematoxylin A. As negativecontrols, adjacent sections were stained with non-immune immunoglobulin G from the same spe-cies using the same dilution as the primary anti-body. No specific signal was noticed in thenegative controls. Sections from normal porcineskin served as positive controls.Image analysis of sections stained for -smoothmuscle actin analysis was performed using whitelight and a low-magnification objective (4). Im-ages were recorded with a color camera (QImag-ing RETIGA EXi FAST; Burnaby, British Colum-bia) attached to a Nikon Eclipse TE2000-5microscope. Vessel density was determined bycounting for vascular structures stained with-smooth muscle actin as outlined in the immu-nohistochemistry procedures above. Tissue areaand number of positively stained lumen-contain-ing vessels were measured using three randomfields per slide viewed at high-magnification(20). The images were viewed with ImageJ soft-ware, and blood vessels in each high-powered fieldwere marked and counted.Tracking Green Fluorescence ProteinLabeledAdipose Stem Cells in Wound Treatment Appli-cationGreen fluorescence proteinlabeled porcineadipose tissue was obtained from transgenic York-shire pigs expressing green fluorescence proteinunder control of the ROSA26 promoter, from theVolume 123, Number 2S Revascularization and Wound Healing59SNational Swine Resource and Research Center(University ofMissouri), followed by adipose stemcell isolation and nonautologous treatment prep-aration as described above. The green fluores-cence proteinadipose stem cell fluorescence wasverified after adipose stem cell isolation and be-fore treatment application. A total of 12 woundswere reserved for the allogeneic transgenic greenfluorescence proteinadipose stem cell treat-ments. Punch biopsy specimens were taken at thesame biopsy time points and underwent immuno-histochemical analysis. The 5-m sections weredeparaffinized and blocked with A/B block (Vec-tor) at room temperature for 15 minutes. Theprimary antibody was a mouse anti-human mono-clonal green f