Yang CHEN,Liang-ping SONG,Yu HE,Ying CHEN,Zheng PENG

1Department of Plastic and Aesthetic Surgery,Fuzhou Dermatology Hospital,Fuzhou City,Fujian Province,350025,China

2The Vaccination Department of Fuzhou International Travel Healthcare Center,Fuzhou City,Fujian Province,350001,China The first author:Yang CHEN,M.D.Prof.chief physician,specialized in basic application of stem cell and tissue engineering,E-mail:cyfuzhou2008@126.com

ABSTRACT Objective To investigate the effects of combination of PRP,PRF and adipose-derived stem cells(ADSC) for wound healing of chronic skin ulcer,and provide evidences for clinical application.Methods Twenty-four SD rats aged 6-8 weeks were used in this study.5 grams of groin adipose tissue were derived from each rat.After collagenase digestion,mesenchyma stem cells derived from adipose tissue (ADSCs) were determined by immunocytochemical method and flow cytometry.After continuous culturing,the 3th passage of ADSCs in good condition was prepared for transplantation.Ten milliliters of whole blood were extracted from one rat for making PRP and PRF backup.Full thickness skin defect model with six wounds with an area of 1.5cm*1.5cm on each side of the spine on the back was established in rats after anesthesia.Then the rats were randomly divided into six groups.Skin wounds were injected with PRF combining ADSC in group A,PRF in group B,ADSC in group C,ADSC combining PRP in group D,PRP in group E,and same amount of normal saline(NS) in group F.Histological analysis was performed to evaluate the velocity and the quality of the wound healing 7 days,14 days,21days and 28 days after treatment,and comparing the regeneration of epidermis,corium,fibroblast,blood vessel and skin appendages respectively.Results The woulds in each group healed well,without symptoms of infection.Skin wounds were almost healed in 21 days,and no significant difference in healing velocity was found among these groups.There were no apparent inflammatory cells in 7 days.The wounds were filled with granulation formed by proliferation of connective tissue with visible new blood capillary and repair cells like fibroblast.Epidermis grew out and became thick,and gradually grew to the wounds,with active proliferation of re-epithelization.Re-epithelization was completed in 14 days,with granulation tissue covered by epidermis and fibrosis showing a significant increase in collagenous fiber,and visible skin appendages like new hair follicle,glandula sebacea,sweat gland and corneum.Appendages like hair follicle,glandula sebacea,sweat gland and corneum increased in 21 days,and granulation tissue gradually formed scar tissue and entered remodeling phase.There was no significant changes from 21 days to 28 days,and the tissues were entering a long remodeling phase.Healing status:Group D,A and C could be observed with more complete epidermis,new blood vessel and new hair follicle.Among them,Group D was most significant,while group A,C,E ,B successively and group F had the slowest epidermal migration and the least new hair follicle.The velocity and the quality of these groups were as follows:D＞A＞C＞E＞B＞F,indicating that ADSC combining PRP can improve quality in wound healing of skin ulcer effectively.Conclusions After the general and pathological observation on the effects,the velocity and the quality for the skin ulcer healing of SD rats on the back were as follows:ADSCPRP＞ADSCPRF＞ADSC＞PRP＞PRF＞NS,indicating that PRP or PRF combining ADSC can improve quality in wound healing of skin ulcer effectively.

KEY WORDS Platelet rich plasma;Platelet rich fibrin;Adipose-derived stem cell;Skin ulcer

Chronic skin ulcers caused by diabetes,wound infection,insufficient arterial blood supply,venous obstruction and compression are common and frequently occurring diseases in clinic[1].Epidemiological surveys have shown that diabetic foot skin ulcers are by far the most common.In China alone,there are more than 10 million diabetic foot patients,most of whom are elderly.Due to the poor ability of tissue regeneration and repair,the ulcer is prolonged and does not heal,or after healing and rapid recurrence,resulting in refractory ulcer.The healing of refractory ulcer wounds has always been a difficult problem in medicine[2,3].The adipose derived stem cells(ADSCs) is a kind of pluripotent stem cells having multiple differentiation potential.ADSCs have become the seed cells commonly used in tissue engineering research with the advantages of abundant sources,easy isolation and culture,no immune rejection in autoapplication and no risk of secondary infectious diseases[4-7].Recent studies have shown that ADSCs can promote the healing and functional recovery of heart,lung,skin,muscle and other tissue wounds[8,9].Plateletrich plasma(PRP) is a concentrated form of platelets obtained by centrifugation of whole blood.When activated,it can release a large number of growth factors with high concentration,stimulating cells to proliferate,differentiate and form new tissues,promoting the repair and healing of wounds[10-13].Platelet-rich fibrin(PRF) is a new biomaterial rich in cytokines and growth factors from autogenous sources,being regarded as a new generation of platelet concentrate.Its molecular structure resembles that of a natural blood clot,providing a place for tissue cells to migrate,multiply and differentiate[14-16].Therefore,we conducted an experimental study on the treatment of skin ulcers by PRP and PRF combined with ADSCs to observe their effect on the repair of skin ulcers,so as to provide scientific basis for their clinical application.

MATERIALS AND METHODS

Experimental Instrumen

Bechtop (Suzhou zhijing purification equipment Co.Ltd,China),CO2Cell culture shelves (Thermo Fisher Scientific,USA),Inverted microscope,Fluorescence microscope (Olympus Corporation,Japan),Rotary Microtome (Leica Microsystems Corporation,Germany),Water bath-slide Drier (tec 2500,Haosilin Corporation,China),Microscope (BX43,Olympus Corporation,Japan),Pipettor (DLAB Scientific Co.Ltd,China),Cellculture dish (Thermo Fisher Scientific,USA),Low speed autobalancing centrifuge (TD Z4-WS,Cence Co.Ltd,China),High speed tabletop refrigerated centrifuge(TGL-16,Cence Co.Ltd,China)),Table-top low speed centrifuge (TDZ4-WS,Cence Co.Ltd,China)),Inverted bioluminescence microscope (IX73,Olympus Corporation,Japan).

Drugs and reagents

Dulbecco’s Modified Eagle Medium (Gibco,Carlsbad,CA,USA),Stem cell complete medium,Trypsin,Collagenase I (Gibco,Carlsbad,CA,USA),Fetal bovine serum (FBS,Gibco,Carlsbad,CA,USA),HaCaT Cell(Hangzhou HIBIO technology Co.Ltd.,China),Fibrin glue (Guangzhou bioseal co.Ltd.,China);Tegaderm(Minnesota Mining and Manufacturing,USA),Antibody(AnaSpec Co.Ltd.,USA);Hematoxylin,Eosin (Sigma,USA),Penicillin&Streptomycin (Meilunbio CO.Ltd.,China),DAPI,D9542 (Sigma,USA),Cytokeratin antibody (Proteintech Group,Inc,USA),Normal Saline(Cisen Pharmaceutical Co.Ltd.,China)

Laboratory animals and breeding

24 SD rats were purchased from Shanghai SLAC Laboratory Animal Co.Ltd (SCXK:2017-0005,Certification:20170005004523).The drinking water was ultra-pure water.The laboratory animal room license number was YXK2015-0008 (Zhejiang).The rats were fed at 20-25°C and 40-70% relative humidity.And the rats were fed adaptively for one week before the experiment.The fodder was provided by the Jiangsu Xietong biological engineering co.Ltd according to the GB14924.3-2010.

Isolation and culture of adipose tissue-derived stem cells (ADSCs) for SD rats

Four 8-week-old SD rats were given anesthetic by intraperitoneal injection according to 10g/L pentobarbital sodium.After having been sterilized by 750ml/L ethanol,5g inguinal adipose tissue from each SD rat was extracted under aseptic conditions,and blood vessels and other tissues were removed thoroughly then.The fat mass was washed repeatedly with PBS containing penicillin &streptomycin.The fat was cut into 1 mm3 and digested for 50 min at 37°C with 12.5ml collagenase type I in 100 r/min horizontal rotators,centrifugated at 800 r/min for 5 min then,and the top layer of shredded fat and supernatant was discarded.The precipitate was made into single-cell suspension using DMEM medium containing 100ml/L fetal bovine serum for cell counting.At the density of 2×105/ml,the cells were inoculated in the culture bottle,placed in a 37°C,50ml/L CO2incubator for routine culture,and the solution was changed after the primary generation for 2 days.When the cells were 80%full,they were digested by trypsin and EDTA for passed culture.

Epidermal induction differentiation and immunofluorescence identification for ADSCs from SD rats in vitro

The third generation of ADSCs cells were digested with Typsin at 0.25% and centrifuged at 1000rpm for 5min.They were counted under the counting board.A 24-well board was laid and 4 holes were selected to add 5×104 cells into each hole,and then they were put into the incubator for static culture.After 80% of ADSCs were grown,two holes were cultured as normal control,and the other two were cultured with medium containing HaCaT cell supernatant and 10% epidermal growth factor (EGF)as experimental group.The ADSCs were cultured for one week,changing the medium every 2d.Then,the medium was removed,and cells were rinsed with PBS 3x3min,fixed by 4% paraformaldehyde at indoor temperature for 20min and rinsed with PBS 3x3min again.Having been ruptured their membranes with 0.1% Triton X-100 for 20 min at indoor temperature,they were rinsed with PBS 3x3min then.Having been blocked with 10% FBS for an hour at indoor temperature and rinsed once with PBS,they were incubated by primary antibodies CK19 with appropriate dilution ratio (1:300) at 4°C overnight and rinsed with PBS 3x3min again.Then,they were incubated with the Donkey anti-Rabbit IgG (HL) ReadyProbes?Secondary Antibody（1:800）at 4°C for an hour avoiding light and rinsed with PBS 3x3min.10ng/ml DAPI was added into and incubated without light for 10min at 4°C,rinsed with PBS 3x3min then.Finally,photographs were taken under an inverted fluorescence microscope,completing immunofluorescence detection.

Lipogenic induction differentiation and staining identification for ADSCs from SD rats in vitro

The third generation of ADSCs cells were inoculated in 6-well plates with glass slides,cultured by DMEM medium containing 100ml/L FBS.After 80% of ADSCs were grown,the medium was replaced by lipid induction liquid (containing 1μmol/L hexadecadrol,10μmol/L insulin,200μmol/L indometacin,0.5μmol/L IBMX and 10%FBS DMEM or F12 medium),observing cell morphology at the same time.After having been cultured for 20d,they were fixed by 4% paraformaldehyde at indoor temperature and stained by Oil red O.

Osteoblastic induction differentiation and staining identification for ADSCs from SD rats in vitro

The third generation of ADSCs cells were inoculated in tissue cultrue plate,cultured by osteogenic induction fluid containing 50μmol/L ascorbic acid,10mmol/Lβ-disodium glycerophosphate,0.01μmol/L Vitamin D and 100ml/L FBS DMEM or F12 medium ),which would be changed once for 3-4 days.When cultured for 10 days,some cell slides were taken for immunocytochemical staining of type I collagen.After culture for 21 days,partial cell slides were taken and stained with Von Kossa to observe the formation of mineralized nodules.

Chondroblastic induction differentiation and staining identification for ADSCs from SD rats in vitro

The third generation of ADSCs cells were inoculated in tissue cultrue plate,cultured by chondroblastic induction fluid containing 10%FBS,10μg/L TGF-β1,6.25mg/L insulin,6.25mg/L transferrin,50μmol/L L-Ascorbate-2-phosphate),which would be changed once for 2 days.After culture for 21 days,alcine blue staining was performed to observe the formation of blue nodules.

Preparation for PRP

10ml venous blood was extracted from SD rats and disodium citrate was used for anticoagulation.The improved Appel method was used to separate and extract PRP for standby,and the whole blood and PRP platelet counts were performed simultaneously to ensure that the number of platelets in PRP was more than 4 times that of the whole blood[17,18].

Preparation for PRF

10ml venous blood was extracted from SD rats and placed in a sterile test tube without antithrombin.Immediately,the test tube was centrifuged at 3000r/min for 10min.After standing still,the blood sample was divided into three layers,the red blood cell fragments at the bottom,the pale-yellow clarifying liquid platelet plasma at the upper fraction,and the pale-yellow gel,platelet-rich fibrin(PRF) in the middle layer was transferred into a new tube.The supernatant and the red blood cells at the base of the gel synthesis had been removed,the primary PRF gel was obtained.Later,it was placed in a dry and sterilized container for 10min to make it contract naturally and release the serum,or to adsorb the serum with sterile gauze.Meanwhile,the platelle-rich fibrin membrane with certain shape,elasticity and toughness was prepared by extrusion molding[19,20].

Preparation and treatment of animal skin ulcer model

After anesthesia,the six full-thickness skin defect ulcer wounds with 1.5cm×1.5cm models were made on both sides of the SD rats’ spine.They were randomly divided into six groups:A,B,C,D,E and F,and were performed medication respectively.Group A was treated with PRF and ADSCs,group B with PRF,group C with ADSCs,group D with PRP and ADSCs,group E with PRP,group F with equal amount of normal saline(NS).Biopsies were taken from ulcer wounds at 7,14,21,and 28 days after treatment for histopathological examination and to observe the rate and quality of ulcer healing,and to compare the regeneration of epidermis,dermis,fibroblasts,blood vessels,and skin accessory structures.

RESULTS

Observation and detection of multidirectional induced differentiation for ADSCs

Microscopically,ADSCs showed long spindle shaped growth with uniform size,similar to fibroblasts(figure 2).After ADSCs had been induced to differentiate by supernatant of HaCaT cell culture with epidermal growth factor(EGF) in vitro,the keratin content of epidermal marker protein increased significantly,indicating successful cell induction(figure 3).ADSCs had been lipidinduced for 20d,the cells were filled with round lipid droplets,showing single or multiple cells,and the oil red O staining showed orange color,indicating mature lipid droplets(figure 4A).Having been osteoblastic-induced for 21d,the cells clustered and opaque nodules formed on its surface.Alizarin red staining showed red indicating the formation of calcium nodules(figure 4B).Having been chondroblastic-induced for 21d,the cells were clustered into clusters.Azin blue staining showed blue nodules,indicating the formation of chondroblast nodules(figure 4C ).All these suggested that ADSCs could differentiate into adipocytes,bone cells and chondrocytes in vitro,with multidirectional differentiation potential and stem cell characteristics.

Healing status of skin ulcer wounds in groups A,B,C,D,E and F

Fig.1 Model of skin ulcer on SD rats’ back

Fig.2 ADSCs show fusiform(×40)

The woulds in each group healed well,without symptoms of infection.Skin wounds were almost healed in 21 days,and no significant difference in healing velocity was found among these groups.There were no apparent inflammatory cells in 7 days.The wounds were filled with granulation formed by proliferation of connective tissue with visible new blood capillary and repair cells like fibroblast.Epidermis grew out and became thick,and gradually grew to the wounds,with active proliferation of re-epithelization(figure 5,6).Re-epithelization was completed in 14 days,with granulation tissue covered by epidermis and fibrosis showing a significant increase in collagenous fiber,and visible skin appendages like new hair follicle,glandula sebacea,sweat gland and corneum(figure 7,8).Appendages like hair follicle,glandula sebacea,sweat gland and corneum increased in 21 days,and granulation tissue gradually formed scar tissue and entered remodeling phase(figure 9,10).There was no significant changes from 21 days to 28 days,and the tissues were entering a long remodeling phase(figure 11,12).Healing status:Group D,A and C could be observed with more complete epidermis,new blood vessel and new hair follicle.Among them,Group D was most significant,while group A,C,E,B successively and group F had the slowest epidermal migration and the least new hair follicle.The velocity and the quality of these groups were as follows:D＞A＞C＞E＞B＞F,indicating that ADSC combining PRP can improve quality in wound healing of skin ulcer effectively.

Fig.3 Epidermal induction differentiation and immunofluorescence identification for ADSCs Note:A1、B1、C1,control group； A2、B2、C2：experimental group

Fig.4 Lipogenic,osteoblastic and chondroblastic induction differentiation and identification for ADSCs Note:A:lipogenic induction differentiation and positive Oil red O staining for ADSCs(×200);B:osteoblastic induction differentiation and positive Alizarin red staining for ADSCs(×200);C:chondroblastic induction differentiation and positive Alcian blue staining for ADSCs(×200)

DISCUSSION

Wound healing is a complex and orderly biological process,showing high integration and network,which involves the mutual coordination and participation of inflammatory cells,repair cells,extracellular matrix,cytokine and many factors regulated by the body.It is generally believed that the four main reasons for that wound repair delays or even stops are as below:1.wound infection or existence of necrotic tissue;2.microcirculation disturbance of wound blood supply;3.the decrease in the quantity of local growth factor and the decline of its activity or the disorder of network regulation of growth factor;4.the change and excessive apoptosis of repair cell scaffold,and the change in receptor structure of cell membrane,which leads to uncoupling between growth factor and receptor.ADSCs are adult stem cells from adipose tissue and have become promising seed cells in regenerative medicine research for the advantages like rich and convenient resource,small wound,easy isolation and culture in vitro,high flexibility,autologous application without immunological rejection and risks of secondary infection,etc.This study finds that ADSC has a certain role in promoting wound healing,indicating that this is probably because that stem cells can replenish and differentiate into multilineage cells needed in wound healing of skin ulcer.Moreover,this study also finds that ADSC can improve the quality in wound healing of skin ulcer,increase the quantity of fibroblasts in granulation tissue in the wound with active function,and increase the vessel density and make the growth of new epidermis more complete and thicker.

Fig.5 Healing of back skin ulcer in six groups of SD rats after 7 days of treatment Note：Group A,PRF and ADSCs;Group B,PRF;Group C,ADSC;Group D,ADSCs and PRP;Group E,PRP;Group F,NS.（HE staining×100）

Fig.6 Pathological biopsy section in six groups of SD rats were examined under light microscope after 7 days of treatment Note：Group A,PRF and ADSCs;Group B,PRF;Group C,ADSC;Group D,ADSCs and PRP;Group E,PRP;Group F,NS.（HE staining×100）

Fig.8 Pathological biopsy section in six groups of SD rats were examined under light microscope after 14 days of treatment Note：Group A,PRF and ADSCs;Group B,PRF;Group C,ADSC;Group D,ADSCs and PRP;Group E,PRP;Group F,NS.（HE staining×100）

Fig.9 Healing of back skin ulcer in six groups of SD rats after 21 days of treatment Note：Group A,PRF and ADSCs;Group B,PRF;Group C,ADSC;Group D,ADSCs and PRP;Group E,PRP;Group F,NS.（HE staining×100）

Fig.10 Pathological biopsy section in six groups of SD rats were examined under light microscope after 21 days of treatment Note:Group A,PRF and ADSCs;Group B,PRF;Group C,ADSC;Group D,ADSCs and PRP;Group E,PRP;Group F,NS.(HE staining×100）

Fig.11 Healing of back skin ulcer in six groups of SD rats after 28 days of treatment Note：Group A,PRF and ADSCs;Group B,PRF;Group C,ADSC;Group D,ADSCs and PRP;Group E,PRP;Group F,NS.（HE staining×100）

Fig.12 Pathological biopsy section in six groups of SD rats were examined under light microscope after 28 days of treatment.Note：Group A,PRF and ADSCs;Group B,PRF;Group C,ADSC;Group D,ADSCs and PRP;Group E,PRP;Group F,NS.（HE staining×100）

PRP is the concentrate of platelet,which can release a large quantity of high concentration of growth factor after activation,like transforming growth factor-β(TGF-β),Bone morphogenetic protein(BMPs),platelet derived growth factor(PDGF),Insulin like growth factor(IGF),vascular endothelial growth factor(VEGF),epidermal growth factor(EGF),and fibroblast growth factor(FGF),etc.These growth factors can induce epidermal cells to migrate,proliferate,differentiate,and promote proliferation of collagen III and IV effectively,and therefore accelerate wound healing.Moreover,the proliferation and differentiation of dermal stem cells have direct and positive correlation with platelet concentration.Cells proliferate and differentiate only when plasma concentration is four to five times normal platelet concentration and only in a good environment.

Current studies prove that the synergetic effect of multiple growth factors contained in PRF can promote the synthesis of collagen I and fibronectin,promote the chemotaxis and proliferation of stromal stem cells,and stimulate the proliferation and differentiation of fibroblasts and vascular endothelium,which plays an important role in regulating the process of tissue healing.The 3D framework of PRF acts as matrix which provides favorable places for the attachment,migration and differentiation of cells,and histiocytic cells and stem cells in circulating blood can grow in it more quickly.Moreover,this structure has good elasticity and large pores,which is favorable to the dispersion of nutrients and oxygen and accelerating healing process.A large quantity of stagnant platelet and grow factor establish chemical bond with fibrin,and this special structure has a strong affinity for many types of grow factor,which results in a slow release of grow factor and extends the actuation duration of PRF in the wound.Studies have also shown that the 3D structure of PRF can drag most leukocyte,regulate inflammation,and release multiple cytokines slowly and sustainedly,which have a positive effect for tissue repair.

This study finds that ADSC alone or PRP,PRF alone can also promote wound healing to some degree,but less effective than combining them.The reason is probably that ADSC can play a role of seed cells in would healing which can differentiate into histiocytic cells induced by the internal environment,while PRP can play a role of nutrition.At the same time,high concentration of active growth factor in PRP can promote the proliferation and differentiation of ADSC and increase the quantity of repair cells,while these repair cells can secret growth factor by autocrine and paracrine which can affect the surrounding cells and themselves.And the 3D framework of PRF acts as matrix which provides favorable places for the attachment,migration and differentiation of cells.Therefore,a positive circulation is constituted,and combination of them has an effect of cooperative remediation,which can promote wound healing and improve healing quality.In addition,this study finds that the velocity and the quality of the wound healing are as follows:ADSCPRF>ADSCPRF>PRP>PRF>NS,indicating that ADSCs play a more important role in wound healing of skin ulcer than PRP and PRF,and PRP is more important than PRF.The specific molecular mechanism still needs further research.

ACKNOWLEDGEMENTS

Supported by Sci-Tech Foundation Project of Fuzhou Health and Family Planning (2016-S-wt10),Sci-Tech Foundation Project of Fuzhou Municipality (2017-S-135-2),Natural Science Foundation Project of Fujian Province(2104J01390),Clinical Medicine Center Construction Program of Fuzhou (2018080309),Key Clinical Specialty Discipline Construction Program of Fuzhou (201807111)