Yn Liu,Shiy Zhou,Yn lin Go,Yinglei Zhi

a Shenyang Pharm aceutical University,No.103,Shenyang 110016,China

b SchoolofPharm acy,Shenyang Pharm aceutica lUniversity,Shenyang 110016,China

c School ofM edical Devices,Shenyang Pharm aceu tical University,Shenyang 110016,China

Key words:Nanof ibers Nanof ibrous d ressing Diabetic foo t u lcer Un iax ia l electrosp inn ing Coaxia l e lectrosp inn ing

A B S T R A C TDiabetes is one of them ost p revalen t d iseases in the w o rld w ith h igh-m orta lity and com p lex com p lication s in clud ing d iabetic foot u lcer(DFU).It has been repo rted that the d iff icu lties in repairing the w ound related to DFU has m uch relation sh ip w ith the w oun d in fection,change of in f lam m atory responses,lack of ex trace llu lar m atrix(ECM),and the failu re of angiogenesis.Fo llow ing the developm en t of m ed ica lm ateria ls and pharm aceu tica l techno logy,nanof ibers has been deve loped by electrosp inn ing w ith huge po rosity,excellen t hum id ity abso rp tion,a better oxygen exchange rate,and som e an tibacteria l activities.That is to say,as a poten tialm ateria l,nanof ibers m ust be a w on derfu l cand idate for the DFU treatm en t w ith so m any bene f its.Carefu l selec tion of po lym ers from natu ra l resou rce and syn thetic resou rce can w iden the nanof ib rous app lication.Popu larm ethods app lied for the nanof ib rous fab rication con sistof un iax ia le lectrosp inn ing and coax ialelectrosp inn ing.Fu rtherm ore,nanof ibers loading chem ical,biochem icalactive pharm aceu tical ingred ien t(API)or even stem cells can be w on derfu l dosage form s fo r the treatm en t of DFU.Th is review sum m arizes the p resen t techn iques app lied in the fabrication of nanof ib rous d ressing(ND)that u tilizes a variety of m ateria ls and active agen ts to offer a better hea lth care for the patien ts su ffering from DFU.

1. Introduction

Acco rd ing to statistics,it is estim ated that there w ere 366m illion peop le w orldw ide w ith d iabetes in 2013,and the num ber w illbe in creased to a lm ost 552m illion by 2030[1].Diabetes is a kind of disease that relates to the dysfun ction of glucose contro l,the destroyed m anagem en t on the p ro tein m etabo lism and lip id m etabo lism[2].Fo llow ing the deterioration of d iabetes,a portion of d iabetic patien ts w ill su ffer from d iabetic foo t u lcer(DFU),one of the com p lications of d iabetes,w h ich possesses the characteristics of long term p rocess of w ound closu re and has the tenden cy to hosp ita lization and even ampu tation in the fu tu re.Un fo rtunate ly,apart from the physica l and m en ta l im pairm en t on d iabetic patien ts,the cost fo r DFU treatm en tm ay be another heavy load.To so lve th is p roblem,w e need to have a clear idea of the reason w hy it is so d iff icu lt to hea l the w ound and w e need tof igu re ou t w hat has p rolonged the hea ling p rocess.Periphera l neu ropathy,de fo rm ity,and m acrovascu lar d isease can be them ain factors that cause the failu re of DFU hea ling p rocess[3].Add itiona lly,new f indings can be the supp lem en ts to the facto rs[3],w h ich in cluding lack ing resistan ce to in fection,changing on m icrocircu lation fun ction,and dam aging to grow th factors(GF)exp ression and activity.Fu rtherm o re,ano ther study is conducted tof ind ou t w hether the periphera l arteria l d isease has a corre lation w ith DFU,and the resu lts ind icate that there is som e co rrelation betw een them,and DFU can be c lassif ied to tw o d isease states acco rd ing to w hether there ex ists the periphera l arteria l d isease[4].Other facto rs,w h ich have sign if ican t contribu tions to DFU in clud ing low p ro liferative capacity of the f ib rob lasts,dow n regu lation of recep to rs,and the absen ce of a su itab le p ro tein m atrix in the derm is[5].In b rief,the m echan ism of th is issue is relative ly com p lex,and there still has a big room for pharm aceu tists to participate in DFU treatm en t.The m ethods of DFU treatm en t varies from ind ividua l sym p tom s and the d isease stages.Severa l therapeu tic app roaches have been reported,g lycem ic regu lation,such as adequate insu lin adm in istration,is essen tial.Debridem en t,skin gra ft and tissue rep lacem en t has re levan tly h igh-e ff icien cy of w ound c losu re[3].Other app roaches su ch as vascu lar reconstru ction,hyperbaric oxygen therapy,and granu locy te-co lony stim u lating factor are also op tions to treat DFU.In m ost cases,the w oun d d ressing is of use to create a benef icia l environm en t fo r the open w ound to ach ieve a better and fasterw ound c losu re[6].Un fortunate ly,conven tiona lw ound d ressings like gauze possess lim ited basic fun ction fo r their defective m ateria l p roperties.W hat is m ore,d ifferen t phases of the d iabetic w ound hea ling have d ifferen t patho logy featu res so that m u ltifun ctiona lw ound d ressing w ith specif ic m ateria l is under the u rgen cy.Cu rren tly,advan ced d ressing techno logy like nanof ib rous d ressing(ND)that can em p loy specif ic m ateria ls tof it specif ic need fo r DFU treatm en t is harvesting la rge interests and atten tion.ND is the co llection of nanof ibers ranging from nanom eters to m icrom eters,as show n in Fig.1,ND is easy to be rem oved[7].The techn ique of nanof ibers fabrication has a long h istory,and it w as f irstly repo rted sin ce 1900[8].Nanof ibers as a p rom isingm atrix have lo ts of advantages such as sm all d iam eter,narrow d iam eter d istribu tion,and h igh-specif ic su rface area.Various po lym ers have been stud ied and deve loped in tofabricate nanof ibers fo r tex tiles,e lectrica l and op tica l com ponen t,senso rs,and f iltration devices[9,10].The p roductsm anu factu red th rough th is techno logy are very soft and h igh ly f lexib le,lacking of sharp co rners and vu lnerab le to tu rn to sheets,tubes,and coatings[11].ND em p loyed by pharm aceu tics show s great benef its and an increased p reva len ce in the d rug delivery system.It u tilizes the excip ien ts o r accesso ries to de liver the therapeu tic agen ts to the site w ith h igh-e ff icien cy,and low-adverse effects.Fo r the DFU treatm en t,it is necessary to pu t em phasis upon the vascu la rization,co llagen accum u la tion and no rm a lphysio logica l fun ctions to con tro l the deterio ration p rocess and even cu re the w ound[12].Kinds of active pharm aceu tica l agen ts like m olecu les and cells p lay the key ro les in th is p rocess;however,them atrix loaded them can a lso be a fun dam en ta l stage for their fun ctiona l rea lization.Basica lly,it has crucia l activities of the abso rp tion of exudation and the exchange of oxygen,w ater,and nu trien t[13].Add itionally,nanof ibersw ith the sim ilar d iam eter to the ex trace llu lar m atrix(ECM)have been p roven to acce lerate the p rocess of ce ll adhesion and p ro liferation.1D-3D nanof ibers p roduction a lso has been investigated in biom aterials f ield[14].

In th is article,w e try to d iscuss the com m on po lym ers used in the fabrication of ND,especia lly fo r the DFU treatm en t.Accord ing to the resou rces,po lym ers are classed in to tw o categories,natu raland syn thetic po lym ers.Bio logicaland physicochem ica l p roperties are a lso d iscussed.The nex t atten tion w ill be paid to the fab rication app roaches in c lud ing un iax ia l and coax ia l e lectrosp inn ing.Attem p t to in trodu ce the device con f igu ration and the p rocess in the nanof ibers fo rm ation has been conducted.In the m ean tim e,underlying facto rs in f luen cing nanof ibers fabrication are listed in detail.An overv iew of the app lication of ND in treating DFU w ill be given in the end.

Fig.1-(A)PLA f ibers e lectrospun by po rtab le e lectrosp inn ing dev ice on hand;(B)the f ina l appea ran ce of a hom ogenou s ND;(C)rem oving the ND w ith a tw eezer from sk in(Rep rodu ced w ith perm ission from[7].).Copy righ t 2015,The Roya l Society of Chem istry.

Tab le 1-Syn thetic po lym ers and natu ra l po lym ers used in ND fab rication and the re levan t pa ram eters set in the stud ies.

2. Po lym ers fo r nanof ibers

There is no un iversa l standard fo r the se lection of the po lym ers app lied for ind ividual nanof ibers,and the choice relies on the desirab le fun ction of the nanof ibers.Differen t po lym ersw ith d ifferen tm o lecu les and sou rcesw ill lead to no tab le d ifferen ces on p roperties such as sp inn ing so lu tion viscosity,nanof ibersm orphology,m echan ical strength,biocom patibility and physicochem ica l characteristics.A large num ber of po lym ers have been developed in the study of nanof ibers u tilized fo r DFU hea ling.They m ay be broad ly c lassif ied in to tw o categories:syn thetic and natu ralpo lym ers.In general,natu ral po lym ers have a better biocom patibility su ch as degradation and low er im m une resistan ce,w hereas the syn thetic po lym ers possess an easier e lectrosp inn ing w ith excellen t m echan ica l strength such as f lex ibility and stiffness.To get the m ax im um benef its from those m ateria ls,taking the b lend ing strategy is advisab le.Fo r exam p le,ch itosan and a lginate w ith the add ition of po lyethy lene oxide(PEO)or polyviny l alcoho l(PVA)has been fab ricated in to nanof ibers[15].Tab le 1 show s various po lym ers and co rrespond ing param eters app lied in the ND fabrication.

2.1. Syn thetic polym ers

Po ly(lactic-co-g lyco lic acid)(PLGA)is a biodegradab le and biocom patible po lym er syn thesized by tw om onom ers:lactic acid and glyco lic acid.Som e artic les[15-18]have show n that PLGA is an app licab le po lym er fo r the nanof ibrous fab rication w ith exce llen tm echan ica l strength and f lex ibility.The ratio of lactic acid to glyco lic acid,m o lecu larw eigh t,crysta llin ity and the m ed ium pH have som e certain in f luen ces on the degradation rate of PLGA.The degradation rate can be slow ed by the redu ction of g lyco lic acid and the im p rovem en t of the m o lecu lar w eigh t[16].How ever,organ ic so lven ts are often used to d isso lve PLGA fo r itsw ater-inso lubility that can cause ce ll toxicity p reven ting the DFU hea ling.Thus,m od if ication on PLGA is favo rab le fo r its app lication in ND.Study p resen ted by Zhou et a l.[19]ind icates that N-carboxyethy l PLGA syn thesized by M ichae l add ition reaction of acry lic acid has a better w ater so lubility.Even though PLGA exh ibits poor so lubility in w a ter,nanof ibers w ith PLGA also has been successfu lly developed in to hybrid sca ffo lds in the d iabetic w ound treatm en t[20].In their study,the add ition of ch itosan and recom binan t hum an p la te let-derived GF in creases the hyd roph ilic characteristics,and w ater con tactangle of the nanof ibersm ade of the tw om ateria ls com pared w ith the pu re PLGA m ateria l p roves it.The area of sim u lative d iabetic w ound d ram atica lly decreases in the in vivo test.Po lym ers like PEO and PVA are recom m ended to app ly for reducing the viscosity of PLGA so lu tion due to its chain en tanglem en ts o r p rom oting the conductivity of Ncarboxyethy l PLGA aqueous so lu tion[19,21].

Po ly(lactic acid)(PLA)is the therm op lastic a liphatic po lyester derived from natu ral resou rces,and it has been w ide ly used in pharm aceu tics and m ed ica lm ed icine,fo r exam p le,d rug delivery system,su tu res,and sten ts.PLA a lso possesses som e activities of biocom patibility and m echan ica l strength,desp ite the app rova l from FDA,there still exists the fact that poo r com patibility can be observed w hen it con tacts the b lood[22].How ever,it does no tm ean that PLA is no tw orthy in the DFU treatm en t,because b lend ing w ith other po lym ers like ch itosanderivatives can m odu late the b lood compatibility[23].Acco rd ingly,as a p latfo rm,it can p rom o te the ce ll attachm en t and p ro lifera tion[24].

Po ly(ε-cap rolactone)(PCL)is a biodegradab le and biocompatib le po lym er w ith a fun ctiona l ester bon d that can be degraded in to non tox ic fractions under the physio logica l environm en t.Even though PCL m im ics the ECM,its capacity of cellattachm en t and p roliferation has been lim ited for the hyd rophobic p roperties[25].As reported in the tex tu re,h ighpo rosity on the su rface of the PCL nanof ibers can overcom e th is d raw back,co-u tilization w ith other hyd roph ilic po lym ers such as gelatin,co llagen,or ch itosan is another good choice[21].Fo r instan ce,PCL w ith ge latin can be e lectrospun in to nanof ibers in tw ofo rm s,a lignm en t and random[14,26],and these topo logies do a ffect the f ib rob lasts behavior.The essen tia l poin t in th is research is that f ib rob lasts can p rodu ce m ore actin attach ing the a lign nanof ibers.In o ther words,the a lignm en t of nanof ibers w ith PCL and gelatin m ust has som e specif ic relationsh ip w ith DFU w ound recovery.Fu rther study abou t the a lignm en t of PCL nanof ibers w ith scann ing e lectron icm icroscope(SEM)and d ifferen tia lscann ing ca lo rim etry(DSC)f inds that a single a ligned nanof iber consists of severa l nanof ibrils and each nanof ibril in c ludes the crystalline and the am o rphous region[27].

Po lyviny la lcoho l(PVA)is aqueous so lub le,non toxic,and no carcinogen ic w ith excellen tm echan ica lp roperties,and chemical resistan ce.Besides,PVA has a fair f lexibility and sw elling capacity.One de fection of PVA is that it tu rns to be unstab le in aqueousm ed ium,so in m any researches,gra fting,crosslinking,and copo lym erization have been done w ith o ther po lym ers like po lyviny lpy rro lidone(PVP),and stercu lia[28].Blending PVA,p lu ron ic,and po lyethy leneim ine(PEI)can ob tain a better stab le nanof ibers em p loyed in the an tibacteria lw oun d d ressing[29].Silveror AgNO3in corporated in nanof ibersm ade by PVA has the ability of an ti-bacteria[15].A resea rch by Zhao et a l.[30]developed an electrosp inn ingm em b rane em p loy ing PVA b lend ing w ith a p ro tein nam ed pnsr16 to cu re Sp rague Daw ley rat skin bu rns,the form ation of granu losa tissue and the con traction ofw ound in test group are better than the negative groups.

Po ly(ethy lene ox ide)(PEO)has been w ide ly used as an ideal hyd roph ilic carrier for active ingred ien ts in nanoscales[31-34].For reason s like w ater in teraction and steric exclusion,the in trodu ction of PEO has little in terferen ce on p la tes and p lasm a p ro tein[35,36].Th is cou ld p rovide a relatively favorable environm en t for the DFU healing.The add ition of PEO to o ther m ateria ls like cyclodex trin and ch itosan w ith poor e lectrosp inn ing p roperties has been repo rted to be use fu l in the long and bead free f ibers fab rication[37,38].M eanw h ile,for fragile biom ateria ls in corpo rated in the co re she ll of coaxia l nanof ibers,m ix ing w ith aqueous PEO so lu tion a lso perform ed w ell to stab le them[39].In ano ther investigation,it can be seen obviously that w ith the ratio of PEO in creased,better m orpho logy of nanof ibers w as found under the SEM im ages[40].M o lecu lar chain orien tation and crysta llin ity of PEO w ere believed to have som e relationsh ip w ith its excellen t perfo rm an ce,fu rtherm ore,other underlying factors like con strained annea ling and so lven ts a lso con tribu ted to it[41].

2.2. Natura l polym ers

Ch itosan is a biodegradab le po lysaccha ride w h ich can be obtained th rough ch itin deacety lation.It has the sam e com ponen t g lycosam inoglycans as in the ECM.Apart from the biocom patibility,biodegradability,and hem ostasis ability,the activity of an ti-bacteria and an ti-fungi is the specia l p roperty of ch itosan[42].Th is advan tage does add assistan ce to the acce leration of DFU recovery.How ever,ch itosan w ith poorm echanica lp roperties lim its its app lication in the DFU treatm en t,and crosslink ing w ith o ther natu ra l po lym ers like ge latin and co llagen can im p rove the m echan ica l p roperties[43].The composite of ch itosan and dextran syn thesized by M ichael add ition reaction a lso has the ability tom ove aw ay from DFU w ithou t ce llu lar adhesion com pared w ith the trad itiona l w ound d ressingm ateria ls[44].It is a tim e-saving and cost-saving app roach to u tilize ch itosan in ND fabrication.How ever,there are indeed som e d iff icu lties tofab ricate un iform u ltra f ine nanof ibersas the strongm o lecu lar in teraction of ch itosan[45].In their research,blend ing strategy w ith PVA show s an ideal w ay to ease the use of ch itosan.On the o ther hand,ch itosan has the effect of loosen ing the tigh t jun ctions betw een cells[46].It m ay be unbene f icia l fo r the DFU w ound closu re,bu t d rugs in corporated in the ND aim ing at the deeper tissue need it.

A lginate is a lso a biodegradab le po lysaccharide ex tracted from seaw eed.Sim ilar to ch itosan,it a lso has the glycosam inoglycan w h ich is the m ajor com ponen t of ECM.The p roperties of b leed ing and exudate abso rp tion m ake a lginate an excellen t w ound d ressing m ateria l.Nevertheless,sim ilar to m ost biopo lym ers,electrosp inn ing p rocessibility of aqueous a lginate is still a cha llenge.To so lve th is p rob lem[47],u tilizing a coso lven t g lycero l added to the aqueous so lu tion cand isrup t the H-bonds betw een in ter and in tra-m o lecu le,consequen tly,new H-bonds establish betw een glycerol and sodium a lginate.Rheo logica l characterization show s that the viscosity of the sod ium a lginate in creases to a good level and the f lex ibility of the ND exh ibits the sam e e ffect.In m ost cases,researchers p refer to use alginate w ith PEO[48,49],PEO in the su itab lem o lecu lar such as 2000 kDa can p rovide the en tanglem en t effect fo r the a lginate to be e lectrospun in to nanof ibers[49].

Co llagen is a critical com ponen tw h ich can be found in the ECM w ith the structu re of f ib rill and th ree-d im ensiona l netw orks.As the biochem ica l activity of co llagen,it is repo rted to stim u late the m igration of the f ibrob lasts and m odu late the m etabo lism of the granu lation tissue[50].It is benef icial for the ce ll attachm en t and p ro liferation du ring the DFU recovery.How ever,it is better to use o ther po lym ers like PCL together due to the instability of co llagen under the h ighvo ltage in the nanof ibers fabrication p rocess.Additionally,sim ilar to PLGA,co llagen canno t be electrospun w ithou t the use of tox ic o rgan ic so lven ts and zein from natu ra l sou rces co-electrospun w ith co llagen can ease the fabrication p rocess.Gela tin is a h igh-biodegradab le po lym er denatu ra lized from co llagens w ith the iden tica l fun ctiona l groups as co llagen.It is be lieved that ge latin a lso canno t be electrospun to nanof ibers w ith aqueous so lu tion.How ever,a research by Li et a l.[51]aim ing at f ind ing the po ten tia l facto rs that in f luen cing the electrosp inn ing p rocessibility of aqueous ge latin ind ica tes that con tro lling the sp inn ing so lu tion tem peratu re at w h ich the gelling behavior is supp ressed can increase the e lectrosp inn ing p rocessibility to get a desirab le ND.

3. M ethod s fo r nanof iber fab rication

Cu rren tly,the setup fo r nanof ibers fab rication is re latively sim p le consisting ofseveralcom ponen ts,such asm icro pum p,sy ringe,sp inneret,vo ltage supp ly,e lectrode m eta l co llector and po lym er so lu tion[52].Tw o cartons rep resen ting the typ ica l facilities used fo r the fabrication of nanof ibers are show n as in the below,Fig.2A exh ibits the apparatus used for un iaxia lelectrosp inn ing and Fig.2B the coax ia lelectrosp inn ing.The m ateria l a ttribu tes and p rocess param eters,fo r exam p le,the d rug to po lym er ratio,the so lu tion v iscosity,and the so lu tion feed rate are crucial to the f inal function of the nanof ibrous p rodu cts.Each w ay of d rug in corpo ration using respective po lym ers and nozzles needs ind ividual m aterial attribu tes and p rocess pa ram eters fo r a desired d rug release p rof ile includ ing im m ed iate re lease and con tro lled re lease[53].

Fig.2-(A)Schem a tic ap pa ratus u sed fo r the un iax ia l e lectrosp inn ing and(B)the coax ia l e lectrosp inn ing.

3.1. Uniaxia l electrospinning

One of the m ost d istingu ished fea tu res of un iaxia l electrosp inn ing abou t the setup is the nozzle con f igu ration.That is to say,it just has one nozzle layer con trast to the coax ia l electrosp inn ing setup w ith tw o con cen tric nozzle layers.The p rocess of un iax ia l e lectrosp inn ing and the consideration on the param eters are relative ly less than the coax ia l e lectrosp inn ing.Firstly,the po lym er so lu tion shou ld be p repared w ith desired d rug-polym er ratio to obtain an ideal viscosity or conductivity.Then,the so lu tion can be ex truded from the sy ringe tube w ith a su itab le fo rce on the sy ringe.The critica l techn ique app lied for th is p rocess is the Ty lo r cone w h ich refers to a cone like ex trusive so lu tion in stead of a trad itiona l cy lin der like solu tion under the vo ltage supp ly on the nozzle.The vo ltage is ex trem e ly h igh to even 27 kV[54].As the reason w hy it becom es a cone like solu tion,a study[55]claim s that the Ty lor cone appears w ith the charge repu lsion force exceed ing the su rface tension of the so lu tion so that a desired nanof ibers w ith ex trem e ly th in,e longated featu res can be observed.Fo llow ing the solu tion going forw ard w ith the p redef ined rate,so lven ts w ill be vo latilized o r evapo rated im m ed iately and the nanof ibers appears on the m eta l co llecto r that is stationary as a foil o r ro tating as a co lum n.In the end,a ND w ith thousands of nanof ibers can be seen.Typ ical featu res of un iax ia l nanof ibers taken by Field-em ission Electron M icroscope show s in Fig.3[56].

Actua lly,m uch atten tion has been paid to the app roach of d rug load ing.As w e know,the in teraction betw een tw o substan ces can be chem ica l o r physica l.In the sam e w ay,the active ingred ien ts also can be loaded chem ically or physically in the po lym er m atrix.A nanof ib rous d rug de livery system con tains therapeu tic agen ts of cip rof loxacin con jugated to the po lym erm atrix of PLA w ith d iam eters ranging from 150 nm to 400 nm and pore size from 62 to 102 nm are developed,the release study show s that the release k inetic can be m odu lated th rough d rug content changing[54].Th is is an excellen t exam p le of the chem ica l link betw een d rug and po lym erm atrix used in nanof ibers.The o ther w ay to load d rugs is physica l in co rporation,sim p le m ix tu re betw een d rug and po lym er is an idea l choice.Zhang et a l.[57]app lied the e lectrosp inn ing techno logy tofabricate the van com ycin ND that is co llected on the a lum inum foil.Consequen tly,the in vivo and in vitro studies ind icate a satisfacto ry an tibacteria lp roperty.Fo llow ing the care fu l selection of the d rug load ing m ethod,con siderations of variab les on the un iaxial nanof ibrous p roperties com e to the nex t.Firstly,it is necessary to pay atten tion to the various fo rces underlying the fo rm ation of nanof ibers.The forces app lied in the p rocess com e from gravity,pum p d riving,su rface strength,and electrical f ield.It is also obvious that the pum p d riving and electrica l f ie ld are the positive facto rs tha t can be he lp fu l to the f ib rous fo rm ation and e longation.The e lectrica l f ield indu ced by vo ltage supp ly is the dom inan t factor for a stab le and con tinuous f ibrous form ation[58,59].The p rim ary featu res of nanof ibers in clud ing f iber size d istribu tion,m o rpho logy,porosity,and d rug release p rof ile are the crucial elem en ts in f luen cing the nanof ib rous fun ction.W ith respect to the f iber size and size d istribu tion,the feed ra te can be one of the m ain factors that a ffect them.The f ibrous d iam eter ranging from nanom eters to som e m illim eters is positively p ropo rtiona l to the so lu tion deliver rate under a rationa l electrica l f ield.How ever,the charge strength from the e lectrica l f ield also has a huge in f luen ce on the f ibrous d iam eter;the f ibrous d iam eterw illbe th inner as the charge strength im p rovesw ith a m ed ium so lu tion deliver rate.There is a m ass ba lan ce underlying the com bined action betw een the deliver rate of the jet solu tion to the nozzle and the rate at w h ich the solu tion is fo rced to be e longated by the e lectric f ie ld[60].Apart from the forces bu rdened on the so lu tion,the characters of the solu tion itse lf a lso determ ine the nanof ibrous d iam eter and the d iam eter d istribu tion.Especially,the conductivity of it is just the th ing;the so lu tion w ith h igh-condu ctivity w ill lead to w ide size d istribu tion[61].W hen it com es to nanof ib rousm o rpho logy,the bead stru ctu re is the com m on phenom enon p rodu ced by in consisten t forces betw een the syringe pum p and the electrica l f ie ld.For exam p le,the beaded stru ctu re can be observed w ith the deliver rate by the sy ringe pum p ex trem ely su rpassing the rate atw h ich the so lu tion is forced to be elongated by the electric f ie ld.Add itiona lly,the excessively poo r condu ctivity of the so lu tion and the re latively h igh-electrica l f ield can be ano ther un favorab le factor toform the hom ogenous nanof ibers,and the charge p re fers accum u lating on the su rface of the jet w ire and m igrate on the co llecto r w ith sho rt d istan ce betw een nozzle and co llecto r tofo rm the bead stru ctu re.Po rosity is another p roperty that p lays a key ro le in the d rug release p rof ile and exudation absorp tion.The porosity can be in creased w hen the so ca lled therm a lly induced phase separation grow s up.It refers to a p rocess that the non-so lven t rich phase separates from the non-so lven t poo r phase.The site of the po re is located at the non-so lven t poo r phase[62].Hum id ity is a lso ano ther e lem en t that in f luen ces the fo rming of po rosity in the p rin cip le of vapo r-in du ced sepa ration or b reathe f igu res w ith non-w ater so lven t evapo ration.The d rug release k inetics is no t on ly in f luen ced by the p rim ary p roperties d iscussed above bu talso the in teraction betw een the d rug and the po lym erm a trix.In a study,ca ffeine is loaded in to the po lym er PCLand the d rug crysta llization induced by the in teraction betw een them has caused the decreased re lease compared w ith the non-crystallization group[63].Another potentia l facto r can be the ion ization state of in co rporated d rugs;h igh-ion ic strength of the d rug w ill lead the d rug to locate to the su rface of the f ibers[53].W hat is m o re,su rface location behavior can also resu lt in bu rst release in som e cases.

3.2. Coaxial electrospinning

As p revious d iscussion,the typ ica lstru ctu re of coax ia le lectrosp inn ing nozzle com p rises tw o con cen tric and separate sp inneret tofo rm a co re-shell arch itectu re nanof ibers.Tw o d ifferen t jet solu tion s w ill be p repared and each is transported to the re levan t sp inneret layers form ing the co re o r shell layer,respective ly.The p rocess of th is techno logy is basica lly similar to the un iax ia l electrosp inn ing.The im ages of a coax ia l nanof ibers taken by transm ission electron m icroscope(TEM)show n in Fig.4[64].

No m atter how the d rug is loaded in to the nanof ibers,the fundam en ta lp rin cip le of th is w ay is equa l to un iax ia le lectrosp inn ing:physical and chem ical lin kage.Even though d rugs app lied in th ism ethod can be d isso lved or suspen ded in the core or shell polym er so lu tion,m ost nanof ibers p refer pu tting the active ingred ien t in the inner layer to keep it from negative factors su ch as m oistu re and oxygen.A study[59]has fab ricated nanof ibers con tain ing f ish oil and zine in the co re layer,the shell layerm ade by PVP is the p rotectan t.A fu rther investigation on the ox idative stability com pared w ith un iaxia lnanof ibers has show n that coaxia lm ethod w as an eff icien t en capsu la tion w ay to p ro tect the active agen t f ish oil against oxidation.Another benef it is con tro lling the release characters of it as im m ed iate release m ode l o r sustained release m odel.Han et a l.[65]deve lop a stim u li-responsive ND w ith coaxia l e lectrosp inn ing techno logy,ou t of the co re layer w h ich comp rises en capsu lated activem o lecu les is the sm art shell layer.It consists of the stim u li-responsive po lym ers,and on ce the shell layer recogn izes the stim u lating signa ls,the depo lym erization of the po lym ers occu rs w ith the active m o lecu les released from the core layer.The fu rther study on the triggered release show s there is no release action w ithou t the stim ulating signa ls.How ever,on ce the signa l is p rovided,the encapsu lated active agen ts a re released sw iftly.Add itiona lly,no m atter w hether there ex ists d rug or no t in the nanof ibers,one layer of the tw o can be a fun ctiona l stru ctu re that assists the other[66].Fo r instan ce,the e lectrosp innab le po lym er so lu tion used in one layer can be fabricated as a sp inn ing aid fo r the inelectrosp innab le in the o ther layer.In the sam e w ay,having a clear idea of the e ffect of variab les can be benef icia l to obtain a desirab le coax ia l nanof ibers.In fact,the variab les d iscussed here are partia lly sim ilar to the un iaxia l.Fo r exam p le,the effect of jet rate,electrica l f ield strength,so lu tion condu ctivity and con cen tration on the d iam eter,d iam eter d istribu tion,and m o rpho logy is para lle l.How ever,the in tegrity w ith the in teraction betw een the tw o jet parts can be one of the greatest cha llenges tofo rm a hom ogeneous nanof ibers.Som etim es,the tw o layersw illseparate from each o ther and the in tegrity d isappears.To som e ex ten t[66],the in terfacial strength p lays a key ro le in the aff in ity of the tw o layers.So it is recom m ended to d isso lve o r suspend the po lym er w ith sim ila r o r even the sam e so lven t and p repa re a harm ony con cen tra tion to ob tain a re latively low-in terfacia l strength.The in terfacia l strength refers to the com patibility am ong the ingred ien ts in clud ing the active agen ts,po lym ers,and so lven ts.The secondary bonds such as electrostatic and hyd rophobic in teraction s have the im p roving effect on com patibilities of the com ponen ts used fo r nanof ib rous fab rication[59].

Fig.4-TEM im ages of coax ia l nanof ibers m ade by PEO and ch itosan.(A)Con cen tric and(B)eccen tric co re and she ll stru ctu res(Rep rodu ced w ith perm ission from[64].).Copy righ t 2012,Am erican Chem ica l Society.

4. Characterization of nanof ibers

Various ana ly tica l instrum en ts w ith d ifferen t m echan ism s have app lied fo r the characterization of the nanof ibers.Optica l m icroscope is a favo rab le choice for the p rim ary exam ination on the p roduction w ith h igh-reso lu tion.First imp ression w ith op tical m icroscope to observe the ou tline of the p rodu ct is necessary,that is to say,beads and non-f ibers can be checked.Another com m on m easu re techn ique is the SEM.To have a better know ledge of the d rug d istribu tion,DSC usually p rovides a m ethod w ith the peak sh ift and the endotherm ic or exo therm ic f lu ctuation[19].Ano ther im portan t characterization item of nanof ibers is w ater con tact ang les,as fo r the ce ll p ro liferation du ring the DFU occ lusion needs hyd roph ilic m atric to support it.W hen d rugs are inco rpo rated in the nanof ibers,their ex isten ce,m o rpho logy,especia lly crysta llin ity,and d istribu tion can a lso be described th rough Fou rier transfo rm in frared spectroscopy(FTIR)[16],and X-ray photoelectron spectroscope[17].The f ina l functiona l testm odelof the nanof ibers in cludes cellm odeland anim a lm odel.The cellm ode l is relative ly sim p le and inexpensive.In the cellm ode l,cells su ch as hum anderm a l f ib rob lasts o r m ouse f ib rob lasts(L929)are cu ltu red w ith the nanof ibers fo r severa l days,then the cy to tox icity,ce ll attachm en t,and ce ll p ro liferation are investigated th rough a ce ll coun ting m ach ine.The an im alm odel u tilizes rabbit or m ouse to directly eva luate the w ound hea ling eff icien cy of ND on DFU.A fter the anesthetization,a specif ic num ber of sim u lated DFU w ound w ith designed d im en sion are created on the loca l sk in of the an im als under the sterile cond itions.To supp ly a betterw ound bed for the fun ctiona lexh ibition of the ND,the ep iderm is,derm is,and perichon d rium shou ld be rem oved.The data abou t the closu re e ffect co llected from them can be ana lyzed w ith the statistical app roach.

5. App lication of ND fo r DUF

5.1. Benefits ofND for DFU w ound hea ling

Prio r to d iscussing the nanof ibrous app lication,it is necessary to have a deep insigh t in to the p rocess of DFU w ound hea ling.No rm a l sk in com p rises ep iderm is,derm is,and the subcu taneous tissue from the ou t layer to the deep layer.Keratinocy tes located in the ep iderm is and f ib rob lasts em bedded in the derm is,they are both in corporated w ith the th ree-d im ensional m atrix consisting of f ibers such as co llagen,f ib rinogen and e lastin.ECM is just the th ree-d im em siona lm atrix.Obviously,vein and lym ph d istribu te am ong them offering requ ired nutrition and im m une activity.One of them ost im portan t ro les of the skin is p rovid ing a natu ra lbarrier p reven ting the harmfu l dam age like m echan ica l im pacts and m icrobia l in fection.Considering the DFU w ound w ith ch ron ic recovery,it needs m ore realization abou t them echan ism ofw ound healing.Fu rtherm o re,re fo rm ing on the w oun d d ressing consisten t w ith the m echan ism is im perative.As w e know,on ce the DFU inju ry deve lops in to the derm is or even the subcu taneous tissue,m any p rocedu res m ay occu r in the w ound recovery.In the in f lam m ato ry phase,there are som e specif ic bio logica l changes as the f lu id leaks from b lood o r lym ph,especia lly w ith the aggregation of p latelets and the nex t clots by it.Keratinocy tesm ay re lease GFo r cy tokines under the stim u lating signa ls from p late lets.Then,im m une ce lls like m acrophages m igrate to the w oun d bed tof igh t against the germ s and fungus,sim u ltaneously,new blood and lym ph vessels are reconstru cted.The nex t phase is p ro lifera tion.Re-ep ithe lia lization occu rs f low ing the p ro liferation of keratinocy tes and m yof ib rob lasts that are critica l fo r the generation of granu lation tissue and ECM.In the f inal m atu ration phase,the rearrangem en t of co llagen and the fo rm of elastin enhan ce the no rm a l p roperties of the renew a lskin.Un fo rtunately,stud ies have ind icated that severe d isorders of the hea ling phase usua lly happen in DFU patien ts[67].Obviously,the healing of the DFU w ound is so com p lex that the app lied w ound d ressing w ith h igh-perfo rm an ce capacities is evitab le.Firstly,hem ostasis of thew ound d ressingm ust be help fu l fo r the clo t fo rm ation[18],ND w ith m arvelous specif ic su rface area just f its th is requirem en t,and the exudate abso rp tion capacity is a lso excellen t.The oxygen exchange ability is a lso needed fo r the b lood vessel renew a l and cell p ro liferation,in add ition,abundan t oxygen has the effect of decreasing the risk to in fection[6].As d iscussed p reviously,ND is the perfect cand idate fo r its huge po rosity to oxygen penetration.Second ly,p rovid ing a su itab le stage fo r the renew a l cells like f ib rob lasts and keratinocy tes to adhere and sp read m ay sha rp ly in crease the DFU w ound occ lusion,fo rtunate ly,ND has been p roven to enhan ce the cells grow th as a good stage[8].Effect of ND fo r d iabetic w ound closu re show s in Fig.5 as below[68].Another capability of substitu tability of an tim icrobia l agen ts is a lso p rof itab le for the perfo rm an ce of the w ound d ressing,using ch itosan is advisab le fo r its activity of inh ibiting the grow th of fungiand bacteria[69].In add ition,superior active pharm aceu tical ingred ien t(API)en trapm en tofw oun d d ressing can ease them od if ication of the re lease k inetic,fo r exam p le,the sustained release and the con tro lled release,as in the coax ia l e lectrosp inn ing chapter,nanof ibers w ith a con tro lled ou t layer m ay rea lize these release p rof ile.

5.2. Active agen ts in the nanofibrous dressing for diabetic foot u lcer

5.2.1. Sm allm olecu les

W ound in fection is a com m on p rogressive sp read ing sym ptom.W ith the dysfun ction of g lucose m etabo lism in DFU patien ts,im m uno logica l response is d isrup ted so that the DFU w ound becom esm o re and m o re suscep tib le to in fection[70].In m ost cond ition s,in fection deterio ration m ay lead to ampu tation.Com pared w ith the w ound in non-d iabetic Yo rksh ire p igs,the d iabetic Yorksh ire p igs show w ound healing de lay and secon da ry in fection by endogenous bacteria[71].Corynebacterium spp is the dom inan t bacteria l genus associated w ith DFU[72].Other bacteria l com m un ities in cluding Streptococcus,Serratia,Staphylococcus and Enterococcus spp are a lso respon sib le for the DFU in fection[71,73].In 2004,IDSA(the In fectious Diseases Society of Am erica)has poin ted ou t that the in fection shou ld be iden tif ied by the bio logical f ind ings instead of the c lin ical criteria avoid ing an tibio tic abuse[74].On ce the severity of the in fection and the types of the bacteria l are iden tif ied,co rrespond ing an tibio tics can be adm in istrated in the ND fo rm s for DFU treatm en t assisting the system ic adm in istration.For exam p le,f luo roqu ino lones su ch as cip rof loxacin,levof loxacin and clindam ycin are recom m ended in non-severe o r severe phase of DFU in fection[75].Severa l an tibiotics have been successfu lly deve loped in to ND fo rm s w ith p roper syn thetic or natu ra l po lym ers.Gen tam ycin as an an ti-Staphylococcus is en trapped in the Eud ragit RS/RL 100 tofabricate ND[76].SEM m icrograph ind icate that sm ooth su rface w ithou t bead and excellen t size d istribu tion p resen ts the ND feasibility ofgen tam ycin.Fu rther therm a l ana lysis w ith DSC and TGA va lidates the in creased therm a lstability of gen tam ycin loaded in the po lym ers.In vivo study dem onstrates that the fabricated ND has the bioactivity of inh ibiting the bacteria l grow th.Ano ther p ractica l an tibio tic in co rpo rated in ND fo r DFU is tetracycline hyd roch lo ride(TCH)w h ich possesses broad spectrum[77].To ob tain a sustained release p rof ile,attem p t of em u lsion electrosp inn ing has been m ade.W ith respect to the em u lsion,TCH d isso lved in w ater as w ater phase and d ib lock po lym er PEG-PLA d isso lved in ch lorofo rm as oil phase.APIw ith f luo rescen t substan ce in corporated in the core of the nanof ibers can be seen from the im age taken by con foca lm icroscope(CM).Under th is bilayer stru ctu re,the bioactivity of the TCH and the sustained release character has been observed in the an tibacteria l test.Add itiona lly,there are a lso m any other an tibio tics under the w ay to be developed in to ND.Cip rof loxacin is an e ff icien t an tibio tics w ith the po ten tia l to be loaded in nanof ibers,and it has been su ccessfu lly app lied in the cu re of S.Aureus in fection[78].

In the in f lam m ato ry phase of the DFU recovery,side e ffects induced by free rad ica l,ce ll deb ris,and cy to toxic enzym e cand istu rb and p ro long the recovery span[79].As d iscussed above,a series of signal cascades and large number of cells in c lud ing p latelets,neu troph ils,keratinocy tes and m acrophages participate in the DUF recovery.Desp ite of the un clearm echan ism of an ti-in f lam m atory agen ts,som e chemical en tities have been p roven to be eff icien t as an APIused in the ND for DFU.Bixin,a p igm en t ex tracted form Bixa Orellana L.Seed,show s a great bioactivity of an ti-in f lam m ato ry,an ti-oxidan t,and hea ling p roperties[80].In a study[79],under the un iaxia l electrosp inn ing techno logy,bixin w ith PCL is fabricated in to an ideal ND.Apart from the rou tinely physica l detection,cell p ro liferation assay and in vivo study in d icates tha t the Bix in-loaded PCL nanof ibers decrease the tim e of w ound c losu re m o re than virgin PCL nanof ibers.Ano ther natu ral com pound cu rcum in derived from tu rm eric has lots of bio logica l activities,fo r exam p le,an ti-tum o r,an ti-ox idan t,and an ti-in f lam m ato ry has been p roven[81-83].A llow ing for the poo r so lubility and low-bioavailability of cu rcum in,researchers select the PCL as a m atrix for the sustained release ND fab rication.To e levate the an ti-in f lam m a to ry effect,IL-6(in terleuk in-6),a rep resen tative m em ber of in f lam m ato ry facto r re leased from m onocy te-m acrophages,is quan tif ied.In the an ti-in f lamm atory experim en t,the in f lamm atoryinduced J774A 1 cells w ith cu rcum in-PCL nanof ibers exp ress low IL-6.Consequen t d iabetic m ouse w ound closu re examination exh ibits decreased tim e fo r d iabetic w ound closu re to be done.Ano ther an ti-in f lam m atory agen t is quercetin.It is a f lavonoid that has been show n an ti-oxidative and an tiin f lam m atory activ ities[84,85].Add itiona l cell adhesion,angiogenesis also has been reported[86].In corporated quercetin by zein fo llow ing the un iax ia l e lectrosp inn ing techn ique show s great benef its fo r neu ropathy in d iabetic w ound.Researchers[87]p repare the quercetin sp inn ing so lu tions in a grad ien t con cen tration to determ ine the best quercetin zein ratio,a set of fab rication param eters like 16 kV vo ltage supp ly,0.2m l/h feed rate,15 cm co llection d istan ce are investigated.Diabetic rats are d ivided in to severa l groups acco rd ing to the grad ien t quercetin con cen tration.Morpho logy analysis dem on strates that the in creased ratio of quercetin to zein has no sign if ican t in f luen ce on the d iam eters.Good APId istribution and po ten tia l hyd rogen bond ing betw een d rug and veh icle are detected by FTIR and XRD(X-ray d iffraction).The detection of quercetin release is conducted in the acetate bu ffer and show s an in creased rate from low to h igh-con cen tration of API.A fu rther investigation on the m o to r fun ction of the sciatic nerve revea ls the favo rab le effect of quercetin for neuropathy in d iabetic w ound.Add itionally,quercetin is also treated as an an ti-carcinogen ic chem ica l[88],no m a tter how b road app lication it possesses,the fun dam en ta lm echan ism fo r DFU treatm en t m ay be relevan t to its po ten t an tiox idan t and an t-in f lamm atory capacities[89,90].

Fig.5-Pho tograph s of the w ound closu re of d iabetic rats on days 0,3,7,14,(A)co llagen/PLGA w ith g lu cophage-loaded g roup,(B)pu re co llagen/PLGA g roup,(C)gauze sponge group(Rep rodu ced w ith perm ission from[68].).Copy righ t 2015,Elsevier.

M etfo rm in is a com m on f irst-line clin ica l d rug fo r hyperg lycem ia,deve loped w ith PLGA under the un iaxia l electrosp inn ing techno logy m etfo rm in suggests a m assive po ten tia l in the app lication of DFU treatm en t[91,92].Metform in is a hyd roph ilic agen t,in o rder to get the aim of p ro longed release character,hyd rophobic and biodegradab le PLGA is the very cand idate to be tested.Sim u ltaneously,the add ition of m etfo rm in to PLGA so lu tion decreases the viscosity of the so lution and in creases the feasibility ofnanof ibers fab rication.The release p rof ile ana lysis ind icates that f irst ou tbu rst of m etform in m ay be relevan t to the API located on the su rface of nanof ibers,the sustained re lease fo llow ing the slow ly degradation of PLGA[91].Study has con f irm ed thatm etfo rm in not on ly regu lates the hepatic glucose ou tpu t,bu t a lso im p roves the insu lin sensitivity[93,94].In fact,m etform in released from nanof ibers just acts as a top ica ld rug delivery system w ith limited absorp tion to system ic body.The exp lanations fo r the e ffectiveness of them etform in on w ound in clude enhan ced endothelial p recu rsor cells circu lating,im p roved angiogenesis,and ba lan ced co llagen from the inh ibiting MMP(m atrix m eta llop ro teinases)[68,95].

As d iscussed in the DFU recovery phase,no rm a l vesse l reconstruction and refunction are critica l for the w ound c losu re.Angiogenesis is one strategy in dea ling w ith th is issue,relevan t bio logica l agen ts in tervened in p ractica l are hypox ia-indu cib le facto r 1α(HIF-α),neu ropep tide Y(NPY),and angiopoietin-1[96-98].In the f ie ld of ND fo r DFU treatm en t,d im ethy loxa ly lglycine(DMOG)-em bedded PCLND has p roved to be feasible for the d iabetic w ound treatm en t[99].W ith the bioactivity of inh ibiting oxygen-dependen t degradation of HIF-α,DMOG exh ibits the angiogenesis fun ction in w ound repairm en t[100].In the study p resen t by Zhang et a l.[99],DMOGand PCLare d isso lved in the d ich lorom ethane(DM)and d im ethy lfo rm am ide(DMF),the f ina l ratio of DMOG to PCL in nanof ibers is con tro lled in 0.1m m o l/g.The e lectric po ten tia l and co llecto r d istan ce are abou t 8 kV and 9 cm respectively.A fter the un iaxialelectrosp inn ing,the ND is cu t in to equal d im ension of square.Organ ic so lven t evapo ration is com p leted by vacuum cham ber and air d ryer.Fiber d iam eter and su rface roughness are eva luated by SEM and CM,respectively.The d iam eter is abou t 300 nm and the su rface roughness 2.13μm;m oreover,there is no sign if ican t d ifferen ce betw een virgin PCL and d rug-loaded PCL.Hom ogeneous API d istribu tion in PCL also has been dem onstrated by f luorescen t p robe m o lecu le Rhodam ine 6G.Com pared w ith the con tro ls o r the virgin PCL groups,DMOG-loaded PCL ND show s acce lerated w ound closu re.In creased CD-31(cluster-d ifferen tiation 31)positive cells observed in the im m unostained analysis in dicates thatangiogenesis is sign if ican t statistica lly.Ano ther p rom ising“d rug”app lied in ND fo r the DFU recovery is the silicate-based bioceram ics(NAGEL,Ca7Si2P2O16),the o rigina l in ten tion of NAGEL app lication is to be an alternative to m etallicm aterials for its h igher biocom patibility[101].Cu rren tly,NAGEL has been intensive ly used in the tissue engineering to p rom o te ce ll attachm en t,grow th and new tissue fo rm a tion[102].W ith respect to w ound healing effect,relevan t reportalso can be seen[12],in v iew ofm odu lating the degradation tim e of the po lym ers app lied in nanof ibers,PCL b lend ing w ith gelatin is p repared to veh icle so lu tion.Coaxia l e lectrosp inn ing techno logy is u tilized w ith NAGEL em bedded in the PCL/ge latin tube.A grad ien t ratio of NAGEL to PCL/gelatin is designed to e levate various physica l and bio logica l in dex,o ther param eters su ch as feed rate,vo ltage supp ly,and co llecto r d istan ce are set to be 0.025m l/m in,10 kV,and 12 cm.The crosslin k for the p rim ary nanof ibers is com p leted by glu tara ldehyde/ethy la lcoho l d isposition.The p ro liferation test of the HUVECS(hum an umbilical vein endothelial cells)seeded on the ND w ith d ifferen t NAGEL con cen tration revea ls NAGEL's no apop tosis.Bu t the cell adhesion ana lysis show s that d rug-loaded nanof ibers is m ore effective than the pu re nanof ibers.Direct quan tif ication and assessm en t of the num ber of neovascu lar in the w ound bed is conducted by the d igita l cam era pho tos,im ages taken from it con f irm that NAGEL does have the capacity to im p rove the vessel especia lly the cap illary form ation com pared w ith the con tro l and virgin group.The h igher level of CD31 is also a specif ic p roof.

5.2.2. Macrom olecu les

Po ten tia lm acrom o lecu les fo r DFU hea ling m ain ly refer to cytok ine o r GF.It consists of d iverse groups of po lypep tides and m ost groups have their ow n d ifferen t fam ily m em bers[103].Platelet-derived grow th facto r(PDGF)[104],basic f ib roblast grow th factors(BFGF),vascu lar endo thelia l grow th facto rs(VEGF)and ep iderm a l grow th facto rs(EGF)are re levan t GF[105,106].PDGF w as f irstly iden tif ied as a d isu lf ide-linked d im er of tw o pep tides[107];it is benef icia l fo r the grow th of specif ic cells[108],the fo rm ation of b lood vesse l,and the early hem atopoiesis.GF def iciency in DFU w ound bed is one of the m ost in du cers fo r the p ro longed w oun d hea ling[109],recombinan t hum an PDGF(RHPDGF-BB)has been su ccessfu lly developed to ND for the in itia l phase of d iabetic w ound treatm en t[110],PLGA as a biodegradable po lym er is selected to load the active RHPDGF-BB,17 kV vo ltage pow er and 12 cm co llector d istan ce are set fo r the fab rication attribu tes.The con cen tration of the tw o m ateria ls is 0.28 g/m l and 0.005 g/m l respectively in the so lven t hexaf luoroisop ropy l alcoho l.SEM im ages of the f ina l ND show that the d rug-loaded PLGA has a th inner f ibers than the virgin PLGA.Even though the tensile strength of the d rug-loaded ND is larger than the pu re,the resu lt of the e longation at b reak is just the opposite.Contact ang les of w ater ind icate that the add ition of RHPDGF-BB is help fu l fo r the in creased hyd roph ilicity of the PLGA.Consequen t w ound repair and h isto logy test also dem onstrate that RHPDGF-BB possesses the featu res of acce lerating the d iabetic w ound hea ling and enhan cing the density of stratum co rneum.These resu lts suggest a substan tia l po ten tia l of PDGF for the DFU treatm en t.Other GF like recom binan t hum an EGF(RHEGF)has been used for the DFU w ith the form of in tra lesiona ladm in istration,the clin ica lp rof ile of th is m ethod exh ibits eno rm ous bene f its[111],in the sam e w ay,RHEGFm ay be a p roper cand idate loaded in ND for the DFU recovery.

5.2.3. Stem cells

Considering the fu ll-th ickness w ound,just as the deterioration of DFU,conven tiona l strategies d iscussed above m ay no t be e ff icien t[112].As an advan ced w oun d treatm en t m oda lity,stem cells therapy has becom e m ore and m ore p reva len t in dea ling w ith the com p licated w ound[113].Com m on stem cells are usua lly d ivided in to em b ryon ic and adu lt stem cells.Various resou rces from bone m arrow,periphera l b lood,and hair follicles con tain the adu lt stem cells.W ith the ability of p ro longing self-renew a l and d ifferen tiation in to targeted tissues like sk in,stem cells has been b road ly investigated for the ch ron ic skin w ound treatm en t[114-117].As fo r the m echan ism of the adu lt stem cells app lied for the skin w ound recovery,tw o aspects are reported,one is repopu lating the lost or in ju red tissue w ith the d ifferen tiated cells and the o ther is recru iting in f lam m atory cells o r tissue p rogen ito r ce lls th rough signal m olecu le release[118].In order to obtain the selection of endo thelia l p recu rso r ce lls(EPCs)en richm en t and the e ff icien cy of EPCs de livery.Mononuclear ce lls derived from m u rine bone m arrow are seeded in to the nanof ibersm ade of PCL/gelatin[119].Regu lar physica l param eters of nanof ibers are determ ined by SEM and TEM;m ononuc lea r ce lls a re extracted from the fem u rs of the m ice,and p rim ary ce llu lar suspen sion is cen trifuged and w ashed to obtain the pu re m ononuc lea r ce lls.A fter seeded in the PCL/ge latin m atrix,the cell starts to d ifferen tiate to EPCs in cubated in specif ic cond ition s,and the characterization of EPCs show s that PCL/ge latin m atrix is a su itable stage for EPCs grow th.Fu rther EPCs adhesion,co lony fo rm ation,and p ro liferation are investigated,and the resu lts ind icate the positive effect of PCL/ge latin m atrix for EPCs.In vivo teston d iabeticm icew oun d revea ls that the EPCsloaded PCL/gelatin noton ly accelerates thew ound closu re bu t a lso im p roves the qua lity.Desp ite of unspecif ic app lication fo r DFU,o ther cells like m esen chym a l stem cells[120,121]and ep iderm al stem cells[122-124]are broad ly u tilized for fu llth icknessw ound treatm en t seeded in p roper nanof ib rousm atrix.

6. Fu rther perspectives

DFU im poses risks of am pu tation and econom ic lost on d iabetic patien ts.Wound d ressing is necessary in p rotecting and accelerating the w ound closu re.ND is an advan ced techn ique w ith characters of th in d iam eters ranging from nanom eters to m icrom eters,consequen t capacities of oxygen penetration and ECM recon stru ction are ob tained.Fu rtherm ore,u tilization of various po lym ers from natu ra l and syn thetic sou rces renders ND a better se lection fo r d iabetic w ound c losu re compared w ith conven tional d ressing types.Add itionally,bioactive sm a llm o lecu les,m acrom o lecu les,and ce lls can be loaded in the nanof ibers to op tim ize the DFU recovery p rocess.Proper selection of po lym ers and p rocess param eters w ill fab ricate desirable ND for DFU treatm en t.How ever,the rheological behav io r that in f luen ces the physicochem ica l p roperties of nanof ibers and the m athem atica lm ode l fo r it in pharm aceutica l cyc le still has a big room for researchers to study and establish.

Con f lict of in terest

The au thors declare that there is no con f lict of in terest.

Acknow ledgm en t

Th is w ork w as f inan cia lly supported by the Nationa l Natu ra l Scien ce Foundation of Ch ina(No.81600353)and the Career Developm en t Program for Young Teachers in Shenyang Pharm aceu tica l Un iversity.

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