Biomaterial Scaffolds for Improving Vascularization During Skin Flap Regeneration

Yunkun PEI,Liucheng ZHANG,Xiyuan MAO,Zhimo LIU,Wenguo CUI ,Xiaoming SUN ,Yuguang ZHANG

Dr.Y.PEI,Dr.L.ZHANG,Dr.X.MAO,Dr.Z.LIU,Dr.X.SUN,and Prof.Y.ZHANG Department of Plastic and Reconstructive Surgery,Shanghai Ninth People's Hospital,Shanghai Jiaotong University School of Medicine,639 Zhi Zao Ju Road,Shanghai 200011,China E-mail:drsunxm@126.com(X.Sun),zhangyg18@126.com (Y.Zhang)

SUMMARY Over the past few decades,biomaterials have made rapid advances in tissue engineering.In particular,there have been several studies on vascularization during skin flap regeneration for plastic surgery.From the perspective of function,the biomaterials used to improve the vascularization of skin flaps are primarily classified into two types:(1)electrospun nanofibrous membranes as porous scaffolds,and (2) hydrogels as cell or cytokine carriers.Based on their source,various natural,synthetic,and semi-synthetic biomaterials have been developed with respective characteristics.For the ischemic environment of the flap tissue,the therapeutic effect of the combination of biomaterials was better than that of drugs,cytokines,and cells alone.Biomaterials could improve cell migration,prolong the efficacy of cytokines,and provide an advantageous survival environment to transplanted cells.

KEY WORDS skin flap; vascularization; biomaterial

INTRODUCTION

Skin flap surgery is commonly performed to repair a skin defect that can be hardly replaced by skin grafting surgery or other types of skin flaps due to its flexibility.However,partial or complete necrosis of distal region of random skin flap,caused by the limitation of length to width ratio of flap and local or systemic metabolic diseases,which leads to insufficiency of blood supply and restricted micro-vascularization of distal region of random flap,remains a great challenge for plastic surgery[1-4].Multiple approaches have been reported for improving skin flap survival,such as vasodilators,sympathetic blocking drugs,and antithrombotic agents.Although these drugs can improve skin flap survival by increasing local blood supply[5,6],systematic treatment could cause low blood pressure or coagulation disorders.On the other hand,growth factors (GFs),such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF),can promote local micro-vascularization,leading to better therapeutic effect on skin flap survival than medicines[7,8].However,the therapeutic effect of GFs is still unsatisfactory due to short half-life period,inefficiency of local application,and potential side effects.For now,delayed surgery is a good supplemental therapy to improve skin flap survival by stimulating the local blood circulation system remodeling.However,the patients may suffer additional pain because of secondary surgery[9].It has been reported that local injection of mesenchymal stem cells(MSCs) could improve capillary formation to promote random flap survival by paracrine effect to increase the levels of vascularization-associated GFs,which leads to good outcome[10].However,the mesenchymal stem cells(MSCs) implanted in the distal region of random flap,which is a highly ischemic environment,are difficult to adapt to the sudden change in microenvironment,leading to the death of massive levels of MSCs postimplantation[11].

MECHANISM OF DISTAL RANDOMPATTERN FLAP NECROSIS

Distal flap necrosis has been a common complication that has bewildered the plastic surgeons for a long period.Although the specific pathological mechanism has not been clear yet,ischemia reperfusion injury,insufficient blood supply,and hemodynamic injury are the most probable causes[4].Hence,increasing microvessel density and blood supply are crucial for flap survival.As a result,the survival rate of random flap is positively correlated with flap blood supply.Hence,flap survival is mainly affected by adequate blood supply as well as good microcirculation.Compared with axial flap,random flap blood supply is mainly dependent on subdermal vascular plexus.This finding showed that vascularization of the wound is more important than vascularization around the flap,which indicates that enhancing flap subdermal vascularization greatly influences random flap survival[18-20].Vascularization of ischemic tissues mainly relies on endothelial cell and GFs associated with vascularization as well as the interaction between them[21-23].Microenvironment of the injured tissue (such as inflammatory factor,oxygen radical,and hypoxia)has a major influence on vascularization process.Therefore,promotion of angiogenesis under specific microenvironment plays an important role in remodeling the blood supply of random skin flap.

FEASIBLE TREATMENT TO PROMOTE VASCULARIZATION IN RANDOM FLAP

Drug application

Many reports have explored different ways to improve random skin flap survival rate,such as systemic or local application of vasoactive agents,radical scavenging drugs and local use of GFs.For example,Xu et al.[24]systematically applied Dilong injection to a McFarlane flap rat model and proved that it could significantly promote vascular density of the flap and improve flap survival.Wang et al.[25]reported that local injection of deferoxamine (DFO),which has been proved to improve neovascularization,could improve random skin flap survival in diabetic mice.In addition,it has been shown that Bezafibrate,Huangqi,Thymosin β4,and other vasoactive drugs could improve random flap survival by increasing microvascular density and local blood flow to improve blood supply in the distal region of the flap[1,26,27].

On the other hand,ischemia-reperfusion injury,which leads to overproduction of free radicals,is another important reason for distal random flap necrosis.Therefore,many researchers used anti-oxidative stress drugs to improve random flap survival.Can et al.significantly improved flap viability on rat by systemic application of Coenzyme Q10 (CQ10),which has antiinflammatory and antioxidant properties[28].Fukunaga et al.found that local injection of free radical scavenger,nitrosonifedipine,could reduce oxidative stress as well as cell apoptosis,thus increasing random flap survival[29].Meanwhile,calcitriol,kaurenoic acid,and diammonium glycyrrhizinate have also been proved to play a role in improving the antioxidative stress ability of random flap,and thus,enhance flap survival[2,30,31].

Therefore,with the development of studies on drugs that can increase vascularization or scavenge free radicals,random flap survival can be improved in some way.However,systemic application of these drugs may cause side effects,such as low blood pressure or coagulation dysfunction.Besides,pharmaceutical effect of drugs can be diluted by circulation and don't last long during local application,which is ineffective in therapeutic.

Cytokine application

It has been revealed that GFs (such as VEGF,bFGF,etc.) exhibit strong pharmacological ability to promote vascularization.Recently,several studies on local application of GFs in improving random flap survival have been reported.For example,Rah et al.employed gene therapy using adenovirus that expressed hepatocyte growth factor to random skin flap to promote local VEGF expression,which enhances skin flap survival[32].Park et al.used heparin-conjugated fibrin (HCF) as a carrier for sustained delivery of fibroblast growth factor 2(FGF2) to skin flap to promote its survival[33].Akimoto et al.found that preoperative injection of VEGF-A164,when combined with a clostridium-derived collagen binging domain (CB-VEGF),could improve random flap survival[34].Although local application of GFs shows a good effect on improving angiogenesis,these therapeutic methods,which are restricted to vascularization promotion only,exhibit certain limitations in terms of therapeutic effects,extensive use for complex gene modification technique,and high expenses.

Cell therapy

With the development of regenerative medicine,several kinds of cells have been found to play a role in vascularization,including endothelial cells (ECs),endothelial progenitor cells (EPCs),and mesenchymal stem cells (MSCs).Nevertheless,ECs are not applicable for tissue engineering and regenerative medicine because of their heterogeneity in phenotype and genotype as well as their low proliferation rate[35-37].

MSCs have been widely applied in regenerative medicine research owing to their pluripotency,secretion of various angiogenic factors,and good immunological tolerance.For instance,Zheng et al.[38]transfected VEGF gene into bone marrow mesenchymal stem cells (BMSCs)to promote VEGF expression by liposome and injected VEGF-transfected BMSCs into random flap region to enhance flap survival.Some researchers employed BMSC sheets or BMSC-containing acellular amniotic membrane matrix to improve flap survival[39-40]; however,this approach is not feasible for clinical application due to limited sources of BMSCs and the trauma caused by getting BMSCs.

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The use of adipose-derived stem cells (ADSCs),the mesenchymal cells derived from stromal vesselassociated adipose tissue,is more advantageous because of their derivation by minimally invasive liposuction causing less trauma compared with other sources of MSCs.In addition,ADSCs can be rapidly cultured and amplified in vitro for that a great amount of ADSCs can be derived at a time[41,42].Many researches have indicated that ADSCs show a satisfactory effect in promoting chronic wound healing mainly via paracrine effect,which leads to production of high levels of GFs and chemokines,such as VEGF,bFGF,epidermal growth factor (EGF),Angiopoietin-1 (Ang-1),and erythropoietin (EPO)[43-45].Moreover,ADSCs can produce large amounts of peroxiredoxins,such as Insulin-like growth factor binding proteins (IGFBPs),granulocyte colony-stimulating factor (G-CSF),platelet derived growth factor-AA (PDGF-AA),superoxide dismutase(SOD2),pigment epithelium-derived factor (PEDF),and hepatocyte growth factor (HGF),which eliminate free radicals and reduce microcirculation damage caused by ischemia-reperfusion[46].Over all,the factors secreted by ADSCs optimize cellular microenvironment of the injured region and significantly accelerate skin flap vascularization in early injury,which improved local blood supply and enhanced flap survival.By local injection of ADSCs to random flap,Lu et al.investigated the effect of ADSCs on random flap survival and found that ADSCs promoted vascularization through paracrine effect and differentiation into endothelial cells[47].Gao et al.observed that local subcutaneous injection of ADSCs increased hypoxia inducible factor-1α (HIF-1α) expression,which significantly improved distal flap reperfusion and ischemic skin flap survival rate in diabetic model[10].In conclusion,ADSCs have a great potential as seed cells for tissue-engineering application.

ELECTROSPUN FIBROUS SCAFFOLD APPLICATION IN VASCULARIZATION

With the development of tissue-engineering,various kinds of functional biomaterial scaffolds have been implanted into animals and have been found to play a role in improving vascularization[48,49].Biomaterial scaffold is an important factor in improving vascularization because that its structure gives support for cell adhesion and is conductive for angiogenesis.

In recent years,electrospinning (Figure 2) has been widely applied in preparing ultrafine fibrous membrane whose diameter ranges from nanometers to microns and mimics extracellular matrix (ECM).The nanofibrous scaffolds made by electrospinning exhibit several advantages,such as high porosity,large specific surface area,fine fiber refinement and homogeneity,and great length-diameter ratio,which are responsible for their wide applications[50,51].It has previously been demonstrated that electrospun nanofiber scaffolds can promote vascularization in distal random flap and reduce the necrosis rate (Figure 3)[52,53].

Electrospun scaffolds made of synthetic materials

Many researchers have used 2D or 3D electrospun fibrous scaffolds,made of polylactic acid (PLLA)or polycaprolactone (PCL),for tissue-engineering vascularization.Ku et al.produced electrospun PCL nanofibers functionalized with catecholamine ad-layer as core support for vascular tissue engineering.However,the results of in vitro experiments showed that it was difficult to construct 3D vascular scaffolds because the pore size of nanofibers were too small,and thus,ECs could only grow on surface[54,55].Besides,some materials whose mechanical hardness is too high are not suitable for soft tissue regeneration and may cause severe inflammatory response in vivo[56].Therefore,development of electrospun fibrous scaffolds with properties of high biocompatibility,suitable pore size for cell migration,and stimulating skin properties is urgently needed.

Electrospun scaffolds made of natural materials

Biomaterials made of natural sources,such as collagen,elastin,gelatin and fibrin,are widely used in preparation of tissue-engineering materials because of their good biological properties.Sun et al.confirmed the advantage of natural material compared to the synthetic material(Figure 3).Biomaterial made from natural materials has been shown to be efficient in promoting cell adhesion and skin flap survival rate[53].These kinds of biomaterial scaffolds have good application potential in tissueengineering vessel production.Zhang et al.prepared silk fibroin scaffolds seeded with human aortic endothelial(HAEC) and human coronary artery smooth muscle cell (HCASMC) and found a favorable interaction between electrospun silk fibroin scaffolds and HAEC as well as HCASMC.They also observed that the seeded ECs formed a complex interconnecting network with identifiable lumens on the surface of materials over a period of seven days[57].In addition,Boland et al.[58]produced electrospun scaffolds from collagen and elastin seeded with smooth muscle cells (SMCs),fibroblasts,and ECs for tissue engineering vascular construct.However,the application of electrospun scaffolds made of natural materials in constructing tissue-engineering vessel is limited due to their poor mechanical strength.

HYDROGEL APPLICATION IN VASCULARIZATION

Hydrogel,with its characteristics of high water content,controllable physical and chemical properties,as well as good simulation of tissue microenvironment,is another biomaterial widely used in tissue engineering[59,60].Besides,its other characteristics,such as ECM-like structure,less stringent preparation condition,and controlled degradation to satisfy vascularization,make it a potential matrix to promote angiogenesis.

GF-containing hydrogel scaffold application in vascularization

According to the reports,several kinds of natural and synthetic hydrogel matrices have been used as media loading vascularization-associated GFs.Hydrogel can be constructed using natural materials,including proteins(such as collagen,gelatin and fibrous protein)[61-63]or polysaccharides (such as hyaluronic acid,alginate,and chitosan)[64-66],and synthetic materials,including polyethylene glycol (PEG) or poly (lactide-glycolide)acid (PLGA)[67-68].

Chitosan,a natural alkaline polysaccharide,obtained by deacetylation of chitin in shrimp and crab shells,is nontoxic and biodegradable and does not cause rejection.Fuji et al.produced a chitosan hydrogel that could control FGF-2 release by adding non-anticoagulant heparin and FGF-2 to viscous water-soluble chitosan (CH-LA),and this hydrogel was applied to wound healing-impaired diabetic mice and ischemic left lower limbs of rats by local injection.They found that this chitosan hydrogel could promote local angiogenesis[66].

Fibrin is the main constituent of a blood clot.Fibrinbased materials with great biocompatibility are beneficial for vascularization and tissue reconstruction.Researchers have also shown that fibrin-based hydrogel also promoted vascularization.For example,Christman et al.injected fibrin-hydrogel into a rat ischemic myocardium model and found that the myocardial function and thickness of necrotic myocardium were both significantly improved[69].There are many other reports about fibrin-based hydrogel carrying vascularization-promoting GFs.The modified fibrin-based hydrogel exhibits better controlled release of GFs and avoids the burst release of GFs.Sacchi et al.dissolved VEGF-164 to a sequence derived from α2-plasmin inhibitor containing coagulation factor XIIIa that allowed VEGF164 covalent cross-linking into fibrin hydrogel and being released only by enzymatic cleavage.The fibrinolysis inhibitor aprotinin was used to control the degradation rate of hydrogel.This fibrin-α2-PI1-8-VEGF164 system had a significant therapeutic effect on wound healing models as well as hind limb models[63].

Hydrogel scaffolds made of synthetic materials are used as the carrier of GFs to enhance local vascularization as well.For example,PEG-based hydrogel constructed by poly (ethylene glycol) diacrylate (PEGDA),whose mechanical properties are better than hydrogel made of natural materials,has been widely applied in preparing tissue-engineering scaffolds.Leslie-Barbick et al.constructed a PEG hydrogel covalently bounded with QK,a VEGF-mimicking peptide,onto the surface and found that PEG-QK hydrogel scaffold showed superior ability to enhance angiogenesis in vitro and promote mouse cornea angiogenesis in vivo[70].

Hydrogel scaffolds made of semi-synthetic materials have gained much attention in recent years because of their physical and chemical characteristics imparted by both natural and synthetic materials.Biomaterials such as hyaluronic acid (HA),gelatin,alginate and others have been widely used for construction of semisynthetic hydrogel[72-74].For example,Peattie et al.[75]incorporated small amounts of modified heparin in thiolmodified HA hydrogels cross-linked by PEG-diacrylate(PEGDA) to control GF release and maintained the bioactivity of released GFs.The study showed that VEGFor angiopoietin-1 (Ang-1)-loaded HA hydrogel performed better in enhancing local angiogenesis than hydrogel alone.

Gelatin methacryloyl (GelMA) hydrogel is a photocrosslinkable hydrogel produced by adding methacrylate group to the amine-containing side groups of gelatin.The photopolymerizable methacrylate group-modified gelatin could not only maintain its own properties but also convert into a solid form via a chemical reaction with methacrylate group.Furthermore,it is easy to control the mechanical properties,degradation,and biological properties of GelMA hydrogel by changing the extent of substitution of methacrylate group[76-78].Prakash Parthiban et al.constructed GelMA hydrogel that was covalently linked with VEGF mimicking peptide (AcQK) and found that the hydrogel system could upregulate vascularspecific genes accelerating micro-vascularization[79].

MSC-carrying Hydrogel scaffold application in vascularization

To improve the therapeutic efficiency of stem cells in ischemic environment,a large number of biomaterials have been used as the carrier in promoting seed cell survival and transplantation efficiency[81,82].Hydrogel is widely used as carrier for stem cell treatment because of its properties.In recent years,many researchers have indicated that various kinds of natural and synthetic hydrogels have been applied in cells with encapsulation coating that are implanted in vivo to improve vascularization[83,84].The advantage of implantation of hydrogel encapsulation cell is that the hydrogel can regulate the implantation microenvironment,increase cell retention,and promote cells survival in ischemic condition.Most importantly,the application of hydrogels can provide favorable physical,chemical,and biological signals for various kinds of GFs and cell-guiding angiogenesis[85].Wang et al.reported a chitosan hydrogel carrying brown adipose tissue derived stem cells (BADSCs)repairing ischemic myocardial tissue and showed that it could improve BADSC survival and differentiation after implantation[86].Another study reported using modified alginate hydrogel implantation cells to promote vascularization.The in vitro study showed that RGDmodified alginate could increase MSC attachment and growth and improve the expression of angiogenic GFs.In vivo implantation of RGD-alginate containing MSCs could improve cell survival in ischemic environment as well as enhance vascularization in order to promote myocardial repair[87].

The methods of hydrogel crosslinking mainly include UV crosslinking,ionic crosslinking,and temperature sensitivity crosslinking.However,these crosslinking methods usually import cytotoxic products from chemicals and crosslinking procedure,which adversely affect stem cell viability[88,89].Therefore,a lessstringent crosslinking procedure is needed for hydrogel preparation for better regulation of stem cell viability and improving the therapeutic effect of stem cells to promote vascularization.

Dextran,a natural polysaccharide composed by glucose,shows significant biocompatibility and nontoxicity and can be easily chemically modified[90,91].In addition,an in situ formation of dextran-based hydrogel,formed by thiol-Michael addition reaction,has been reported whose preparation protocol extends only for 2 minutes,which could help in avoiding cell toxicity derived from traditional crosslinking,such as UV light,heating,and pH.Furthermore,the dextran-based hydrogel remains stable at pH ranging from 7.0 to 7.8 and its pores can be regulated by changing the pH during hydrogel preparation pH.Moreover,the reaction can be conducted under physiological conditions.Therefore,dextran-based hydrogel could be an ideal biomaterial for encapsulating cells and cell-based transplantation.After encapsulating mouse embryonic fibroblasts or rat BMSCs with hydrogel,the researchers found that a high viability of hydrogel encapsulated cells of more than 80%,which remained up to 70% when cultured 14 days in vitro[92].Dextran-based hydrogel exhibits great potential in promoting ischemic tissue vascularization.

In conclusion,hydrogel scaffolds,not only as the cell adhesion structure but also as the carrier for cells promoting in vivo vascularization implantation,exhibit unique advantages in improving vascularization.Therefore,it is quite important to prepare nano-fibrous scaffolds under nontoxic crosslinking condition on which the cells can grow in 3D or hydrogel scaffolds that can encapsulate cells and maximize stem cell function.These biomaterials could prove to be beneficial in promoting tissue angiogenesis,and thus,improving random flap survival,which could be instrumental in solving the problem of ischemic random skin flap and improving the method of local application of the cells.

CONCLUSION AND PROSPECT

In recent years,biomaterials have been widely applied in improving ischemic skin flap regeneration by providing adequate micro-environment for tissue-engineering vascularization as well as by being the carrier of vasoactive or radical scavenging drugs,GFs,or certain cells to speed up local vascularization.Among these,hydrogel shows a high potential as an angiogenesis promoting matrix.However,there are still some limitations in its application,since hydrogel scaffolds that are made of natural materials are deficient in mechanical properties while those that are made from synthetic materials may not have adequate biocompatibility.The currently available biomaterial scaffolds show great potential in improving skin flap survival but they still exhibit certain limitations.Therefore,preparing a desirable biomaterial scaffold with appropriate properties,which refers to a biomaterial scaffold that can better simulate normal tissue microenvironment with good mechanical properties,high biocompatibility,less stringent preparation condition as well as controllable degradation and release of GFs or cells,to enhance angiogenesis and ameliorate skin flap ischemia might be beneficial in treating ischemic random skin flap,which could provide certain progress in flap transplantation.

ACKNOWLEDGEMENTS

Yunkun Pei and Liucheng Zhang contributed equally to this work.This work was supported,in part,by the National Natural Science Foundation of China (81772099,81701907,81801928,and 81772087),Shanghai Sailing Program (18YF1412400),Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20171906),Shanghai talent development fund (2018099),Shanghai Municipal Health and Family Planning Commission (201840027),and Shanghai Jiao Tong University “Medical and Research” Program(ZH2018ZDA04).We also thank Y.Wang for the helpful discussion and technical assistance.

COMPETING INTERESTS

The authors declare no competing interests.