pereira et al. (2013) - advanced biofabrication strategies for skin regeneration and repair

8/11/2019 Pereira Et Al. (2013) - Advanced Biofabrication Strategies for Skin Regeneration and Repair

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ISSN 1743-588910 2217/NNM 13 50 2013 Future Medicine Ltd Nanomedicine (2013) 8(4) 603621

Advanced biofabrication strategies for skin

regeneration and repair

Skin damage & wound healingSkin is the largest organ of the body, servingprimarily as a protective barrier against theenvironment [13]. It also helps to prevent bodydehydration and constitutes a physical barrier,limiting the penetration of potentially harmfulagents to internal organs. The skin has a three-layer structure composed of epidermis, dermisand hypodermis (FIGURE1) [46]. The epidermis,the supercial layer, is mainly composed of kera-tinocytes but also contains other cell types, suchas Langerhans cells and melanocytes [5,7], provid-ing a barrier against infection and moisture loss.The dermal layer, situated below the epidermis,is responsible for the elasticity and mechanicalintegrity of the skin. It contains vascularizedextracellular matrix (ECM) rich in collagen, elas-tin and glycosaminoglycans [3,5,6]. The cellular

components of the dermal layer include bro-blasts, endothelial cells, smooth muscle cells andmast cells [3,5]. The hypodermis, located belowthe dermis, is mainly composed of adipose tis-sue and collagen, and mainly acts as an energysource [4,5,7].

Skin damage is mainly caused by burn injuries,chronic wounds (venous, pressure and leg ulcers),excision of skin, tumors and other dermatologicalconditions [2,8]. According to WHO, 300,000deaths are annually attributed to burn injuries,while 6 million patients worldwide suffer from

burns every year [4]. Additionally, more than

6 million individuals suffer from chronic skinulcers [4]. In the USA alone more than 3 millionpatients suffer from chronic wounds [9].

Skin has a natural healing ability that gener-ates, at the moment of injury, a complex cascadeof highly integrated and overlapping events ofhemostasis, inammation, migration, prolif-eration and maturation, as illustrated in FIGURE2[1012]. This complex, dynamic and continuousprocess relies on the interaction between cel-lular components, growth factors and cyto-kines, acting in concert to repair the damagedtissue [13,14].

Treatment of skin lesionsThe treatment of skin lesions is a critical issue inhealthcare, requiring the need to consider severalparameters affecting the healing process, such

as the wound type (e.g., burn, ulcer, acute andchronic wound), the wound depth (e.g., epi-dermal, superficial partial-thickness, deeppartial-thickness and full-thickness wounds),the patients health (e.g., diabetes and persistentinfections) and the level of the exudate [14]. Cur-rently, a great variety of wound-care products areavailable for the treatment of different types ofskin lesions (TABLE 1).

Solutions, creams & ointmentsLiquid and semi-solid formulations, such as

solutions, creams and ointments, have been

Skin is the largest organ of human body, acting as a barrier with protective, immunologic and sensorialfunctions. Its permanent exposure to the external environment can result in different kinds of damagewith loss of variable volumes of extracellular matrix. For the treatment of skin lesions, several strategiesare currently available, such as the application of autografts, allografts, wound dressings and tissue-engineered substitutes. Although proven clinically effective, these strategies are still characterized by keylimitations such as patient morbidity, inadequate vascularization, low adherence to the wound bed, theinability to reproduce skin appendages and high manufacturing costs. Advanced strategies based on bothbottom-up and top-down approaches offer an effective, permanent and viable alternative to solve theabovementioned drawbacks by combining biomaterials, cells, growth factors and advanced

biomanufacturing techniques. This review details recent advances in skin regeneration and repairstrategies, and describes their major advantages and limitations. Future prospects for skin regenerationare also outlined.

KEYWORDS: bottom-up approach in situbiofabrication nanoscale structureskin regeneration tissue engineering top-down approach wound healing

Rben F Pereira1,2,3,

Crisna C Barrias2,

Pedro L Granja2,3,4

& Paulo J Bartolo*1

1Centre for Rapid & Sustainable

Product D evelopment, Polytechnic

Instute of Leiria, Portugal2Instuto de Engenharia Biomdica,

Universidade do Porto, Porto, Portug3Instuto de Cincias Biomdicas Abe

Salazar, Universidade do Porto, Porto

Portugal4Faculdade de Engenharia da

Universidade do Porto, Departament

de Engenharia Metalrgica &

Materiais, Porto, Portugal

*Author for correspondence:

paulo.bartolo@ipleir ia.pt

SPECIAL FOCUS Advanced nanobiomaterialsfor tissue engineering and regenerative medicine

part of


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REVIEW Pereira, Barrias, Granja & Bartolo

widely used for wound heal ing, disinfection,cleaning and debridement [1521]. However, thephysical form of these products limits their per-manency in the wound for acceptable clinicalperiods [13]. Consequently, these agents havebeen replaced by advanced products that includewound dressings and skin substitutes.

Wound dressingsSkin lesions involving the loss of high amountsof skin require the immediate use of a dress-ing primarily to protect the wound [22]. Wound

dressings have been widely used due to theirrelative low cost, ease of use, and effectivenessto clean, cover and protect the wound fromthe external environment. An ideal wounddressing should provide a moist environmentto the wound bed, remove the excess of exu-date, avoid maceration, minimize scar forma-tion, protect the wound from infection andmaintain an adequate exchange of gases [2325].Wound dressings must be exible, permeableto water vapor, t the lesion region, and exhibitgood adhesion to the wound bed and adequate

mechanical properties [6,2327]. Some dressings

also act as drug delivery systems, incorporatingand releasing therapeutic agents into the woundbed [24,2833]. Wound dressings can be classiedaccording to different parameters, including thetype of material, function in the wound, physi-cal form or ability to provide a moist environ-ment to the wound. They can a lso be classiedas traditional and modern dressings [13].

Traditional dressings, such as bandages,gauzes or cotton wool, represent the mostconventional products used for the treatmentof skin lesions, providing a protective barrier

against bacteria and microbes [26]. When appliedto the wound, they absorb large amounts ofexudate, drying the wound bed and avoidinga moist wound environment, which is widelyrecognized to promote the healing process [6,31].Thus, the use of these materials may inhibitthe healing process [6,32,33]. Owing to the highrate of uid absorption, these dressings may alsoadhere to the wound bed, making its removaldifcult and consequently causing pain to thepatient [13].

Modern dressings were developed to address

the limitations of traditional ones. They are

Dermis

Epidermis

Hair shaft

Sweat gland pore

Sweat gland

Keratinocytes

Melanocyte

Elastin

Fibroblast

Artery

Adipose tissue

Vein

Collagen

Hypodermis

Figure 1. Skin structure showing the three layers, the skin appendages and the maincellular constituents.


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Advanced biofabrication strategies for skin regeneration & repair REVIEW

able to create and maintain a warm, moistenvironment, providing optimal conditionsfor an improved healing process [6,13], contraryto traditional dressings. Modern dressings canbe obtained from either natural [23,24,3437]orsynthetic polymers [28,3841], or a combination

of both [30,4244]. They are available as thinlms, foams or gels, and can be classied ashydrocolloid dressings, alginate dressings andnonalginate dressings (TABLE 2)[13].

Autografts & allograftsIn the case of patients with skin lesions extendedto the dermis or hypodermis regions, a complextreatment procedure is required. In these cases,the clinical gold standard procedure relieson the use of split-thickness skin autografts orallografts, which contain all of the epidermis

and marginal parts of the dermis[7,45]

. However,autografts are naturally limited in their extent,inducing scarring at donor sites and lengthy hos-pital stays, while allografts present ethical andsafety issues related to disease transmission, andmay lead to immune rejection [2,3,5,46,47]. There-fore, alternatives providing permanent solutionsare required.

Tissue-engineered skin substitutesTissue engineering has emerged as a new andpromising eld for the treatment of skin lesions,combining scaffolds, cells and biomolecularsignals, such as growth factors. It is a multi-disciplinary eld requiring the combined effort

of clinicians, cell biologists, engineers, materialscientists and geneticists toward the developmentof biological substitutes to restore, maintain orimprove tissue function [48].

Despite recent developments in manufac-turing processes and biomaterials, both auto-grafts/allografts and wound dressings have sig-nicant limitations for skin regeneration, whichwere previously mentioned. The main draw-backs of wound dressings are the low adhesionto the lesion, impossibility of reproducing skinappendages and the inability to replace the lost

tissue, particularly the dermis, after severe burns[2,13]. In order to address these limitations andsolve the problem of the donor graft shortage,both cellular (e.g., Apligraf; Organogenesis,MA, USA) and acellular (e.g., Alloderm;Biohorizons, AL, USA) tissue-engineered skinsubstitutes were developed. Skin regenerationis, in fact, among the few elds where tissue

Fibrin clotPlateletMacrophage

InflammationHemostasis

Migration and proliferation Maturation

MacrophageNeutrophilLymphocyteMast cell

Late granulation tissueKeratinocyteEpithelial cellFibroblast

Granulation tissue

Figure 2. Phases of the wound healing process.Hemostasis involves the formation of a fibrin clot occurring simultaneously with theinflammatory phase, in which inflammatory cells are responsible for wound cleaning. During the migration and proliferation phases,epithelial cells and fibroblasts migrate to the wound site in order to synthesize the constituents of the extracellular matrix, leading to theformation of a granulation tissue. In the maturation phase, the composition and properties of the granulation tissue are continuouslyremodeled in order to achieve values proximal to the healthy skin.Adapted with permission from [4].


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engineering commercial products are alreadyavailable and under clinical utilization. Acellularconstructs are made of natural or syntheticbiomaterials, and can be used in combinationwith autografts [2]. Cellular constructs containbiomaterials and cells, obtained from differentorigins including autologous, al logenic or xeno-geneic [7]. Clinically available skin substitutescan be broadly divided into epidermal, dermaland dermoepidermal constructs, as shown inTABL E3 [4]. However, available skin substitutesoften suffer from a range of problems including

poor integration due to inadequate vasculariza-tion, inefcient adhesion to the wound bed,scarring at graft margins or the lack of skinappendages [5,49].

Epidermal substitutesEpidermal substitutes, used for the treatmentof lesions affecting the epidermis, contain auto-logous keratinocytes that are generally culturedin the presence of murine broblasts [7]. Toproduce epidermal substitutes, the rst step isa skin biopsy with an extension in the range of

25 cm2

. The epidermis is then separated from

the dermis, and the keratinocytes isolated andcultured using an appropriate medium. As thisprocedure takes approximately 3 weeks, thewounds are primarily treated with temporarydressings that protect the lesion and stimulatethe healing [2,7]. Major limitations of epidermalconstructs are the fabrication time, high pro-duction costs, variable engraftment rates anddifcult handling, as a result of the thin andfragile nature of the constructs [7]. To overcomethe long fabrication times associated with theseepidermal substitutes, cell populations obtained

from a biopsy can alternatively be directlysprayed into the lesion (ReCell; Avita Medical,Perth, Australia; and SprayXP; Graco, MN,USA), allowing the local delivery of epidermalcells [7,50,51]. This strategy allows a faster epithe-lialization and epidermal maturation. However,it is not suitable for the treatment of third-degreeburn wounds [7]. Zhu et al.investigated the clini-cal efcacy of a surface-functionalized medical-grade polymer disc that enables the delivery ofcultured autologous keratinocytes into burn andchronic wounds [52]. Skin lesions treated with

this strategy showed improved healing rates and

Table 1. Strategies for the treatment of skin lesions: main advantages and limitations.

Treatment of skinlesions

Advantages Limitations

Autografts Golden standard in skin regeneration; good adhesion to

the wound bed; provides pain relief; reduced rejection

Limited availability of donor sites; induce scar

formation; patient morbidity; lengthy hospital stays

Allografts Temporary prevention of wound dehydration andcontamination; incorporate into deep wounds Limited availability; may lead to immune rejection;transmission of diseases

Creams, solutions and

ointments

Ease of use; provide disinfection, cleaning and

debridement; not expensive in general

Limited skin regeneration; short residence time on the

wound (require frequent administrations)

Traditional dressings Not expensive; provide a protective barrier against the

penetration of exogenous microbes

High absorption capacity; do not provide a moist

environment; adhere to the wound bed; may inhibit

the healing process

Modern dressings Create and maintain a moist wound environment; can be

made from a wide range of materials with different

properties; ability to hydrate the wound and remove

excess exudate

More expensive; low adhesion to the wound bed;

inability to promote the regeneration of lost skin, in

particular the dermal layer

Tissue-engineered

skin substitutes

Promote the regeneration of dermis and epidermis;

prevent fluid loss and provide protection from

contamination; may deliver extracellular matrix

components, cytokines, growth factors and drugs to the

wound bed, enhancing the healing process; can be used

in combination with autografts

High manufacturing costs; difficult handling; poor

adhesion to the wound bed; possibility of immune

rejection and transmission of diseases (allogeneic skin

cells); inability to promote the regeneration of

full-thickness wounds; poor vascularization;

impossibility of reproducing skin appendages

In situbiofabrication

of skin substitutes

Provide immediate and effective relief to the patient;

enable the direct fabrication of skin substitutes fitting to

the anatomical shape of the defect; allow the controlled

deposition of cells and biomaterials; may eliminate the

use of bioreactors for growing and maturing the tissue

ex vivo; may solve the need for vascularization through

the controlled deposition of endothelial cells

Biofabrication techniques need to be adapted for

in situbiofabrication; may require integration with

imaging techniques to print the skin substitute with

appropriate anatomical shape; requires the use of

printable materials exhibiting adequate biological and

mechanical properties

Data taken from [2,13].


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a signicant reduction in ulcer size. Recently,Hu et al.developed a skin substitute composedof human EGF gene-modified HaCaT cells(an immortal keratinocyte cell line) [53]. Theauthors investigated the ability of the constructto heal burn wounds created in a rat model.

Results indicated that the substitute signicantlyimproved epidermal morphogenesis along withthe formation of thicker and compact completeepidermis. Other clinical trials were performedto evaluate the effectiveness of epidermal sub-stitutes for the regeneration of different skinlesions such as chronic wounds [54,55]and ulcers[56,57]. However, further clinical investigation isnecessary to evaluate its effectiveness and safety.

Dermal substitutesThe treatment of full-thickness lesions, affect-

ing both epidermis and dermis, requires the useof an allograft skin or, alternatively, the use ofdermal constructs [58]. Dermal substitutes canbe produced by using either natural or syntheticmaterials [59]. These substitutes prevent woundcontraction, provide good mechanical stability,and are available in different thicknesses andcompositions [2,58]. Dermal substitutes must be

covered by a permanent epidermal substitute,usually an autologous split-thickness skin graft[58]. The substitutes undergo colonization andvascularization by the underlying cells, leadingto the formation of an autologous neodermis [7].Dermal substitutes are used for the treatment

of skin ulcers [60,61]and burns [6264]; althoughmore controlled clinical trials are required,involving a signicant number of patients, toconrm its clinical effectiveness. Recently, Guoet al.developed a bilayer dermal substitute com-posed of a covalently crosslinked collagenchi-tosan sponge and a silicone membrane, whichacts as a temporary protection layer [65]. Thedermal substitute was loaded with plasmid DNAencoding VEGF-165/N,N,N-trimethyl chitosanchloride complexes, and tested for the regen-eration of full-thickness burn wounds in a pig

model. In vitroresults showed that the humanumbilical vein endothelial cells remained viableafter culture into the dermal substitute, express-ing high levels of VEGF. Full-thickness burnwounds treated with the N,N,N-trimethyl chito-san chloride/pDNAVEGF dermal substituteshowed the fastest regeneration of the dermisand vascularization rate. Furthermore, results

Table 2. Commercially available wound dressings.

Wounddressing type

Properties and application Product Manufacturer

Hydrocolloiddressings

Promote debridement, angiogenesis and a moist environment;do not adhere to the wound bed; useful for low-to-moderate

exuding wounds

Granuflex; Aquacel Conva Tec (NJ, USA)

Tegasorb 3M Healthcare (MN, USA)

Comfeel Coloplast (Humlebk,

Denmark)

Alginate

dressings

Hydrophilic gel that maintains a moist environment and

protects the wound; absorbent; partial dissolution; indicated

for moderate-to-heavy exuding and bleeding wounds

Sorbsan Maersk (Copenhagen,

Denmark)

Kaltostat Conva Tec

Tegagen 3M Healthcare

Nonalginate

dressings

Ability to hydrate the wound bed; low absorption capacity;

promote autolytic debridement; indicated for dry, necrotic and

thick sloughy wounds

Nu-Gel Johnson & Johnson (NJ, USA)

Purilon Coloplast

Intrasite

Smith & Nephew Healthcare(London, UK)

Semi-permeable

films

Good adhesion with the healthy skin; can be applied as a

secondary dressing; high transparency and flexibility; promote

a moist environment; indicated for low-exuding wounds

Opsite Smith & Nephew Healthcare

Cutifilm Smith & Nephew Healthcare

Bioclusive Johnson & Johnson

Tegaderm 3M Healthcare

Foam dressings High absorption; provide thermal insulation; appropriate for

wounds with moderate-to-high levels of exudate

Lyofoam Conva Tec

Allevyn Smith & Nephew Healthcare

Antimicrobial

dressings

Useful for infected wounds; can be applied in both dry and

exuding woundsActicoat Smith & Nephew Healthcare

Inadine Johnson & Johnson

Data taken from [3,13,26,31].


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also indicated the positive effect of the con-struct on angiogenesis, leading to signicantlyhigher numbers of newly formed and matureblood vessels. Similarly, Philandrianos et al.compared the efcacy of ve acellular dermal

skin substitutes (Integra, Integra Life Sciences,NJ, USA; ProDerm, UDL Laboratories Inc.,IL, USA; Renoskin, Symatese, Ivry-le-Temple,France; Matriderm2 mm, Ideal Medical Solu-tions, Wallington, UK; and HyalomatrixPA,Addmedica, Paris, France) to heal full-thicknesswounds created in a porcine model [58]. Thestudy was conducted in a two-step procedurethrough the implantation of the skin substitutesin the wound, followed by the reconstruction ofthe epidermis using an autologous split-thicknessskin graft or cultured epithelial autograft (after

21 days). Results indicated signicant differ-ences between the skin substitutes in terms ofdermis incorporation and early wound contrac-tion. However, no signicant differences werenoted in the long-term wound retraction. Inrespect to long-term macroscopic and micro-scopic scar qualities, no signicant differenceswere observed between the wounds treated withthe dermal substitutes and the control group.

Dermoepidermal substitutesConstructs that mimic epidermal and der-

mal layers of the skin are the most advanced

substitutes currently available for clinical use.These constructs, consisting of keratinocytesand broblasts incorporated into constructs toform a temporary cover [2], have the potential topromote the regeneration of dermal and epider-

mal layers, resembling the skin structure. Themain limitations of the dermoepidermal substi-tutes are the high production costs and failureto close the wound permanently, due to rejectionof allogeneic cells [2].

Mazlyzam et al. developed an equivalentbilayer human skin substitute consisting ofhuman plasma-derived brin, broblasts andkeratinocytes [66]. For the fabrication of thebilayer construct, broblast cells were embed-ded into a human brin matrix (dermal layer),and subsequently a layer of human keratino-

cytes within human brin (epidermal layer) wasdeposited on the top of the construct. Substi-tutes were implanted at the dorsal part of athy-mic mice for 4 weeks to evaluate their ability topromote skin regeneration. Results showed theformation of a continuous epidermaldermaljunction between the stratied epidermal anddermal layers, mimicking the organization ofnative human skin.

Although widely recognized that full-thick-ness wounds more than 1 cm in diameter needskin grafting to promote healing and prevent the

formation of excessive scarring, the skin grafts

Table 3. Commercially available skin substitutes.

Substitute Product Graft type Cell source Manufacturer

Epidermal

substitutes

Epicel Cell based Autologous keratinocytes Genzyme Biosurgery (MA, USA)

CellSpray Cell based Autologous keratinocytes Avita Medical (Perth, Australia)

Myskin Scaffold containing cells Autologous keratinocytes CellTran Ltd (Sheffield, UK)

Laserskin Scaffold containing cells Autologous keratinocytes Fidia Advanced Biopolymers

(Abano Terme, Italy)

ReCell Autologous epidermal cell suspension Autologous keratinocytes Avita Medical

Dermal

substitutes

Integra Cell free Integra NeuroSciences (NJ, USA)

AlloDerm Cell free LifeCell Corp. (NJ, USA)

Hyalomatrix PA Cell free Fidia Advanced Biopolymers

Dermagraft Scaffold containing cells Neonatal allogeneic

fibroblasts

Advanced BioHealing (CT, USA)

TransCyte Scaffold containing cells Neonatal allogeneic

fibroblasts

Advanced BioHealing

Hyalograft 3D Scaffold containing cells Autologous fibroblasts Fidia Advanced BiopolymersDermoepidermal

substitutes

OrCel Natural-based scaffold containing cells Allogeneic keratinocytes

and fibroblasts

Ortec International (GA, USA)

Apligraf Natural-based scaffold containing cells Allogeneic keratinocytes

and fibroblasts

Organogenesis Inc. (MA, USA)

PolyActive Synthetic scaffold containing cells Autologous keratinocytes

and fibroblasts

HC Implants BV (Leiden,

The Netherlands)


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currently available are not capable of promotingthe regeneration of these lesions [2]. Tissue-engineered skin substitutes are still characterizedby both poor vascularization and the impossi-bility of reproducing skin appendages such ashair follicles, sebaceous glands and sweat glands

[2,3,7]. To induce the formation of skin append-ages, recent works are exploiting the culture ofspecic cell lines (e.g., Schwann cells, hair fol-licle cells and melanocytes) within scaffolds.Sriwiriyanont et al. used an engineered skinsubstitute model composed of collagenglycos-aminoglycan, containing murine dermal papillacells expressing greenuorescent protein, humandermal papilla cells and/or human broblasts,to investigate the hair regeneration [67]. In thiswork, chimeric hair follicles were successfullygenerated in the engineered skin substitute con-

taining combinations of murine dermal papillacells and unmodied or genetically modiedhuman epidermal keratinocytes overexpressingstabilized b-catenin. However, the formed chi-meric hair follicles were anatomically decient.Several other works demonstrate the potential oftissue-engineered skin substitutes for skin regen-eration [6668], although further clinical evidenceis necessary to assure its clinical efciency andmarket costeffectiveness.

Although the eld of skin engineering hasdelivered a limited number of products to themarket, there have been several product fail-

ures. Dermoepidermal substitutes Orcel(OrtecInternational, GA, USA) and PolyActive(HCImplants BV, Leiden, The Netherlands) haveboth failed, and dermal substitute Transcyte(Advanced BioHealing Inc., CA, USA) hasfailed to penetrate the market. These failureshave resulted from several factors, includingpoor performance and a prohibitive cost, result-ing in failed costeffectiveness for medicalreimbursement.

Advanced skin regeneration

strategiesAdvanced skin regeneration strategies offer aneffective, permanent and viable alternative tosolve the major drawbacks of currently avail-able skin grafts. These strategies combine bio-materials, cells, growth factors and advancedbiomanufacturing techniques for the fabricationof constructs that mimic skin anatomy, and pro-mote the regeneration of healthy and vascular-ized tissues. Two fundamental strategies can beconsidered, based on bottom-up or top-downapproaches [48,6973]. These two approaches are

adaptable to computer control, allowing the

realization of several operations in an automaticway, such as the deposition of cells aggregates,scaffold fabrication and cell seeding. Their maindifference consists of the use of a scaffold as asupporting material.

The bottom-up approach employs different

techniques for creating modular tissues thatare then assembled into engineered tissues withspecic microarchitectural features [74]. Tissuemodules can be created through self-assembledaggregation, microfabrication of cell-ladenhydrogels, fabrication of cell sheets or directprinting. The ability of cell aggregates to fuse isbased on the concept of tissue uidity, accordingto which embryonic tissues can be considered asliquids [75]. The major drawback of this approachis that some cell types are unable to producesufficient ECM, migrate or form cellcell

junctions[71]

.The top-down or scaffold-based approach isbased on the use of a temporary scaffold thatprovides a substrate for implanted cells and aphysical support to organize the formation ofthe new tissue [48,69,76]. In this approach, trans-planted cells adhere to the scaffold, proliferate,secrete their own ECM and stimulate new tissueformation.

Bottom-up approaches for skinregeneration & repairBottom-up approaches are based on the use of

cells or cell aggregates to produce tissue-engi-neered constructs without the use of scaffoldsas supporting matrices [48,7779]. Usually, theseapproaches comprise three key elements: a bio-ink containing the cell suspension to be printed,a biopaper, which provides a temporary supportfor the deposited bio-inks, and a bioprinter orrobotic dispenser [79,80].

Bottom-up approaches have the potential toimprove the vascularization of the 3D constructsthrough the fabrication of intraorgan vasculartrees to perfuse the constructs and ensure their

viability [81,82]. Additionally, they also allowthe deposition of multiple cell types with 3Dorganization [48,83], which is relevant for skinregeneration due to its inherent multilayeredstructure.

Different laser-assisted techniques, such aslaser-guided direct writing [84,85]and modiedlaser-induced forward transfer [86,87], or jet-basedprocesses, such as inkjet printing systems [88,89]and microdispensing techniques [90,91], havebeen developed to print different biomaterials,cells and growth factors with high precision and

reproducibility.


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Modified laser-induced forward transferprocesses comprise two main techniques: thematrix-assisted pulsed-laser evaporation directwrite or matrix-assisted pulsed-laser direct write,and the biological laser printing or absorbingfilm-assisted laser-induced forward transfer

[89,92,93]. Both processes use a pulsed focusedbeam to print cells from a transfer layer or res-ervoir onto a substrate with micron-scale resolu-tion. The cell printing can be performed throughthe deposition of cells onto an ECM-like layerwith variable thickness (substrate) or by printingbio-inks consisting of cells embedded in ECM-like printable biomaterials such as hydrogels [87].The main difference between them is the num-ber of layers of the target. Biological laser print-ing comprises one additional layer (conversionlayer or laser absorption layer) that eliminates the

interaction of the laser with the cells, protectingthem from the direct exposure of incident UVlight [92]and improving process reproducibility[94]. Recently, Koch et al.used a biological laserprinting process to produce 3D skin grafts, con-sisting of broblasts and keratinocytes embed-ded in collagen (F IGURE3 ) [95]. After 10 days of

culture, cells proliferated well and remained via-ble without mixing. The formation of skin tissuewas conrmed by the formation of basal laminabetween the broblasts and keratinocytes, andthe existence of intercellular adherens junctionsbetween the cells.

Top-down approaches for skinregeneration & repairTop-down approaches are based on the fabrica-tion of porous, biocompatible and biodegrad-able scaffolds, in which donor cells with or with-out growth factors are seeded, followed by thematuration of this 3D assembly in a bioreactor[48,78,96]. Scaffolds can be made of natural or syn-thetic materials, as well a combination of both,mimicking the function of the natural ECMpresent in the human body [48,77,97,98]. They are

critical elements for the regeneration of largedefects, where the isolated use of cell therapiescompromise the healing, due to the high volumeof lost ECM [46]. Ideal scaffolds for skin regen-eration should actively guide tissue formation,prevent scarring, and retain and deliver cells andgrowth factors (TABLE 4).

Laser absorbing layerLaser pulse

Gel with cells

Figure 3. Biological laser printing process to produce skin grafts.(A)The laser printingprocess. (B)Layer-by-layer printed construct composed of fibroblasts (green) and keratinocytes (red).(C & D)Histologic cryosection of seven alternating layers of red and green fluorescence-labeledkeratinocytes. Each layer contains four sublayers. The construct has a height of approximately 2 mmand a square base area of 10 10 mm. Scale bars: 500 m.Reproduced with permission from [95].


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Biomanufacturing represents a group ofunconventional fabrication techniques recentlyintroduced in the medical eld for the produc-tion of biological structures for tissue engineer-ing applications. These techniques use additivetechnologies, biodegradable and biocompatiblematerials, cells and growth factors, to producecomplex 3D scaffolds in a layer-by-layer patternfrom a computer-aided design model, providingprecise control over the pore size and shape, the

percentage of porosity and the pore interconnec-tivity [48,69,99]. As some of these processes oper-ate at room temperature, they have been used tofabricate carriers containing cells and growth fac-tors without signicantly affecting cell viability[100]. Biomanufacturing comprises a wide rangeof processes such as vat photopolymerization pro-cesses, powder bed fusion processes, extrusion-based processes, inkjet printing processes andelectrospinning [48,69,77,100,101].

Vat photopolymerization processes produce3D solid objects in a multilayer procedure,

through the selective photoinitiated curing reac-tion of a liquid photosensitive material contain-ing a low-molecular weight prepolymer, additivesand photoinitiators [48,69,102,103]. The curing reac-tion is induced by a light source that irradiates theliquid polymer and supplies the necessary energyto bond a large number of small molecules, form-ing a highly crosslinked polymeric structure[104]. Vat photopolymerization processes havebeen used to induce the alignment of NIH/3T3mouse embryonic broblasts on 3D hydrogelspatterned with acryl-bronectin [105], and for the

production of 3D constructs, enabling study of

the interactions between different cell types [106].As a result of their resolution and precision, vatphotopolymerization processes can potentially beused to reproduce the microenvironment of skin,allowing for the study of the interaction betweendifferent types of cells, such as broblasts, kerati-nocytes and endothelial cells, leading towards thedevelopment of vascularized constructs.

Several extrusion-based techniques such as 3Dber deposition [107], precision extruding depo-

sition [108], the bioextruder [109]and the biocellprinting system [110]were developed for tissueengineering applications. Generally, extrusion-based processes comprise multiple reservoirscontaining different materials and cells, a move-able nozzle with variable diameter (501000 m)controlled by a computer-aided design/manufac-turing system, and a platform that collects theprinted material [90,91,111114]. Lee et al.developeda method for the fabrication of 3D skin cell layersusing a multichannel robotic cell printing system(FIGURE4)[91]. In this method, instead of printing

the biomaterialcell suspension into a solutioncontaining the crosslinking agent, the hydrogellayers are crosslinked by using a nebulized aque-ous sodium bicarbonate solution before and afterprinting each layer. These authors fabricated 3Dmultilayer cellhydrogel constructs through thedeposition of a collagen hydrogel precursor, bro-blasts and keratinocytes. The constructs wereprinted into a planar tissue culture dish and ina poly(dimethylsiloxane) mold containing 3Dsurface contours as a wound model. Fibroblastsand keratinocytes printed on planar surfaces pre-

sented good proliferation after 4 days of culture,

Table 4. Growth factors for skin regeneration.

Growthfactor

Function Ref.

EGF Stimulates the migration of keratinocytes, the proliferation and differentiation

of fibroblasts and the synthesis of granulation tissue; induces the proliferation

and differentiation of fibroblasts, epithelial cells and mesenchymal cells

[4,53,96,146]

IGF-1 Glycoprotein synthesized by the liver and expressed by fibroblasts that

stimulates the migration and proliferation of keratinocytes

[4,96]

bFGF Induces angiogenesis and formation of granulation tissue; regulates cellular

proliferation

[98,147]

KGF Mediates the proliferation and differentiation of epithelial cells; induces

keratinocyte proliferation and differentiation

[148]

PDGF Stimulates fibroblasts to produce extracellular matrix; stimulates proliferation

and migration of endothelial and smooth muscle cells

[4,96]

VEGF Stimulates the migration and proliferation of endothelial cells; promotes

angiogenesis and re-epithelialization during the healing process

[4,5,96]

TGF-b Most potent growth factor involved in wound healing; induces migration and

proliferation of fibroblasts; stimulates collagen synthesis, neovascularizationand formation of granulation tissue

[4,149,150]


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while the hydrogelcell layers printed on non-planar surfaces presented a nonhomogeneousdistribution, showing good proliferation.

Despite great advances in both the devel-opment of tissue-engineered skin substi-tutes and the use of either bottom-up or top-down approaches to produce macroscale andmicroscale constructs for skin regeneration,these products do not yet replicate the multiscaleorganization of skin, failing in the reproductionof the nanoscale of ECM components. The com-ponents of natural ECM include structural pro-teins such as collagen and elastin, specializedproteins such as bronectin, brillin and lam-inin, and proteoglycans [115], well organized in

the different skin layers. For instance, the basal

lamina in the epidermis contains an ECM berlayer composed of type IV collagen, laminin,bronectin and heparin sulfate proteoglycan

with a well-dened microtopography and nano-topography, while the dermis comprises a 3Dbrillar network with diameters in a nanoscalerange (30130 nm) that provides structuralintegrity and mechanical strength to the skin[116119]. In order to promote the regenerationof skin with biological and mechanical prop-erties similar to the healthy skin, constructsshould be able to reproduce the microscale andnanoscale organization of natural ECM com-ponents, providing an optimal environment forcell attachment, proliferation and differentiation

[120]. These constructs should also enable the

10987654321

Collagen FBs KCs

Two layers of collagen

Embedded KCs

Six layers of collagen

Embedded FBs

Two layers of collagen

Figure 4. Bioprinting system and fabrication procedure of 3D hydrogel constructs for skinregeneration.(A)The multichannel robotic cell printing system: (1)syringes containing cellsuspensions and hydrogel precursors, (2)array of four channel dispensers, (3)target substrate,(4)horizontal stage, (5)vertical stage, (6)range finder, (7)vertical stage heater/cooler and(8)optional independent heating/cooling unit for the dispenser. (B)Fabrication procedure ofmultilayer cellhydrogel constructs.FB: Fibroblast; KC: Keritanocyte.Reproduced with permission from [91].


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incorporation/attachment of biological signal-ing molecules, such as growth factors and celladhesive peptides, to stimulate cellular activity[121,122]. Several strategies have been explored toproduce constructs at the nanoscale, includingself-assembly, electrospinning, template synthe-

sis and phase separation [116,123]. Owing to itsoperational simplicity, versatility, wide rangeof processable materials and ability to producenanobers resembling the collagen structure ofthe ECM, there is a great interest on the useof electrospinning for fabricating nanoscalestructures for skin regeneration.

Electrospinning is the most relevant electro-chemical process to produce nanoscale meshesfor tissue engineering [10,69,101,124,125]. Nanoberswith diameters ranging from a few nanometersto several micrometers can be produced using

either an electrostatically driven jet of a polymersolution (solution electrospinning) or a polymermelt (melt electrospinning) [69,101,126128]. Thebasic requirements of an electrospinning appa-ratus are illustrated in FIGURE5A, including a capil-lary tube with a needle or pipette, a high-powervoltage supply, electrical wires connecting thehigh-power voltage supply to both the capillarytube, and the collector or target. The collectorcan be moved in the vertical direction and inthe xy plane, enabling electrospinning to be anadditive technology.

Nanopatterned electrospun fiber meshesare very attractive for skin regeneration due totheir high surface-to-volume ratio, porosity andstructural similarity to the meshwork of ECMbers of the dermis [127,129,130]. As a result of thehigh surface area ratio, these constructs also pro-

mote better cellular interaction and improvedvascularization, while the porosity allows thedrainage of exudate and oxygen permeation[10,119,124]. Additionally, the dimension of theelectrospun nanobers are in the range of thestructural components of natural ECM and themolecules involved in the wound healing process(1100 nm) [4,120]. The main limitations are thepoor mechanical properties, nonuniform thick-ness distribution and poor integrity [124]. A widerange of electrospun natural materials [125,131133],synthetic materials [22,127,120,134]and combina-

tions of both material types[1,129,130,135,136]

havebeen used to produce electrospun nanobersmeshes for skin regeneration applications.

For the regeneration of dermal wounds,Chong et al.used the electrospinning techniqueto produce poly(caprolactone)/gelatin nano-brous meshes on top of a commercial poly-urethane wound dressing (Tegaderm; 3MHealthcare, MN, USA) [136]. Human dermalbroblasts cultured in these constructs for 7 daysshowed good proliferation rates. Yang et al.pro-duced poly(ethylene glycol)poly(d,l-lactide)

V

Emulsification

High-voltage generator

Collector

High-voltag

generator

EmulsionWater phase

Emulsifier

Oil phase

Syringe needle

Capillary tube

Needle

CollectorHigh-voltagepower supply

Nanomedicine Future Science Group (2013)

Figure 5. Electrospinning processes to produce fiber meshes for skin regeneration.(A)Solution electrospinning process and(B)emulsion electrospinning process.


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bers with a coresheath structure containingbFGF through an emulsion electrospinningprocess (F IGURE 5B) [127]. The in vitro deliv-ery studies showed a gradual release of thegrowth factor for 4 weeks. The constructs werealso seeded with mouse embryo broblasts,

which showed good adhesion, proliferat ionand ECM production. Once implanted intodorsal wounds created on diabetic rats, thebFGF/poly(ethylene glycol)poly(d,l-lactide)meshes promoted a faster healing process withcomplete re-epithelialization and regenerationof skin appendages such as hair and sebum. Jinet al. produced poly(l-lactic acid)-co-poly(e-caprolactone) nanobrous scaffolds, with andwithout collagen, to invest igate the dif feren-tiation of bone marrow-derived mesenchymalstem cells into the epidermal lineage (keratino-

cytes)[135]

. Better results were observed in thecase of poly(l-lactic acid)/poly(caprolactone)constructs with collagen.

Electrospun nanober meshes can also beused as drug delivery systems for the release ofbioactive molecules, including natural or syn-thetic drugs and growth factors [122,127,137,138].Shalumon et al. prepared sodium algi-nate/poly(vinyl alcohol) meshes containingdifferent concentrations of zinc oxide (ZnO)nanoparticles as an antibacterial agent [137].Mouse broblasts cultured on meshes contain-ing 0.5 and 1.0 wt% ZnO nanoparticles showed

good adhesion and spreading. For high concen-trations of ZnO nanoparticles (2 and 5 wt %),a decrease in cell spreading was observed as aresult of the toxicity effect of these particles.The antibacterial activity of the nanobers wasevaluated through diffusion disc tests, usingStaphylococcus aureusand Escherichia coli. Theseresults conrmed the antibacterial activity of thenanobers, which was improved by increasingthe concentration of ZnO nanoparticles.

Despite the growing interest on the use ofnanoscale structures for skin regeneration, the

medical effectiveness of these materials is notyet conrmed, and only a few studies focus ontheir in vivoevaluation [44,127,139].

In situbiofabrication of skinsubstitutesThe development of in situ biomanufactur-ing strategies, such as in situskin fabrication,represents one of the major challenges in tissueengineering [69,77,140143]. Contrary to the tradi-tional biofabrication approaches, in which con-structs are produced based on predesigned mod-

els and then cultured in in vitroconditions for

subsequent implantation into the patient, in situbiofabrication involves the fabrication of substi-tutes for tissue repair and regeneration directlyin the lesion of the patient. In this case, bio-fabrication systems are combined with real-timeimaging techniques and path-planning devices,

enabling the controlled deposition of biomateri-als with or without encapsulated cells into thelesion site. In situbiofabrication has great poten-tial for clinical applications, due to its minimallyinvasive nature, the possibility of eliminatingthe need for postprocessing operations, abilityto fabricate patient-specic biological substitutesand reduced intervention time. Theoretically,this new concept can be applied for the regen-eration of different tissues, but recent works areonly focused on osteochondral [141], bone [142]and skin [140]defects. Owing to its layered struc-

ture and multiple cell composition, the regenera-tion of healthy and vascularized skin remains ahuge challenge. The current strategies for skinregeneration still present major pitfalls such asinadequate vascularization, poor adherence tothe wound bed, inefcient elasticity, inability toreproduce hair follicles, sweat, sebaceous glandsand pigmentation, the possibility of rejectionand high manufacturing costs. Some of theselimitations could be addressed by the develop-ment of an in situbiofabrication device, enablingthe direct deposition of cell-laden constructs in alayer-by-layer fashion, in this way mimicking the

structural and compositional organization of theskin tissue. The printed skin substitute coversthe wound to prevent contamination, providesadequate moisture to avoid dehydration, givesimmediate and effective relief to patients, andinduces skin regeneration, due to its functionalcomposition and cell combination. Recently,Sofokleous et al.developed a portable electro-hydrodynamic multineedle system and proposedits use for the in situ fabrication of polymericmeshes in the damaged skin site of a patient(FIGURE6)[143]. These meshes can be coated, thus

encapsulating drugs, and therapeutic and clot-ting agents. Poly(lactic-co-glycolic) acid mesheswith an average diameter of 2.3 0.5 m werereported. The use of a coaxial needle deviceallowed the fabrication of bimaterial bers (aver-age diameter of 3.9 0.7 m), with a core struc-ture made by polymethysilsesquioxane coatedwith a thin layer of poly(lactic-co-glycolic) acid.

Binder et al.developed a portable inkjet deliv-ery system for the in situprinting of skin cells intothe lesion site [140]. The potential of the systemto induce the skin regeneration was evaluated

through the printing of human keratinocytes


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and broblasts into full-thickness skin lesions(3 2.5 cm) created on mice. Results showed thecomplete closure of the wound after 3 weeks, aswell the formation of skin with similar propertiesto the healthy skin. Histological analysis revealedthat the new skin contained organized dermal

collagen and a fully formed epidermis.Despite the tremendous potential of in situ

biofabrication of skin substitutes, this con-cept is still in its infancy. To be effective, greatchallenges need to be addressed: the integra-tion between biomanufacturing technologiesand imaging techniques; the development ofdedicated software to control the deposition ofdifferent biomaterials and cells, and planningthe surgical intervention; and the developmentof advanced multifunctional biomaterials,capable of entrapping different cells types and

maintaining their viability and functionality.Currently, several strategies are being exploredto promote skin regeneration. The most com-monly used are autografts and allografts, wounddressings and tissue-engineered skin substitutes.Although these strategies result in products withdistinct characteristics, they have been used incombination to improve the healing process. Forexample, wound dressings, either traditional ormodern and tissue-engineered skin can beapplied on top of autografts and allografts, pro-viding a better adhesion to the defect site andprotecting it from uid loss and contamination[144,145]. Another approach involves the fabrica-tion of electrospun meshes on top of commercialwound dressings in order to obtain a nanobrousscaffold with morphological and architecturalfeatures similar to the natural ECM in the skin[136]. Despite the great potential of the in situbiofabrication of skin substitutes, this strategymay also require the combined use of dressingsor cell-free tissue-engineered skin substitutesto ensure initial adhesion to wound bed andprevent biomaterial (hydrogel) dehydration.

Conclusion & future perspectiveDespite recent advances in the development ofbiomaterials and fabrication strategies for skinrepair and regeneration, their clinical use stillrequires several drawbacks to be addressed.Numerous wound-care products were developed,such as lotions, dressings and tissue-engineeredskin substitutes, and are currently commer-cialized. Until now, the gold standard for skinregeneration still relies on the use of autograftsand allografts, which present signicant limita-tions as previously discussed. Advanced strate-

gies, combining additive biomanufacturing

processes, biomaterials, cells and growth factors,have emerged as a group of techniques withhigh potential for skin regeneration applica-tions. These techniques allow printing of skinsubstitutes in a precise and automated manner,enabling the production of skin replacementswith properties that resemble both the structureand the function of native skin. These techniquesenable the development of bottom-up approachesto fabricate 3D constructs that mimic the orga-

nization of healthy skin and can be used forthe regeneration of full-thickness wounds, forwhich an efcient dermoepidermal substitute isnot available.

In order to achieve effective and clinically rele-vant skin regeneration, relevant challenges to beaddressed in the future include:

The insufcient vascularization of the regener-ated skin. This requires the development ofsophisticated printing systems to producemultihydrogel constructs with multiple cell

types (e.g., stem cells and endothelial cells) andthe controlled release of angiogenic growthfactors;

Development of advanced in situcrosslinkingmultifunctional hydrogels, mimicking the bio-signaling and mechanical properties of theskin, and allowing the incorporation ofmultiple cell types;

The inability of currently available skin graftsto reproduce all skin structures, such as hair and

glands. This requires a deeper understanding of

Figure 6. Electrohydrodynamic multineedle system printing a polymeric

mesh directly into the lesion site on the patient.Reproduced with permission from [143].


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Executive summary

Skin damage & wound healing

Skin is a multilayer organ with a natural ability to promote regeneration after injury.

The healing of a wound comprises five integrated and overlapping phases, namely hemostasis, inflammation, migration, proliferation

and maturation.

The healing process, involving the interaction between cells, growth factors and cytokines, is highly dependent on the extension of

the lesion and the number of affected layers.

Treatment of skin lesions

The gold standard for skin regeneration remains the use of autografts and allografts, with important limitations such as patient

morbidity, risk of immunogenic rejection and disease transmission.

Solutions, creams and ointments are widely used due to their ability to provide disinfection, cleaning and debridement.

Wound dressings are used as an alternative to the autografts and allografts. Although proven clinically effective, these productshave low adherence to the wound bed and an inability to reproduce skin appendages and promote regeneration of the lost tissue, in

particular the dermis.

Tissue-engineered skin substitutes

Principles of tissue engineering are used in the development of cellular and acellular epidermal, dermal and dermoepidermal skin

substitutes.

Tissue-engineered skin substitutes are designed by combining natural/synthetic polymers with cells, enabling the regeneration of the

different skin layers.

The main limitations of these products rely on the inability to regenerate full-thickness wounds, poor vascularization and the

difficulty of reproducing skin appendages.

Further research is necessary to investigate the clinical efficiency and market costeffectiveness of tissue-engineered skin

substitutes.

Advanced skin regeneration strategies Advanced skin regeneration strategies provide a clinical route for the development of biological substitutes for skin regeneration, by

combining biomaterials, cells, growth factors and biomanufacturing techniques.

Bottom-up and top-down approaches are used to produce well-organized 3D multilayer skin substitutes containing fibroblasts and

keratinocytes.

Nanoscale structures are very attractive for skin regeneration, due to their high surface/volume ratio, porosity, improved

vascularization and dimensions in the range of the structural components of the natural extracellular matrix.

There is a need for clinical trials to investigate the medical effectiveness of these materials.

In situbiofabrication of skin substitutes

In situbiofabrication is a novel concept involving the fabrication of skin substitutes directly in the patient, using a bioprinting device.

It comprises the direct deposition of cell-laden constructs in a layer-by-layer fashion, mimicking the structural and compositional

organization of the skin tissue.

This strategy aims to regenerate healthy and vascular skin, providing immediate relief to the patient.

skin biology and the use of more complex cell-ular approaches, including the use of geneticallyengineered cells;

Applying nano-, micro- and macro-fabricationtechnologies for enhancing efcacy and preci-sion and to reproduce the natural hierarchy

that characterizes the skin;

Increasing the level of automation and indus-trialization, as traditional laboratory pro-cesses are often characterized by a high degreeof manual handling operations;

Enhancing multidisciplinarity, linking clini-cians, biologists and engineers to facilitatefurther developments and the clinicaltranslation of the products being investigated;

Scaling up the fabrication strategies underdevelopment toward clinical application.

Financial & competing interests disclosure

The authors are supported by the Strategic Projects

PEST-OE/EME/UI4044/2011 and PEST-C/SAU/

LA0002/2011, and financed by European RegionalDevelopment Fund through the Programa Operacional

Factores de Competitividade COMPETE and by

Portuguese funds through Fundao para a Cincia e a

Tecnologia. The authors have no other relevant affiliations

or financial involvement with any organization or entity

with a financial interest in or financial conflict with the

subject matter or materials discussed in the manuscript apart

from those disclosed.

No writing assistance was utilized in the production of

this manuscript.


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