Cell therapy for severe burn wound healing

Cell therapy for severe burn wound healing Cell therapy has emerged as an important component of life-saving procedures in treating burns. Over past decades, advances in stem cells and regenerative medicine have offered exciting opportunities of developing cell-based alternatives and demonstrated the potential and feasibility of various stem cells for burn wound healing. However, there are still scientific and technical issues that should be resolved to facilitate the full potential of the cellular devices. More evidence from large, randomly controlled trials is also needed to understand the clinical impact of cell therapy in burns. This article aims to provide an up-to-date review of the research development and clinical applications of cell therapies in burn wound healing and skin regeneration. Keywords: Skin, Burn injuries, Stem cells, Cell therapy, Wound healing, Skin regeneration Background autologous progenitor cells that could replicate to regen- Burns remain one of the most traumatic injuries causing erate skin tissues for permanent wound closure. In past significant health, social and economic consequences [1]. decades, cell-based therapies have emerged as popular Globally, severe burns lead to about 180,000 deaths an- choices in conjunction with standard skin grafting tech- nually and millions of patients suffering from non-fatal niques for burn wound healing and regeneration of skin burns experiencing substantial and life-long physical and structure and functions. This article aims to provide an psychological morbidities. up-to-date review of the research development and clin- Severe burn wound is characterised by the destruction ical applications of cell therapies in severe burn wound of skin structures, functions and more importantly the healing. loss of the progenitor cell populations that are essential for regenerating and restoring the structures and func- Review tions [2]. Until now, autologous skin grafting remains a Development of cell therapy for burn wound healing standard practice in treating severe burns. However, its Cell therapy which also called cellular therapy or effectiveness is often challenged in treating severe burn cytotherapy involves delivering an autologous or allo- patients with limited donor sites for skin graft harvest- genic cellular component into a patient to repair or re- ing. Consequently, the patients could experience a sig- generate the damaged tissue due to injuries or diseases, nificant delay in wound closure, detrimental infection, to rectify the diseased conditions associated with the scarring or even death. damages or deficiency of the unique cell population and To overcome the autograft shortage, a variety of alterna- to restore the physiological functions. tives for autologous skin grafts including allogeneic skin, Skin as the multi-functional and protective barrier in xenografts and synthetic skin substitutes have been widely human contains essential stem cell population and vari- adopted in burn wound care [3, 4]. While those alternative ous cellular types that are critical for renewing and devices provide temporary wound coverage and deliver maintaining its structural integrity and functions. Re- various bio-factors to facilitate the angiogenesis and search on skin cell transplantation for wound healing granulation of wound bed for further surgery, they could was first reported by Billingham and Reynolds in 1952 never replace the skin autograft delivering the essential [5]. In a guinea pig model, they harvested both epider- * Correspondence: zhe.li1@sydney.edu.au mal sheets and epidermal cell suspension by trypsin di- Burns Unit, Concord Hospital, Concord, New South Wales 2139, Australia gestion and then transplanted them to surgically created Skin Laboratory, NSW Statewide Burns Service, Concord, New South Wales, wounds to evaluate their possible application in plastic Australia Full list of author information is available at the end of the article surgery, experimental pathology, wound healing and © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li and Maitz Burns & Trauma (2018) 6:13 Page 2 of 10 contracture release. However, the era of cultured cell transient-amplifying keratinocytes and migrating to- therapy for burn injuries was only opened up after wards skin surface to form multiple keratinocytes Rheinwald and Green revolutionised the cell culture layers [11]. They gradually lose the capacity in popula- technique in 1975 allowing the isolation and serial tion doublings through differentiation [10–15]. KSC cultivation of strains of human keratinocytes from a skin populations with more active, proliferative and regen- biopsy [6]. For the first time, epithelial sheets could be erative capacities are observed in young people com- produced using cultured keratinocyte clones [7]in paring to aged peoples [16]. laboratory and cultured epithelia autografts (CEAs) were The keratinocytes can be easily isolated and ex- successfully transplanted in severe burns for wound panded in numbers under in vitro cell culture condi- healing [8]. The encouraging report further sparked tions. Keratinocytes produce various bio-factors and worldwide research activities on cell therapy in burn re- cytokines including interleukin (IL)-1, IL-6, IL-7, IL-8, search. Progresses as described in the following sections IL-10, IL-12, IL-15, IL-18, and IL-20 and tumour ne- have been achieved over past decades to understand crosis factor alpha (TNF-α)[17], which are important various cell types and their potential roles in burn for regulating skin regeneration and wound healing. wound healing. All these properties make them the most important and widely used cells for therapeutic application in Different cell types with therapeutic potentials burn wound care. Wound healing and restoration of skin structures and A population of circulating keratinocyte progenitor functions depend on many factors including the avail- cells (KPCs) was identified with p63 in peripheral ability of essential progenitor cells, dermal extracellular blood, which differentiated into keratinocytes with matrix (ECM), and bio-factors and cytokines for angio- expression of the cytokeratins, involucrin and filag- genesis and regulation of cell-matrix and cell-cell inter- grin [18]. actions. The following cell types have demonstrated potentials as therapeutic devices in skin wound healing and tissue regeneration. Many autologous and allogeneic Dermal fibroblasts cell products were developed using cells of skin and Dermal fibroblast cells play an important role in normal non-skin origins for therapeutic application in burn skin and skin wound healing post-burn. They produce wound management. the key ECM proteins in the dermis including laminins, fibronectins, collagens, elastic fibres, non-collagen mole- Keratinocyte stem cells cules and bio-factors to regulate cell function, migration The epidermis is mainly comprised of keratinocytes and the cell-matrix and cell-cell interactions in normal which are renewed and sustained by keratinocyte skin homeostasis and wound healing [19–21]. Dermal stem cell (KSC) populations anchored at the fibroblast cells produce many growth factors and cyto- membrane in the epidermal-dermal junction, the hair kines including vascular endothelial growth factor follicle bulge and the sebaceous gland [9, 10]. KSCs, (VEGF), basic fibroblast growth factor (bFGF), hepato- expressing K5, K14 and p63, are well known for regu- cyte growth factor (HGF), platelet-derived growth factor lating epithelial stratification, hair folliculogenesis and (PDGF)-AA, transforming growth factor-beta1 (TGF-β1) wound repair [10–12]. , keratinocyte growth factor (KGF), IL-6 and IL-8 and Based on molecular biomarkers, regenerative cap- tissue inhibitors of metalloproteinases [19–21]. Cryopre- ability and the status of cellular differentiation, kerati- served cultured dermal substitute maintained sufficient nocytes in human skin could be classified as KSCs, fibroblast cell viability and the ability to proliferate and transient-amplifying keratinocytes and differentiated release a significant amount of VEGF [22]. Epidermal- keratinocytes [13]. The KSCs, making about 4% to 8% derived pro-inflammatory cytokines IL-1α and TNF-α of total keratinocyte population in skin [14], are local- mediate synergistically the secretion of wound-healing ised at dermal-epidermal junction, hair follicles and mediators (with the exception of sST2) from fibroblasts in the surrounding zones of skin appendages. They repli- dermal substitutes. Studies demonstrated a synergistic en- cate themselves by asymmetric division to maintain a hancement of the expression of bio-factors including IL- stable population of undifferentiated stem cells at the 1α, IL-6, bFGF, CCL2, CXCL1, CXCL8, and sST2 if KSCs basal region and are essential for epidermal regener- and dermal fibroblast cells were co-cultured in a dermal ation or repair due to skin injuries. Beta4 integrin is regenerative scaffold [21, 23, 24]. required for hemidesmosome formation, cell adhesion Skin-derived precursor (SKP) cells have also been isolated and cell survival [15]. During normal epidermal from dermal papillae and can be differentiated into adipo- homeostasis or repair, the KSCs go through asymmet- cytes, smooth myocytes and neurons in vitro [25, 26]. Fi- ric division, exiting their niche, transforming into broblasts from adult mouse and human can be induced Li and Maitz Burns & Trauma (2018) 6:13 Page 3 of 10 into pluripotent stem cells with potentials for regeneration pluristratified epidermis [47–51]. Immortalised keratino- of various tissues [27, 28]. cyte lines [52] and fibroblasts [53] were also derived from hESCs. The hESCs-derived fibroblasts were ob- Mesenchymal stem cells served to direct development and repair of three dimen- Mesenchymal stem cells (MSCs), a unique popula- sional (3D) human skin equivalents [53]. tion of multipotent cells with self-renewal and regen- Although hESCs could be developed to provide tem- erative capacity can be derived from autologous or porary skin substitutes for burn patients awaiting autolo- allogenic tissues including bone marrow, adipose tis- gous grafts, the potential applications are however sue, nerve tissue and umbilical cord and cord blood, complicated due to the ethical issues in relation to using and dermis. MSCs express surface CD markers in- human embryos. cluding CD44+, CD73+, CD90+ and CD105+ and The Nobel Prize winning discovery of induced pluripo- are distinguished from haematopoietic cells by a lack tent stem cells (iPSCs) by Yamananka and his team of CD34, CD14, CD45, CD11b/CD79 and CD19/ brought an alternative source of stem cells with pluripo- HLA-DR [29–32]. tent potentials for therapeutic applications [27, 28]. MSCs produceand releasebio-factors[33–37]such While avoiding the ethical concerns in using hESCs, as VEGF, stromal cell-derived factor-1, epidermal they induced adult mouse or human dermal fibroblasts growth factor, KGF, insulin-like growth factor and into pluripotent stem cells by transforming them with matrix metalloproteinase-9 promoting angiogenesis; Sox2, Oct4, Klf4 and c-Myc transcription factors using a recruit endogenous progenitor cells; and direct cell retroviral system. The generated iPSCs are, although not differentiation, proliferation and ECM formation dur- identical, extremely similar to embryonic stem cells be- ing wound repair. MSCs also secrete cytokines includ- ing capable of generating all cell lineages of different tis- ing interferon-λ,TNF-α,IL-1α and IL-1β, and nitric sues including skin [49, 54]. iPSC-derived cells could oxide to modulate host immune response in wound secrete proteins including VEGF, FGF-2, TGF-β, healing [33, 36, 37]. S100A4, GRO, GM-CSF, MCP-1, IL-6 and IL-8 that pro- Studies demonstrated that both autologous and mote increased proliferation, contraction and migration allogenic MSCs, administered both systemically or and [55]. iPSCs as patient specific or allogenic devices dem- locally, exhibit therapeutic potentials promoting cuta- onstrated potential values for clinical applications, re- neous wound healing and tissue regeneration via the search and drug discoveries in laboratory research. Their paracrine bio-factors and cytokines and the multi- safety and efficacy are still to be assessed mainly due to potency in tissue regeneration [31, 32, 38–40]. Under the use of retroviral vectors, associated risk, genetic in- specific niche conditions and molecular stimulations, stability and potential immunogenicity [56]. To avoid MSCs could be induced to differentiate into multiple the risks of mutagenesis and oncogenic transformation tissue-specific cell lineages including osteoblasts, adi- associated with retroviral vector, iPSCs are also gener- pocytes, chondrocytes, tenocytes, myocytes, endothe- ated with non-integrative reprogramming strategies lial cells, vascular smooth muscle cells, keratinocytes using plasmid vectors, episomal plasmid vectors, modi- and sweat gland-like structures [35, 37, 41]. MSCs fied/microRNA or even direct delivery of reprogram- could produce anti-fibrotic factors and modulate ming protein factors [57–59]. The non-integrative development of hypertrophic scarring [42]. Adipose- reprogramming strategies were considered safer for derived stem cells (ADSCs) and ADSC-conditioned therapeutic purpose. However, the efficiency of iPSC in- medium appear highly effective in promoting hair duction by original plasmid vector was low although it growth [43, 44], and development of functional sweat could be enhanced by essential factor for episomal amp- gland-like structures was observed from transplanting lification of the vector [58]. differentiated bone marrow mesenchymal stem cells (BM-MSCs) [45]. MSCs promote angiogenesis Clinical research and applications of cell-based therapies and vascular stability that are critical for delivering the in burn wound care essential nutrient supply for tissue regeneration and Cultured epithelial autografts wound healing [46]. The key technique that Rheinwald and Green established in the 1970s for serial culture of keratinocytes opened Induced pluripotent stem cells the era of research and therapeutic uses of cultured au- Human embryonic stem cells (hESCs), derived from hu- tologous keratinocytes in burn wound healing. Under la- man embryos are well known for their pluripotent cap- boratory conditions, KSCs could be isolated from a acities in tissue regeneration. Research demonstrated small skin biopsy and cultivated in the presence of that hESCs could be induced to efficiently differentiate growth-arrested 3T3 mouse fibroblasts as feeder cells to into functional basal keratinocytes that regenerate a form epidermal sheets that could be transplanted back Li and Maitz Burns & Trauma (2018) 6:13 Page 4 of 10 to the same patient for treatment. The innovative grafting wide meshed autograft together with acellular technology quickly gained global attention and dermal matrix in some cases [83] and were observed to emerged as a popular way to generate alternative auto- improve skin texture of burn scars after resurfacing the grafts to facilitate permanent wound closure when wounds [84]. The use of CEA in treating a full-thickness treating severe burns. Since 1981, both cultured living skin wound in severely burned patients results in keratinocytes and epithelial autograft sheets (CEAs), favourable quality of scars and was reported to show either produced in house or by commercial compan- good potential to save lives by providing epidermal cover ies, have been used as clinical devices in treating large [85]. Early excision followed by temporary coverage with burns [8, 60–67]. autograft, which is allowed to engraft, was found to be Cultured keratinocytes were used for treating burns in associated with a low infection rate and a higher rate of various ways. Commonly, the cells from a skin biopsy CEA take [86]. CEAs regenerate a stable normal epider- are cultivated, screened and passaged in a cell culture mis and are capable of inducing dermal regeneration system to expand the numbers of KPCs. In the presence from wound bed connective tissue [68, 84]. Better out- of feeder fibroblast cells, the keratinocytes could prolif- comes in aesthetic appearances and scar formation were erate and differentiate to form an epidermis-like tissue also reported in burn patients grafted with CEAs [87]. in cell culture. The process of CEA cultivation usually In addition, the procedure can be initiated with a small requires about 3 weeks depending on many factors in- skin biopsy causing minimum donor site morbidity. This cluding patient age, health condition and severity of would be particularly important for patients with exten- burn injuries. The colony-forming efficiency of keratino- sive burns as a life-saving device. CEAs deliver living au- cytes from freshly trypsinised skin was reported very tologous epidermal stem cells for permanent wound low, between 0.15% and 3.8% in the primary cell culture closure without eliciting immunological rejection. CEAs [68–70] but was increased to 26% to 90% at passage 2 to could also function as biological wound dressings deliv- 3 cultures and to over 16% in CEA sheets [70], being ering the much needed biological factors and niche en- significantly higher than that (4–8%) in normal skin vironment for wound healing. [14]. Cultured epidermis can be harvested as a sheet graft for clinical application. Cultured human sole-derived keratinocyte grafts re- Cultured keratinocyte suspension express site-specific differentiation after transplantation In 1952, Billingham and Reynolds examined the poten- [71]. The formation of hair follicles and development of tials of transplanting sheets of pure epidermal epithe- normal epidermal microarchitecture were observed lium and non-cultured epidermal cell suspensions for when epidermal cells were transplanted together with wound healing in guinea pigs [5]. In treating burn pa- cells of dermal origin [72]. tients, the burn wound areas often require early exci- Very often, CEAs were used in combination with sions and then immediate skin grafting. While a skin widely meshed skin grafts for wound closure. CEAs sample could be taken for growing CEAs, a 3-week wait- could be transplanted directly to the wound area [8, 73] ing time for CEA growth would be too long for many with undifferentiated progenitor cells attached to the re- patients as it unnecessarily delays the surgical interven- generated or freshly debrided wound bed [74]. CEAs tion. Consequently, the keratinocytes were cultured only were of significant value in severe burn management, to sub-confluent stage or be grown on carrier material providing wound coverage, facilitating wound epithelisa- to allow early harvesting and application as cell suspen- tion and permanent closure, and improving survival sion or sub-confluent sheet [88, 89]. rates among patients with massive burn wounds [75– Autologous keratinocytes isolated from any available 78]. CEA demonstrated a very high beneficial value in donor site area including sole and scalp [90] could be managing burns > 60% total body surface area (TBSA), isolated and cultured for clinical application. The deliv- being a life-saving treatment [79]. Although being ex- ery of cultured keratinocytes in suspensions, directly to pensive compared to standard skin autograft, the en- the wound or in combination with skin grafting, acceler- graftment rates and survival ratio results make CEAs ated epidermal wound healing in animal models and excellent alternative wound coverage when donor sites burn patients [90, 91]. The transplantation of cultured are limited in severe burns [80]. Studies reported an keratinocytes were found to facilitate the reformation of average graft take of 72.7% with a 91% overall survival organised epidermal structure [69] and to promote per- rate in treating critically burns using CEA, indicating manent epithelialisation in the burn wound [92, 93] and cultured keratinocytes as an excellent alternative or ad- accelerated epithelial maturation in an in vivo wound junct to conventional split-thickness skin grafting [81, model [94]. Permanent repigmentation of piebaldism 82]. The CEAs were reported to enhance the take of a was also reported in patients treated by erbium:YAG wide meshed autograft in massive burns and allow for laser and autologous cultured epidermis [95]. Li and Maitz Burns & Trauma (2018) 6:13 Page 5 of 10 To ensure the effectiveness of living keratinocytes for Scaffold-guided co-culture and skin regeneration burn wound healing, the cells were delivered in many Severe burns usually involve detrimental damages to the ways. Co-spray of cultured keratinocytes and autologous dermal matrix niche that is essential for structural fibrin sealant helped retaining the cells in wound and support, cell attachment, migration, proliferation and dif- appeared an effective means of keratinocyte delivery for ferentiation. While CEAs, cultured keratinocytes or epi- wound healing [96, 97]. Grafting of a fibrin-based cul- dermal cell suspensions facilitate wound healing and ture improved adhesion and development of the epider- epithelialisation, they showed limitations in treating deep mis and graft take onto the artificial dermis [98]. burn wounds that lack dermal foundation. Artificial der- Cultured keratinocytes sped up the epithelisation when mal regenerative templates such as Integra and Matri- used in combination with the Meek technique in Derm [4] are commonly used for dermal regeneration in achieving wound closure in the severely burned treating deep burns. They are porous bio-scaffolding paediatric patients [99] and led to permanent burn structures made of biodegradable polymers including pro- wound coverage when being grafted with allodermis teins and proteoglycans. They provide structural supports in burn wound [100, 101]. and niche matrix for cell attachment, migration and pro- Sub-confluent autologous keratinocytes were also liferation, and more importantly, they allow the concur- grown on polymer materials and successfully transferred rent application of multiple cell types, cell-cell and cell- to achieve would closure [102–105]. The technique sig- matrix interactions for engineered skin tissue regeneration nificantly reduced the grafting time to within 5–7 days and therapeutic purposes. Dermal scaffolds together with of receipt of biopsy and ensured the cell delivery. High cultured keratinocytes and fibroblasts enable in vitro gen- yields of proliferating autologous keratinocytes can be eration of living, durable skin substitutes within several grown on gelatin microbeads and delivered to epithelia- weeks, with reconstructed epidermis and dermis of close lise cutaneous wound [106–108]. Less wound contrac- to normal histological appearance [116, 117]. Scaffold- tion was reported when keratinocytes on carrier beads guided co-culture of different skin cells synergistically en- were delivered for wound healing [107]. hance the production of bio-factors and cytokines that benefit wound healing. Increased expression of integrins Non-cultured epidermal cell suspension and decreased apoptosis correlate with increased mel- A quick procedure using non-cultured autologous epi- anocyte retention in cultured skin substitutes [118]. dermal cells for wound healing was also reported [5, The autologous living skin substitutes were proved to 109]. It involved isolating the epidermal cells from a be clinically effective in severe burn management, small skin sample by enzymatic digestion, preparing a resulting in permanent wound closure with improved basal cell-enriched suspension and redistributing the aesthetic outcomes and minimising donor site cas- cells by spraying or dripping to the debrided or granu- ualty [115]. In addition, other cell types including lated wound area to facilitate permanent wound closure endothelial cells, melanocytes, MSCs could also be or to correct the pigmentation loss. The isolated living seeded to produce engineered skin equivalents with cells were then re-distributed immediately to a larger microvascular network, skin appendages and pigmen- wound surface area at 1:80–100 expansion ratio for fast tation, which share more structural and functional wound epithelisation [110]. similarities to natural skin [119–122]. The in vitro The use of non-cultured epidermal suspension is a fast pre-microvascularisation of engineered skin substitute procedure that involves minimal tissue manipulation could inosculate with host vasculature to enhance in and can be completed within a couple of hours in surgi- vivo graft survival. 3D Skin Equivalents were also cal settings. The non-cultured epidermal cells can be reconstituted from human iPSCs [54]. Co-culture of used alone or in combination with meshed grafts for KSCs and dermal fibroblast cells with dermal regen- wound healing. It was reported to promote wound erative scaffold enable the development of living skin epithelialisation and reduce the required surgical inter- substitute with structures comparable to natural skin. vention and total length of stay in treating burns and It indicated that the full-skin substitute has a greater donor site wounds [109, 111]. However, wound re- potential to stimulate wound healing than epidermal epithelialisation and the number of keratinocyte colonies or dermal substitutes alone. were significantly less in wounds transplanted with non- cultured keratinocytes compared to wounds with cul- tured keratinocytes [91]. MSC- and iPSC-based therapies Due to the existence of melanocytes in epidermal har- MSCs including BM-MSCs and ADSCs have demon- vests, the non-cultured epidermal cell suspension helped strated great potentials in acute and chronic wound in correcting the skin hypopigmentation post burns or healing and skin repair [123–125]. They promote wound repigmenting vitiligo [112–115]. healing through enhanced cell migration, differentiation Li and Maitz Burns & Trauma (2018) 6:13 Page 6 of 10 and release of signalling cytokines and bio-factors to in long-term investigations for any associated cancer regulate the process of angiogenesis [126, 127]. risk, epigenetic memory retained from parent cells, gen- The subcutaneous adipose tissue from debrided skin etic instability and potential immunogenicity [56, 144]. in burn surgery can be a rich source of ADSCs for many Until now, cultured keratinocytes and non-cultured applications such as being grafted as cutaneous wound epidermal isolates are the most used devices for treating therapy or for tissue engineering [128]. Autologous burns. Cultured epidermis and skin substitutes are only ADSC-enhanced healing of cutaneous radiation syn- structurally similar but will never be identical to natural drome was observed in a mini-pig model [129]. skin as evidenced by the differences in their gene expres- BM-MSCs were reported to enhance wound healing sion profiles [145]. They are fragile and more susceptible quality and facilitate skin regeneration after full- to mechanical shearing forces and are more sensitive to thickness injury, with the grafted cells transdifferentiat- infections due to an insufficient level of beta-defensins, ing into cell types including vascular endothelial cells, the antimicrobial peptides [146]. CEAs also displayed sebaceous duct cells and epidermal cells [122]. poor adherence, poor stability and delay in rete ridge Allogenic human umbilical cord mesenchymal stem formation and elastin filament development [147] and cells (hUCMSCs) and BM-MSCs are being trialled clin- lacking or delayed basement membrane [117] post graft- ically for treating acute or second-degree burns [130, ing. Although the potential and feasibility for the use in 131] but no results have been reported until now. BM- the treatment of burns were demonstrated, the data MSCs in hydrogels could be a promising therapeutic were mainly generated from laboratory analysis, animal strategy for curing chronic wound including diabetic ul- studies, case reports and observations of small patient cers [124]. Autogenic BM-MSCs transferred with colla- cohorts lacking of proper controls. More reliable and gen membrane were seen to repair full-thickness randomly controlled trials in a large scale are still cutaneous deficiency in porcine model [132]. ADSCs needed to assess the efficacy and to understand the full were reported to modulate development of hypertrophic capacities of each cellular device in burn wound healing, scarring in a red Duroc porcine model [42] and attenu- scarring and skin repair. ate scar formation during wound healing [133, 134]. More research and development should be carried out Patient-specific and gene-corrected iPSCs, expressing to address the following technical problems and factors collagen 7, were developed with the inherent capacity to that could affect the outcomes of cell therapy. For ex- treat recessive dystrophic epidermolysis bullosa, a severe ample, the time needed for generating autologous CEAs and often lethal condition caused by mutations in the or scaffold-guided living skin substitutes is often too long COL7A1 gene-encoding type VII collagen [135]. Very re- for managing acute burns. Technical improvement is cently, the entire human epidermis could be generated needed to ensure the timely supply of cultured cellular using transgenic stem cells for the genetic disease [136]. products for burn wound healing when early debridement was carried out in treating burns. The use of cultured cell Considerations and future directions suspension as well as non-cultured cells significantly re- The evidence from laboratory, animal and clinical stud- duced the preparation time for cellular products but came ies demonstrate that cell-based therapy is becoming an across other issues following their delivery. The cloning important alternative or conjunctive treatment for burn efficiency is more of a concern with non-cultured epider- wound management. It is important to point out that mal cells as the freshly isolated cells showed very low effi- cell therapy devices and associated procedures are now ciency in cloning or replicating themselves [63, 68, 70]. highly regulated by global authorities to ensure their The run-off issue of cell suspension from the wound sur- bio-safety and efficacy. While animal sourced reagents face could further decrease the number of cells actually could cause concerns, unresolved issues also exist in the retained in the wound after delivery. In addition, other potential applications using MSCs and iPSCs. It is also factors including wound depth, wound bed preparation, important to understand that MSCs from different and wound infection further complicate the application of sources could have different immunomodulation cap- cell therapy and lead to variable outcomes. As timing is al- abilities and varied capacities to proliferate and differen- ways critical for surgical intervention in treating burns, tiate to various cells. If allogeneic MSCs are used, their the successful use of cell therapy also depends on the immunogenicity could have impact on their in vivo dur- well-coordinated collaboration between clinical scientists, ability. More studies are needed to define those issues as surgeons and nursing staff. they could potentially affect the therapeutic outcomes [137]. Also, MSCs were reported to enhance tumour for- Conclusions mation and growth in vivo and metastasis [138–143]. Cell therapy has emerged as an important component of The biosafety of retroviral vectors for transcriptional fac- life-saving and wound healing procedures in treating tor delivery in making iPSCs also need to be examined burns. Although progresses have been made to Li and Maitz Burns & Trauma (2018) 6:13 Page 7 of 10 demonstrate the potential and feasibility of various stem 19. Kubo K, Kuroyanagi Y. A study of cytokines released from fibroblasts in cultured dermal substitute. Artif Organs. 2005;29(10):845–9. cells for burn wound healing, there are still scientific 20. Sorrell JM, Baber MA, Caplan AI. Site-matched papillary and reticular human and technical issues that should be resolved to facilitate dermal fibroblasts differ in their release of specific growth factors/cytokines the full potential of the cellular devices. More evidence and in their interaction with keratinocytes. J Cell Physiol. 2004;200(1):134–45. 21. Spiekstra SW, Breetveld M, Rustemeyer T, Scheper RJ, Gibbs S. 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Cell therapy for severe burn wound healing

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Medicine & Public Health; Traumatic Surgery; Emergency Medicine
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Abstract

Cell therapy has emerged as an important component of life-saving procedures in treating burns. Over past decades, advances in stem cells and regenerative medicine have offered exciting opportunities of developing cell-based alternatives and demonstrated the potential and feasibility of various stem cells for burn wound healing. However, there are still scientific and technical issues that should be resolved to facilitate the full potential of the cellular devices. More evidence from large, randomly controlled trials is also needed to understand the clinical impact of cell therapy in burns. This article aims to provide an up-to-date review of the research development and clinical applications of cell therapies in burn wound healing and skin regeneration. Keywords: Skin, Burn injuries, Stem cells, Cell therapy, Wound healing, Skin regeneration Background autologous progenitor cells that could replicate to regen- Burns remain one of the most traumatic injuries causing erate skin tissues for permanent wound closure. In past significant health, social and economic consequences [1]. decades, cell-based therapies have emerged as popular Globally, severe burns lead to about 180,000 deaths an- choices in conjunction with standard skin grafting tech- nually and millions of patients suffering from non-fatal niques for burn wound healing and regeneration of skin burns experiencing substantial and life-long physical and structure and functions. This article aims to provide an psychological morbidities. up-to-date review of the research development and clin- Severe burn wound is characterised by the destruction ical applications of cell therapies in severe burn wound of skin structures, functions and more importantly the healing. loss of the progenitor cell populations that are essential for regenerating and restoring the structures and func- Review tions [2]. Until now, autologous skin grafting remains a Development of cell therapy for burn wound healing standard practice in treating severe burns. However, its Cell therapy which also called cellular therapy or effectiveness is often challenged in treating severe burn cytotherapy involves delivering an autologous or allo- patients with limited donor sites for skin graft harvest- genic cellular component into a patient to repair or re- ing. Consequently, the patients could experience a sig- generate the damaged tissue due to injuries or diseases, nificant delay in wound closure, detrimental infection, to rectify the diseased conditions associated with the scarring or even death. damages or deficiency of the unique cell population and To overcome the autograft shortage, a variety of alterna- to restore the physiological functions. tives for autologous skin grafts including allogeneic skin, Skin as the multi-functional and protective barrier in xenografts and synthetic skin substitutes have been widely human contains essential stem cell population and vari- adopted in burn wound care [3, 4]. While those alternative ous cellular types that are critical for renewing and devices provide temporary wound coverage and deliver maintaining its structural integrity and functions. Re- various bio-factors to facilitate the angiogenesis and search on skin cell transplantation for wound healing granulation of wound bed for further surgery, they could was first reported by Billingham and Reynolds in 1952 never replace the skin autograft delivering the essential [5]. In a guinea pig model, they harvested both epider- * Correspondence: zhe.li1@sydney.edu.au mal sheets and epidermal cell suspension by trypsin di- Burns Unit, Concord Hospital, Concord, New South Wales 2139, Australia gestion and then transplanted them to surgically created Skin Laboratory, NSW Statewide Burns Service, Concord, New South Wales, wounds to evaluate their possible application in plastic Australia Full list of author information is available at the end of the article surgery, experimental pathology, wound healing and © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li and Maitz Burns & Trauma (2018) 6:13 Page 2 of 10 contracture release. However, the era of cultured cell transient-amplifying keratinocytes and migrating to- therapy for burn injuries was only opened up after wards skin surface to form multiple keratinocytes Rheinwald and Green revolutionised the cell culture layers [11]. They gradually lose the capacity in popula- technique in 1975 allowing the isolation and serial tion doublings through differentiation [10–15]. KSC cultivation of strains of human keratinocytes from a skin populations with more active, proliferative and regen- biopsy [6]. For the first time, epithelial sheets could be erative capacities are observed in young people com- produced using cultured keratinocyte clones [7]in paring to aged peoples [16]. laboratory and cultured epithelia autografts (CEAs) were The keratinocytes can be easily isolated and ex- successfully transplanted in severe burns for wound panded in numbers under in vitro cell culture condi- healing [8]. The encouraging report further sparked tions. Keratinocytes produce various bio-factors and worldwide research activities on cell therapy in burn re- cytokines including interleukin (IL)-1, IL-6, IL-7, IL-8, search. Progresses as described in the following sections IL-10, IL-12, IL-15, IL-18, and IL-20 and tumour ne- have been achieved over past decades to understand crosis factor alpha (TNF-α)[17], which are important various cell types and their potential roles in burn for regulating skin regeneration and wound healing. wound healing. All these properties make them the most important and widely used cells for therapeutic application in Different cell types with therapeutic potentials burn wound care. Wound healing and restoration of skin structures and A population of circulating keratinocyte progenitor functions depend on many factors including the avail- cells (KPCs) was identified with p63 in peripheral ability of essential progenitor cells, dermal extracellular blood, which differentiated into keratinocytes with matrix (ECM), and bio-factors and cytokines for angio- expression of the cytokeratins, involucrin and filag- genesis and regulation of cell-matrix and cell-cell inter- grin [18]. actions. The following cell types have demonstrated potentials as therapeutic devices in skin wound healing and tissue regeneration. Many autologous and allogeneic Dermal fibroblasts cell products were developed using cells of skin and Dermal fibroblast cells play an important role in normal non-skin origins for therapeutic application in burn skin and skin wound healing post-burn. They produce wound management. the key ECM proteins in the dermis including laminins, fibronectins, collagens, elastic fibres, non-collagen mole- Keratinocyte stem cells cules and bio-factors to regulate cell function, migration The epidermis is mainly comprised of keratinocytes and the cell-matrix and cell-cell interactions in normal which are renewed and sustained by keratinocyte skin homeostasis and wound healing [19–21]. Dermal stem cell (KSC) populations anchored at the fibroblast cells produce many growth factors and cyto- membrane in the epidermal-dermal junction, the hair kines including vascular endothelial growth factor follicle bulge and the sebaceous gland [9, 10]. KSCs, (VEGF), basic fibroblast growth factor (bFGF), hepato- expressing K5, K14 and p63, are well known for regu- cyte growth factor (HGF), platelet-derived growth factor lating epithelial stratification, hair folliculogenesis and (PDGF)-AA, transforming growth factor-beta1 (TGF-β1) wound repair [10–12]. , keratinocyte growth factor (KGF), IL-6 and IL-8 and Based on molecular biomarkers, regenerative cap- tissue inhibitors of metalloproteinases [19–21]. Cryopre- ability and the status of cellular differentiation, kerati- served cultured dermal substitute maintained sufficient nocytes in human skin could be classified as KSCs, fibroblast cell viability and the ability to proliferate and transient-amplifying keratinocytes and differentiated release a significant amount of VEGF [22]. Epidermal- keratinocytes [13]. The KSCs, making about 4% to 8% derived pro-inflammatory cytokines IL-1α and TNF-α of total keratinocyte population in skin [14], are local- mediate synergistically the secretion of wound-healing ised at dermal-epidermal junction, hair follicles and mediators (with the exception of sST2) from fibroblasts in the surrounding zones of skin appendages. They repli- dermal substitutes. Studies demonstrated a synergistic en- cate themselves by asymmetric division to maintain a hancement of the expression of bio-factors including IL- stable population of undifferentiated stem cells at the 1α, IL-6, bFGF, CCL2, CXCL1, CXCL8, and sST2 if KSCs basal region and are essential for epidermal regener- and dermal fibroblast cells were co-cultured in a dermal ation or repair due to skin injuries. Beta4 integrin is regenerative scaffold [21, 23, 24]. required for hemidesmosome formation, cell adhesion Skin-derived precursor (SKP) cells have also been isolated and cell survival [15]. During normal epidermal from dermal papillae and can be differentiated into adipo- homeostasis or repair, the KSCs go through asymmet- cytes, smooth myocytes and neurons in vitro [25, 26]. Fi- ric division, exiting their niche, transforming into broblasts from adult mouse and human can be induced Li and Maitz Burns & Trauma (2018) 6:13 Page 3 of 10 into pluripotent stem cells with potentials for regeneration pluristratified epidermis [47–51]. Immortalised keratino- of various tissues [27, 28]. cyte lines [52] and fibroblasts [53] were also derived from hESCs. The hESCs-derived fibroblasts were ob- Mesenchymal stem cells served to direct development and repair of three dimen- Mesenchymal stem cells (MSCs), a unique popula- sional (3D) human skin equivalents [53]. tion of multipotent cells with self-renewal and regen- Although hESCs could be developed to provide tem- erative capacity can be derived from autologous or porary skin substitutes for burn patients awaiting autolo- allogenic tissues including bone marrow, adipose tis- gous grafts, the potential applications are however sue, nerve tissue and umbilical cord and cord blood, complicated due to the ethical issues in relation to using and dermis. MSCs express surface CD markers in- human embryos. cluding CD44+, CD73+, CD90+ and CD105+ and The Nobel Prize winning discovery of induced pluripo- are distinguished from haematopoietic cells by a lack tent stem cells (iPSCs) by Yamananka and his team of CD34, CD14, CD45, CD11b/CD79 and CD19/ brought an alternative source of stem cells with pluripo- HLA-DR [29–32]. tent potentials for therapeutic applications [27, 28]. MSCs produceand releasebio-factors[33–37]such While avoiding the ethical concerns in using hESCs, as VEGF, stromal cell-derived factor-1, epidermal they induced adult mouse or human dermal fibroblasts growth factor, KGF, insulin-like growth factor and into pluripotent stem cells by transforming them with matrix metalloproteinase-9 promoting angiogenesis; Sox2, Oct4, Klf4 and c-Myc transcription factors using a recruit endogenous progenitor cells; and direct cell retroviral system. The generated iPSCs are, although not differentiation, proliferation and ECM formation dur- identical, extremely similar to embryonic stem cells be- ing wound repair. MSCs also secrete cytokines includ- ing capable of generating all cell lineages of different tis- ing interferon-λ,TNF-α,IL-1α and IL-1β, and nitric sues including skin [49, 54]. iPSC-derived cells could oxide to modulate host immune response in wound secrete proteins including VEGF, FGF-2, TGF-β, healing [33, 36, 37]. S100A4, GRO, GM-CSF, MCP-1, IL-6 and IL-8 that pro- Studies demonstrated that both autologous and mote increased proliferation, contraction and migration allogenic MSCs, administered both systemically or and [55]. iPSCs as patient specific or allogenic devices dem- locally, exhibit therapeutic potentials promoting cuta- onstrated potential values for clinical applications, re- neous wound healing and tissue regeneration via the search and drug discoveries in laboratory research. Their paracrine bio-factors and cytokines and the multi- safety and efficacy are still to be assessed mainly due to potency in tissue regeneration [31, 32, 38–40]. Under the use of retroviral vectors, associated risk, genetic in- specific niche conditions and molecular stimulations, stability and potential immunogenicity [56]. To avoid MSCs could be induced to differentiate into multiple the risks of mutagenesis and oncogenic transformation tissue-specific cell lineages including osteoblasts, adi- associated with retroviral vector, iPSCs are also gener- pocytes, chondrocytes, tenocytes, myocytes, endothe- ated with non-integrative reprogramming strategies lial cells, vascular smooth muscle cells, keratinocytes using plasmid vectors, episomal plasmid vectors, modi- and sweat gland-like structures [35, 37, 41]. MSCs fied/microRNA or even direct delivery of reprogram- could produce anti-fibrotic factors and modulate ming protein factors [57–59]. The non-integrative development of hypertrophic scarring [42]. Adipose- reprogramming strategies were considered safer for derived stem cells (ADSCs) and ADSC-conditioned therapeutic purpose. However, the efficiency of iPSC in- medium appear highly effective in promoting hair duction by original plasmid vector was low although it growth [43, 44], and development of functional sweat could be enhanced by essential factor for episomal amp- gland-like structures was observed from transplanting lification of the vector [58]. differentiated bone marrow mesenchymal stem cells (BM-MSCs) [45]. MSCs promote angiogenesis Clinical research and applications of cell-based therapies and vascular stability that are critical for delivering the in burn wound care essential nutrient supply for tissue regeneration and Cultured epithelial autografts wound healing [46]. The key technique that Rheinwald and Green established in the 1970s for serial culture of keratinocytes opened Induced pluripotent stem cells the era of research and therapeutic uses of cultured au- Human embryonic stem cells (hESCs), derived from hu- tologous keratinocytes in burn wound healing. Under la- man embryos are well known for their pluripotent cap- boratory conditions, KSCs could be isolated from a acities in tissue regeneration. Research demonstrated small skin biopsy and cultivated in the presence of that hESCs could be induced to efficiently differentiate growth-arrested 3T3 mouse fibroblasts as feeder cells to into functional basal keratinocytes that regenerate a form epidermal sheets that could be transplanted back Li and Maitz Burns & Trauma (2018) 6:13 Page 4 of 10 to the same patient for treatment. The innovative grafting wide meshed autograft together with acellular technology quickly gained global attention and dermal matrix in some cases [83] and were observed to emerged as a popular way to generate alternative auto- improve skin texture of burn scars after resurfacing the grafts to facilitate permanent wound closure when wounds [84]. The use of CEA in treating a full-thickness treating severe burns. Since 1981, both cultured living skin wound in severely burned patients results in keratinocytes and epithelial autograft sheets (CEAs), favourable quality of scars and was reported to show either produced in house or by commercial compan- good potential to save lives by providing epidermal cover ies, have been used as clinical devices in treating large [85]. Early excision followed by temporary coverage with burns [8, 60–67]. autograft, which is allowed to engraft, was found to be Cultured keratinocytes were used for treating burns in associated with a low infection rate and a higher rate of various ways. Commonly, the cells from a skin biopsy CEA take [86]. CEAs regenerate a stable normal epider- are cultivated, screened and passaged in a cell culture mis and are capable of inducing dermal regeneration system to expand the numbers of KPCs. In the presence from wound bed connective tissue [68, 84]. Better out- of feeder fibroblast cells, the keratinocytes could prolif- comes in aesthetic appearances and scar formation were erate and differentiate to form an epidermis-like tissue also reported in burn patients grafted with CEAs [87]. in cell culture. The process of CEA cultivation usually In addition, the procedure can be initiated with a small requires about 3 weeks depending on many factors in- skin biopsy causing minimum donor site morbidity. This cluding patient age, health condition and severity of would be particularly important for patients with exten- burn injuries. The colony-forming efficiency of keratino- sive burns as a life-saving device. CEAs deliver living au- cytes from freshly trypsinised skin was reported very tologous epidermal stem cells for permanent wound low, between 0.15% and 3.8% in the primary cell culture closure without eliciting immunological rejection. CEAs [68–70] but was increased to 26% to 90% at passage 2 to could also function as biological wound dressings deliv- 3 cultures and to over 16% in CEA sheets [70], being ering the much needed biological factors and niche en- significantly higher than that (4–8%) in normal skin vironment for wound healing. [14]. Cultured epidermis can be harvested as a sheet graft for clinical application. Cultured human sole-derived keratinocyte grafts re- Cultured keratinocyte suspension express site-specific differentiation after transplantation In 1952, Billingham and Reynolds examined the poten- [71]. The formation of hair follicles and development of tials of transplanting sheets of pure epidermal epithe- normal epidermal microarchitecture were observed lium and non-cultured epidermal cell suspensions for when epidermal cells were transplanted together with wound healing in guinea pigs [5]. In treating burn pa- cells of dermal origin [72]. tients, the burn wound areas often require early exci- Very often, CEAs were used in combination with sions and then immediate skin grafting. While a skin widely meshed skin grafts for wound closure. CEAs sample could be taken for growing CEAs, a 3-week wait- could be transplanted directly to the wound area [8, 73] ing time for CEA growth would be too long for many with undifferentiated progenitor cells attached to the re- patients as it unnecessarily delays the surgical interven- generated or freshly debrided wound bed [74]. CEAs tion. Consequently, the keratinocytes were cultured only were of significant value in severe burn management, to sub-confluent stage or be grown on carrier material providing wound coverage, facilitating wound epithelisa- to allow early harvesting and application as cell suspen- tion and permanent closure, and improving survival sion or sub-confluent sheet [88, 89]. rates among patients with massive burn wounds [75– Autologous keratinocytes isolated from any available 78]. CEA demonstrated a very high beneficial value in donor site area including sole and scalp [90] could be managing burns > 60% total body surface area (TBSA), isolated and cultured for clinical application. The deliv- being a life-saving treatment [79]. Although being ex- ery of cultured keratinocytes in suspensions, directly to pensive compared to standard skin autograft, the en- the wound or in combination with skin grafting, acceler- graftment rates and survival ratio results make CEAs ated epidermal wound healing in animal models and excellent alternative wound coverage when donor sites burn patients [90, 91]. The transplantation of cultured are limited in severe burns [80]. Studies reported an keratinocytes were found to facilitate the reformation of average graft take of 72.7% with a 91% overall survival organised epidermal structure [69] and to promote per- rate in treating critically burns using CEA, indicating manent epithelialisation in the burn wound [92, 93] and cultured keratinocytes as an excellent alternative or ad- accelerated epithelial maturation in an in vivo wound junct to conventional split-thickness skin grafting [81, model [94]. Permanent repigmentation of piebaldism 82]. The CEAs were reported to enhance the take of a was also reported in patients treated by erbium:YAG wide meshed autograft in massive burns and allow for laser and autologous cultured epidermis [95]. Li and Maitz Burns & Trauma (2018) 6:13 Page 5 of 10 To ensure the effectiveness of living keratinocytes for Scaffold-guided co-culture and skin regeneration burn wound healing, the cells were delivered in many Severe burns usually involve detrimental damages to the ways. Co-spray of cultured keratinocytes and autologous dermal matrix niche that is essential for structural fibrin sealant helped retaining the cells in wound and support, cell attachment, migration, proliferation and dif- appeared an effective means of keratinocyte delivery for ferentiation. While CEAs, cultured keratinocytes or epi- wound healing [96, 97]. Grafting of a fibrin-based cul- dermal cell suspensions facilitate wound healing and ture improved adhesion and development of the epider- epithelialisation, they showed limitations in treating deep mis and graft take onto the artificial dermis [98]. burn wounds that lack dermal foundation. Artificial der- Cultured keratinocytes sped up the epithelisation when mal regenerative templates such as Integra and Matri- used in combination with the Meek technique in Derm [4] are commonly used for dermal regeneration in achieving wound closure in the severely burned treating deep burns. They are porous bio-scaffolding paediatric patients [99] and led to permanent burn structures made of biodegradable polymers including pro- wound coverage when being grafted with allodermis teins and proteoglycans. They provide structural supports in burn wound [100, 101]. and niche matrix for cell attachment, migration and pro- Sub-confluent autologous keratinocytes were also liferation, and more importantly, they allow the concur- grown on polymer materials and successfully transferred rent application of multiple cell types, cell-cell and cell- to achieve would closure [102–105]. The technique sig- matrix interactions for engineered skin tissue regeneration nificantly reduced the grafting time to within 5–7 days and therapeutic purposes. Dermal scaffolds together with of receipt of biopsy and ensured the cell delivery. High cultured keratinocytes and fibroblasts enable in vitro gen- yields of proliferating autologous keratinocytes can be eration of living, durable skin substitutes within several grown on gelatin microbeads and delivered to epithelia- weeks, with reconstructed epidermis and dermis of close lise cutaneous wound [106–108]. Less wound contrac- to normal histological appearance [116, 117]. Scaffold- tion was reported when keratinocytes on carrier beads guided co-culture of different skin cells synergistically en- were delivered for wound healing [107]. hance the production of bio-factors and cytokines that benefit wound healing. Increased expression of integrins Non-cultured epidermal cell suspension and decreased apoptosis correlate with increased mel- A quick procedure using non-cultured autologous epi- anocyte retention in cultured skin substitutes [118]. dermal cells for wound healing was also reported [5, The autologous living skin substitutes were proved to 109]. It involved isolating the epidermal cells from a be clinically effective in severe burn management, small skin sample by enzymatic digestion, preparing a resulting in permanent wound closure with improved basal cell-enriched suspension and redistributing the aesthetic outcomes and minimising donor site cas- cells by spraying or dripping to the debrided or granu- ualty [115]. In addition, other cell types including lated wound area to facilitate permanent wound closure endothelial cells, melanocytes, MSCs could also be or to correct the pigmentation loss. The isolated living seeded to produce engineered skin equivalents with cells were then re-distributed immediately to a larger microvascular network, skin appendages and pigmen- wound surface area at 1:80–100 expansion ratio for fast tation, which share more structural and functional wound epithelisation [110]. similarities to natural skin [119–122]. The in vitro The use of non-cultured epidermal suspension is a fast pre-microvascularisation of engineered skin substitute procedure that involves minimal tissue manipulation could inosculate with host vasculature to enhance in and can be completed within a couple of hours in surgi- vivo graft survival. 3D Skin Equivalents were also cal settings. The non-cultured epidermal cells can be reconstituted from human iPSCs [54]. Co-culture of used alone or in combination with meshed grafts for KSCs and dermal fibroblast cells with dermal regen- wound healing. It was reported to promote wound erative scaffold enable the development of living skin epithelialisation and reduce the required surgical inter- substitute with structures comparable to natural skin. vention and total length of stay in treating burns and It indicated that the full-skin substitute has a greater donor site wounds [109, 111]. However, wound re- potential to stimulate wound healing than epidermal epithelialisation and the number of keratinocyte colonies or dermal substitutes alone. were significantly less in wounds transplanted with non- cultured keratinocytes compared to wounds with cul- tured keratinocytes [91]. MSC- and iPSC-based therapies Due to the existence of melanocytes in epidermal har- MSCs including BM-MSCs and ADSCs have demon- vests, the non-cultured epidermal cell suspension helped strated great potentials in acute and chronic wound in correcting the skin hypopigmentation post burns or healing and skin repair [123–125]. They promote wound repigmenting vitiligo [112–115]. healing through enhanced cell migration, differentiation Li and Maitz Burns & Trauma (2018) 6:13 Page 6 of 10 and release of signalling cytokines and bio-factors to in long-term investigations for any associated cancer regulate the process of angiogenesis [126, 127]. risk, epigenetic memory retained from parent cells, gen- The subcutaneous adipose tissue from debrided skin etic instability and potential immunogenicity [56, 144]. in burn surgery can be a rich source of ADSCs for many Until now, cultured keratinocytes and non-cultured applications such as being grafted as cutaneous wound epidermal isolates are the most used devices for treating therapy or for tissue engineering [128]. Autologous burns. Cultured epidermis and skin substitutes are only ADSC-enhanced healing of cutaneous radiation syn- structurally similar but will never be identical to natural drome was observed in a mini-pig model [129]. skin as evidenced by the differences in their gene expres- BM-MSCs were reported to enhance wound healing sion profiles [145]. They are fragile and more susceptible quality and facilitate skin regeneration after full- to mechanical shearing forces and are more sensitive to thickness injury, with the grafted cells transdifferentiat- infections due to an insufficient level of beta-defensins, ing into cell types including vascular endothelial cells, the antimicrobial peptides [146]. CEAs also displayed sebaceous duct cells and epidermal cells [122]. poor adherence, poor stability and delay in rete ridge Allogenic human umbilical cord mesenchymal stem formation and elastin filament development [147] and cells (hUCMSCs) and BM-MSCs are being trialled clin- lacking or delayed basement membrane [117] post graft- ically for treating acute or second-degree burns [130, ing. Although the potential and feasibility for the use in 131] but no results have been reported until now. BM- the treatment of burns were demonstrated, the data MSCs in hydrogels could be a promising therapeutic were mainly generated from laboratory analysis, animal strategy for curing chronic wound including diabetic ul- studies, case reports and observations of small patient cers [124]. Autogenic BM-MSCs transferred with colla- cohorts lacking of proper controls. More reliable and gen membrane were seen to repair full-thickness randomly controlled trials in a large scale are still cutaneous deficiency in porcine model [132]. ADSCs needed to assess the efficacy and to understand the full were reported to modulate development of hypertrophic capacities of each cellular device in burn wound healing, scarring in a red Duroc porcine model [42] and attenu- scarring and skin repair. ate scar formation during wound healing [133, 134]. More research and development should be carried out Patient-specific and gene-corrected iPSCs, expressing to address the following technical problems and factors collagen 7, were developed with the inherent capacity to that could affect the outcomes of cell therapy. For ex- treat recessive dystrophic epidermolysis bullosa, a severe ample, the time needed for generating autologous CEAs and often lethal condition caused by mutations in the or scaffold-guided living skin substitutes is often too long COL7A1 gene-encoding type VII collagen [135]. Very re- for managing acute burns. Technical improvement is cently, the entire human epidermis could be generated needed to ensure the timely supply of cultured cellular using transgenic stem cells for the genetic disease [136]. products for burn wound healing when early debridement was carried out in treating burns. The use of cultured cell Considerations and future directions suspension as well as non-cultured cells significantly re- The evidence from laboratory, animal and clinical stud- duced the preparation time for cellular products but came ies demonstrate that cell-based therapy is becoming an across other issues following their delivery. The cloning important alternative or conjunctive treatment for burn efficiency is more of a concern with non-cultured epider- wound management. It is important to point out that mal cells as the freshly isolated cells showed very low effi- cell therapy devices and associated procedures are now ciency in cloning or replicating themselves [63, 68, 70]. highly regulated by global authorities to ensure their The run-off issue of cell suspension from the wound sur- bio-safety and efficacy. While animal sourced reagents face could further decrease the number of cells actually could cause concerns, unresolved issues also exist in the retained in the wound after delivery. In addition, other potential applications using MSCs and iPSCs. It is also factors including wound depth, wound bed preparation, important to understand that MSCs from different and wound infection further complicate the application of sources could have different immunomodulation cap- cell therapy and lead to variable outcomes. As timing is al- abilities and varied capacities to proliferate and differen- ways critical for surgical intervention in treating burns, tiate to various cells. If allogeneic MSCs are used, their the successful use of cell therapy also depends on the immunogenicity could have impact on their in vivo dur- well-coordinated collaboration between clinical scientists, ability. More studies are needed to define those issues as surgeons and nursing staff. they could potentially affect the therapeutic outcomes [137]. Also, MSCs were reported to enhance tumour for- Conclusions mation and growth in vivo and metastasis [138–143]. Cell therapy has emerged as an important component of The biosafety of retroviral vectors for transcriptional fac- life-saving and wound healing procedures in treating tor delivery in making iPSCs also need to be examined burns. Although progresses have been made to Li and Maitz Burns & Trauma (2018) 6:13 Page 7 of 10 demonstrate the potential and feasibility of various stem 19. Kubo K, Kuroyanagi Y. A study of cytokines released from fibroblasts in cultured dermal substitute. Artif Organs. 2005;29(10):845–9. cells for burn wound healing, there are still scientific 20. Sorrell JM, Baber MA, Caplan AI. Site-matched papillary and reticular human and technical issues that should be resolved to facilitate dermal fibroblasts differ in their release of specific growth factors/cytokines the full potential of the cellular devices. More evidence and in their interaction with keratinocytes. J Cell Physiol. 2004;200(1):134–45. 21. Spiekstra SW, Breetveld M, Rustemeyer T, Scheper RJ, Gibbs S. 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Burns & TraumaSpringer Journals

Published: May 28, 2018

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