Bioact Mater.
2022 Oct; 16: 187–203.doi:
10.1016/j.bioactmat.2022.02.023PMCID:
PMC8965724
PMID: 35386328
aBiopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM), University of Granada, Granada, Spain
bDepartment of Human Anatomy and Embryology, Faculty of Medicine, University of Granada, Granada, Spain
cBioFab i3D Lab - Biofabrication and 3D (bio)printing Laboratory, Granada, Spain
dExcellence Research Unit “Modeling Nature” (MNat), University of Granada, Granada, Spain
eBiosanitary Research Institute of Granada (ibs.GRANADA), University Hospitals of Granada ‐ University of Granada, Granada, Spain
Find articles by Paula Pleguezuelos-Beltrán
fR&D Human Health, Bioibérica S.A.U, Barcelona, Spain
gDepartment of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada, Granada, Spain
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bDepartment of Human Anatomy and Embryology, Faculty of Medicine, University of Granada, Granada, Spain
cBioFab i3D Lab - Biofabrication and 3D (bio)printing Laboratory, Granada, Spain
dExcellence Research Unit “Modeling Nature” (MNat), University of Granada, Granada, Spain
eBiosanitary Research Institute of Granada (ibs.GRANADA), University Hospitals of Granada ‐ University of Granada, Granada, Spain
hComplex Tissue Regeneration Department, MERLN Institute for Technology Inspired Regenerative Medicine, Universiteitssingel 40, 6229ER Maastricht, the Netherlands
Find articles by Daniel Nieto-García
aBiopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM), University of Granada, Granada, Spain
bDepartment of Human Anatomy and Embryology, Faculty of Medicine, University of Granada, Granada, Spain
cBioFab i3D Lab - Biofabrication and 3D (bio)printing Laboratory, Granada, Spain
dExcellence Research Unit “Modeling Nature” (MNat), University of Granada, Granada, Spain
eBiosanitary Research Institute of Granada (ibs.GRANADA), University Hospitals of Granada ‐ University of Granada, Granada, Spain
Find articles by Juan Antonio Marchal
aBiopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM), University of Granada, Granada, Spain
cBioFab i3D Lab - Biofabrication and 3D (bio)printing Laboratory, Granada, Spain
dExcellence Research Unit “Modeling Nature” (MNat), University of Granada, Granada, Spain
eBiosanitary Research Institute of Granada (ibs.GRANADA), University Hospitals of Granada ‐ University of Granada, Granada, Spain
iDepartment of Health Sciences, University of Jaén, Jaén, Spain
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Author information Article notes Copyright and License information PMC DisclaimeraBiopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM), University of Granada, Granada, Spain
bDepartment of Human Anatomy and Embryology, Faculty of Medicine, University of Granada, Granada, Spain
cBioFab i3D Lab - Biofabrication and 3D (bio)printing Laboratory, Granada, Spain
dExcellence Research Unit “Modeling Nature” (MNat), University of Granada, Granada, Spain
eBiosanitary Research Institute of Granada (ibs.GRANADA), University Hospitals of Granada ‐ University of Granada, Granada, Spain
fR&D Human Health, Bioibérica S.A.U, Barcelona, Spain
gDepartment of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada, Granada, Spain
hComplex Tissue Regeneration Department, MERLN Institute for Technology Inspired Regenerative Medicine, Universiteitssingel 40, 6229ER Maastricht, the Netherlands
iDepartment of Health Sciences, University of Jaén, Jaén, Spain
Juan Antonio Marchal: se.rgu.og@lahcramj
Elena López-Ruiz: se.neaju@ziurle
∗Corresponding author. Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM), University of Granada, Granada, Spain.Corresponding author. Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM), University of Granada, Granada, Spain. se.neaju@ziurle
∗∗Corresponding author. Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM), University of Granada, Granada, Spain.Corresponding author. Biopathology and Regenerative Medicine Institute (IBIMER), Centre for Biomedical Research (CIBM), University of Granada, Granada, Spain. se.rgu.og@lahcramj
Copyright © 2022 The AuthorsThis is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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To date, skin wounds are still an issue for healthcare professionals. Although numerous approaches have been developed over the years for skin regeneration, recent advances in regenerative medicine offer very promising strategies for the fabrication of artificial skin substitutes, including 3D bioprinting, electrospinning or spraying, among others. In particular, skin sprays are an innovative technique still under clinical evaluation that show great potential for the delivery of cells and hydrogels to treat acute and chronic wounds. Skin sprays present significant advantages compared to conventional treatments for wound healing, such as the facility of application, the possibility to treat large wound areas, or the homogeneous distribution of the sprayed material. In this article, we review the latest advances in this technology, giving a detailed description of investigational and currently commercially available acellular and cellular skin spray products, used for a variety of diseases and applying different experimental materials. Moreover, as skin sprays products are subjected to different classifications, we also explain the regulatory pathways for their commercialization and include the main clinical trials for different skin diseases and their treatment conditions. Finally, we argue and suggest possible future trends for the biotechnology of skin sprays for a better use in clinical dermatology.
Keywords:
Skin, Spray, Tissue engineering, Fibrin, Hydrogel
Abbreviations:
ATMP, advanced therapy medicinal product; BLA, Biologic License Application; CAT, Committee for Advances Therapies; CEA, cultured epithelial autograft; CFR, Code of Federal Regulations; CHMP, Committee for Medicinal Product for Human Use; CTD, Common Technical Document; DMEM, Dulbecco's Modified Eagle's medium; ECM, extracellular matrix; EMA, European Medicines Agency; EU, European Union; FDA, Food and Drug Administration; GAGs, glycosaminoglycans; GLP, Good Laboratory Practice; GMP, Good Manufacturing Practice; HA, hyaluronic acid; HCT/Ps, human cells, tissues, and cellular and tissue-based products; ISO, International Organization for Standardization; MA, marketing authorization; NP, Notified body; OTAT, Office of Tissues and Advanced Therapies; PCL, polycaprolactone; PEG, polyethylene glycol; PHSA, Public Health Service Act; PMA, Premarket Approval; PRF, platelet rich fibrin; PRP, platelet rich plasma; PU, polyurethane; QMS, Quality Management System; TE, tissue engineering; USA, United States of America
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Skin sprays represent a promising technique for wound healing applications.
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Skin sprays can deliver cells and hydrogels with great facility over large wounds.
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Many skin spray products have been studied, only a few have been commercialized.
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Numerous clinical trials study spray products for skin diseases like psoriasis.
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Improved spraying devices should be developed for different materials and cells.
Skin is the largest organ in the human body and one of the most important defense mechanisms against pathogens and thermal, mechanical and chemical hazards, given that it is the outermost protecting sheath and is in direct contact with the external environment [[1], [2], [3], [4], [5], [6]]. The skin is comprised of three main layers, a superficial epidermis, and a deeper dermis, followed by the subcutaneous hypodermis. The epidermis consists of several layers of keratinized epithelium, which is dynamic and in continuous self-renewal. Keratinocytes are the major cell type in the epidermis, and they play a crucial role in skin function thanks to their ability to differentiate and synthesize a variety of structural proteins and lipids that are important for epidermal regeneration [5,7]. Other cells within the epidermis are melanocytes, which provide pigmentation, and Langerhans' cells, which contribute to immune surveillance [8]. Corneocytes (enucleated dead cells) are found embedded in a lipid matrix in the uppermost layer of the epidermis, the stratum corneum, that plays a role against absorption of components and water loss [5]. The dermis is a thicker layer of connective tissue, consisting mainly of cells (like fibroblasts, endothelial cells, smooth muscle cells, and mast cells), extracellular matrix (ECM) and structural components like collagen and elastin fibers, which provide mechanical strength and elasticity, as well as a vascular plexus for skin nutrition [4,8]. Between the epidermis and the dermis is the basal membrane, a highly specialized ECM structure, composed of different glycoproteins and proteoglycans, that provides a stabilizing but dynamic interface and a diffusion barrier between these two layers, and where skin appendages such as hair follicles, sweat glands, and nerves are all embedded [4,5,9]. These skin appendages maintain the body's hydration and constitute a protective layer that helps to defend the body from hostile environment, among other functions [3]. Connecting the dermis to the underlying structures is the hypodermis, the connective tissue which includes fibroblasts and adipose tissue for fat storage and protection, serving as the skin's shock-absorber and the body's heat insulator [4,5,9]. The thickness of the human skin is approximately 1.5–2.5 mm, but it varies depending on its anatomical localization and its function in supporting the weight of the body [2].
Skin wounds or injuries may result from burns and other physical and chemical traumas, genetic irregularities, skin diseases (such as diabetic foot ulcers, perianal fistulae, or epidermolysis bullosa), or removal of skin during surgery [5]. Depending on the depth of the injury, skin wounds can be categorized as epidermal, superficial partial-thickness, deep partial-thickness, and full-thickness wounds [10]. For optimal treatment, wounds must be diagnosed early, although some cases may be difficult to classify accurately with an early evaluation [11,12]. Thanks to the self-healing functions of the skin, epidermal and superficial partial-thickness wounds can usually be regenerated without requiring therapeutic intervention [5].
However, in deep partial and full-thickness wounds, the skin's regenerative components are destroyed, which makes it impossible for the skin to regenerate on its own. Therefore, these wounds need immediate treatment to avoid a late re-epithelialization, which could facilitate infections, produce worse functional and esthetic healing outcomes (like contracture and scarring) and extend the patient's stay at the hospital [5,[11], [12], [13]]. A large surface area wound, coupled with extensive fluid loss, could even result in death [13]. Fast therapeutic interventions can allow mobility for the patient and can help regain the skin's structure and function, although full recovery of its function may not be possible unless all skin cell types are restored [1,6,14,15].
Conventional skin grafts have been used for decades for the regeneration of deep wounds, although they present some disadvantages and limitations [1,2]. However, the development of tissue engineering (TE) and regenerative medicine has offered multiple therapeutic strategies for the regeneration of skin lesions, using diverse types of skin substitutes, such as cell suspensions, hydrogels, or 3D scaffolds [16]. Moreover, another promising solution that is still not commonly used in clinical practice, but represents a potential therapeutic strategy for wound healing applications, is the use of skin sprays, given their numerous advantages such as their versatility to deliver different cell types and materials.
In this review, we discuss and summarize the main approaches and techniques for skin regeneration, and particularly focus on the latest advances in the technology of skin sprays, including both investigational and currently available commercialized acellular and cellular products, and an explanation of their regulatory pathways to be marketed. Finally, we include an exhaustive search for clinical trials using sprays to treat a variety of skin conditions and propose future trends for the biotechnology of skin sprays with a more feasible clinical application.
The use of topical sprays as an application method for hydrogels and/or cell suspensions to treat acute and chronic wounds has been of interest in clinical practice for the past few decades, thanks to their advantages such as the possibility to treat large wounds or spray over areas with unfavorable topography, the reduced time for application, and the homogeneous distribution of the sprayed suspensions [21,27,118,119].
When using spray devices to deliver biomaterials and living cells for medical applications, it is important to pay attention to the spraying parameters, such as the spraying pressure, the distance from the tip of the spray device to the receiving surface, the angle, or the sprayed volume, because these parameters can vary results significantly [13,27]. For example, in clinical practice, it has been reported that at least a 10 cm distance from the spray device to the body is necessary for a safe application without risking an air embolus [13].
For cellular sprays, it is also important to determine the amount of cells required to treat a certain wound area [27]. In the case of autologous cell spray, Esteban-Vibes et al. (2016) proposed equations to calculate the minimum skin area needed from a donor site, to obtain enough cells to treat a certain burn wound area, obtaining ratios of 1:80 to 1:100 (donor site area:wound area) [11].
Moreover, sprayed cells are expected to be damaged when they impact the target surface, with cell survival depending on numerous variables such as the viscosity of the transporting fluid, nozzle diameter, distance, velocity of the sprayed droplet, or the characteristics of the receiving surface [20,120,121]. It has been found that post-aerosolization cell viability significantly decreases with higher pressure, smaller nozzle diameter, high transporting fluid viscosity, high spraying velocity, and stiffness of the receiving tissue surface. On the contrary, cell viability is positively affected by a larger cell-containing droplet diameter [20,120,121]. Numerous studies have demonstrated the viability of epidermal cells after aerosolization [27,[121], [122], [123]]. For example, Harkin et al. (2006) demonstrated that cell viability was unaffected with air pressures up to 10 psi, but metabolic activity was significantly reduced at higher pressures. They also determined the approximate cell density required for re-epithelialization, concluding that, under the conditions tested, 0.2 mL bursts of 1.5·106 cells/mL sprayed from a height of 10 cm should theoretically epithelialize an area of approximately 10–15 cm2 within 7 days [27]. Denman et al. (2007) developed a mathematical model to simulate the growth patterns of cell colonies after being applied using an aerosolized technique. This model could be used as a decision support tool for clinicians, allowing them to predict the evolution of wound coverage over time, depending on different parameters such as the number of sprayed cells and their spatial distribution, the size of initial colonies, or the wound size [124].
However, it is not sufficient to only consider post-aerosolization viability. Proliferative capacity must be assessed too, because cells experience three types of stress while being sprayed (hydrostatic, shear, and elongation stresses), which may damage the cells without disrupting their membranes, with the duration of applied stress also being a key factor on the caused impairment [125].
Acellular skin sprays generally consist of hydrogels that form thin layers when they are sprayed over the wound, acting as dressings and protecting the wound against infection and fluid loss. In addition, some hydrogels may help to enhance the wound healing process, and some have antibacterial properties or can act as a drug delivery system. Fibrin is the most widely used hydrogel for skin sprays and is often sprayed directly over less severe wounds to act as a hemostatic dressing or over grafts to promote adhesion and take rates [126].
To date, there have been several studies that apply different experimental hydrogels over the wounds, shielding them from infections and avoiding water loss. They are summarized in . These specific studies were only performed in vitro or in animal models, generally using a simple spray pump as the spraying device, and most of the spraying parameters were not specified.
In 1981, Neumann et al. formulated a gelatin foam that is able to cool burn wound surfaces and wash and disinfect them when it is sprayed, and once it dries it adheres to the wound, creating a foam layer that reduces the rate of evaporative water loss and prevents infection [127].
Quinn et al. (2000) used an octylcyanoacrylate-adhesive spray bandage to treat traumatic abrasions in pigs. Octylcyanoacrylate polymerizes with the moisture on the wound surface and dries within 30 s, producing a thin, flexible dressing that is non-toxic, hemostatic and waterproof, it has antimicrobial activity and sloughs off with the wound eschar as epithelialization occurs [128].
Jáuregui et al. (2009) immobilized papain enzyme in a peptin gel, which forms a thin, even and smooth film when aerosolized. This gel promotes wound healing better than untreated wounds, and it stays in place on the wound bed without dripping off [129].
More recently, Catanzano et al. (2015) used a spray-by-spray deposition technique to deliver a tea tree oil microemulsion in an in situ-forming cross-linked alginate hydrogel. Results showed that this microemulsion is homogeneously distributed in the resulting transparent hydrogels and has potential as an advanced dressing for infected wounds, given that it has antimicrobial and anti-inflammatory properties, as well as good thermodynamic and kinetic stability [130].
Among the commercial acellular sprays, plenty of them use fibrin for their products. One of the most popular is TISSEEL® by Baxter (licensed under the trademark of TISSUCOL® in some countries), a fibrin glue that has been widely used since the early 90s as an adjunct to hemostasis and sealing in open surgery and laparoscopy, derived from human pooled plasma. It offers both drip and spray applicators, that are attached to a double syringe (one for the fibrinogen solution and the other for the thrombin solution), allowing surgeons to select the right tools to address individual patient needs [126,131]. As a spray, numerous studies have used TISSEEL® fibrin sealant in vitro and in vivo, alone or alongside cells. Sometimes TISSEEL® is delivered using TISSOMAT®, a pressurized spray module (air compressor) by Baxter, whose Spray Kit has a dual syringe that supports the co-application of other materials like cell suspensions [27,123,132]. Studies using TISSEEL® (summarized in ) reported that when fibrin was added to a cellular treatment (e.g. autologous keratinocytes and fibroblasts [123], or autologous mesenchymal stem cells [132]), wound healing was accelerated and wounds contracted less in comparison with wounds treated with cells alone. TISSEEL® fibrin sealant is an excellent liquid cell delivery vehicle that rapidly polymerizes upon addition of thrombin, forming an adherent network of cells embedded in a fibrin meshwork that still preserve their ability to grow and differentiate [25,27,123,[132], [133], [134]].
ARTISS® is another fibrin sealant by Baxter, used to adhere autologous skin grafts to the wound beds in burns and to adhere tissue flaps during facial rhytidectomy surgery, but, in contrast to TISSEEL®, ARTISS® is not employed as an adjunct to hemostasis [135]. Earnshaw et al. (2020) used ARTISS® in 50 patients undergoing lateral selective neck dissections, showing a significant reduction in the length of the hospital stay of the patients, in the drain retention time, and in the total volume drained. Moreover, they reported an easier use of ARTISS® in comparison to TISSEEL® [136] ( ).
EVICEL® by Johnson & Johnson is a fibrin sealant very similar to TISSEEL®, also derived from human pooled plasma, used as an adjunct to hemostasis for use in patients undergoing surgery, with the difference that its airless spray accessory does not need an external gas source, reducing setup time [137]. Reddy et al. (2017) conducted an observational prospective study to evaluate the efficiency of EVICEL® for the adherence of skin grafts. They reported better hemostasis and graft adhesion, and a reduction in surgical time [138] ( ).
Vivostat® has different products, with a system that prepares autologous fibrin sealant or platelet rich fibrin (PRF) from the patient's own blood without requiring additional thrombin and without using cryoprecipitation. The fibrin is shown to act as a hemostatic, as well as a glue for graft fixations in burn surgery or to adhere cells to the burn wound. Vivostat® PRF has also been successfully used to treat diabetic foot ulcers, venous ulcers, and pressure ulcers. The Spraypen is a sterile, disposable, hand-held device that delivers the fibrin sealant or PRF solution to the tissue; but besides the Spraypen, the Vivostat® system offers different types of applicators (e.g. the Endoscopic Applicator). Vivostat® also has a Co-Delivery system that makes it possible to co-deliver a desired substance, such as a cell suspension, along with Vivostat® Fibrin Sealant or Vivostat® PRF [2,24,139]. Studies using Vivostat® fibrin sealant (see ) reported that its rate of polymerization is significantly faster than that of other sealants that require thrombin, allowing immediate adhesion of cells onto the wound; and since this system produces completely autologous fibrin, any risk of disease transmission is eliminated [2,24].
On the other hand, there is a wide variety of antiseptic sprays for wound cleansing in superficial wounds, for example, Prontosan® Wound Spray by B. Braun and Hansaplast® Wound Spray, composed of polyaminopropyl biguanide (polihexanide), or The Wonder Spray®, whose key ingredient is hypochlorous acid. It is worth mentioning that there are other commercial acellular spray products for healing that form waterproof, thin, transparent films or dressings, which cover the wound and protect it against infections, such as URGO® Spray Dressing, Nobecutan® by Inibsa Hospital or Hansaplast® Spray Dressing.
A variety of cell types have been utilized in cellular sprays for in vitro wound healing studies, but autologous epidermal cells are mainly used for in vivo studies and clinical trials. Cell spray autografting is an innovative technique still under clinical evaluation, consisting of taking autologous skin cells from a small biopsy of undamaged skin of the patient and either spraying the cells directly over the wound or culturing them to amplify their number before spraying. In comparison to traditional sheet autografts, cell spray autografting is a simple and cost-effective method, it avoids blister formation and needs a much smaller donor site, therefore reducing healing time and minimizing complications [12,140].
Epithelial cells can be sprayed uniformly, remain viable and proliferate on the wound bed, achieving a fast re-epithelialization, which is important for a good functionality and esthetic outcome, particularly in large wounds and in joint areas [12,21,23,140].
Moreover, compared to cultured autografts, non-cultured cell spray autografting has the advantage that grafting can be carried out in situ, immediately after cell isolation, avoiding a prolonged culture waiting time and the need to cover the wound with another type of graft or skin substitute during the culture time. Not culturing the cells also avoids progenitor and stem cells being lost because of their differentiation, an usual consequence of in vitro culture expansion [12]. Moreover, for deeper wounds, cell spray can be applied in combination with mesh grafting [20,21,141,142].
Cell suspensions, normally using lactate or normal saline solutions as carriers, can run off when applied over convex structures of the body, leading to an uneven coverage and distribution of living cells, reducing the number of viable cells that stay in contact with the wound [2,13,143]. Hence, they can be sprayed in combination with a hydrogel, such as fibrin, to ensure that cells remain attached to the wound surface. The first use of a combination of sprayed autologous cultured keratinocytes and sprayed autologous fibrin sealant for full-thickness wounds in pigs was reported by Grant et al. in 2002 [24]. Since then, many studies have combined epidermal cells with fibrin to be sprayed for different applications, and several spray products have been developed.
The delivery of cells via spray or aerosol has been evaluated in many studies. collects the main studies that utilized experimental cell-spray suspensions for burns and wound healing applications, normally making use of syringes with spray nozzles or airbrushes as the delivery devices.
The in vitro studies showed in demonstrated the feasibility of sprays as a delivery system for fibroblasts and keratinocytes. In these cases, cells maintained high viability and good proliferative capacity after being sprayed, although viability decreased as spraying pressure increased [13,121,125].
Navarro et al. carried out two studies using skin sprays in pigs. In one study, a greater wound re-epithelialization (in terms of a more complete confluence, epithelial coverage, and basal cell thickness) and a more organized dermal-epidermal junction were observed in wounds treated with non-cultured autologous pig keratinocytes when compared to wounds treated without cells. In the second study, they demonstrated the feasibility of delivering melanocytes (which are necessary for the repigmentation of full-thickness wounds) to the wound via spray, but they were not able to demonstrate macroscopic pigmentation, and further work was required [141,144].
The studies in patients used autologous keratinocytes to treat burns. Complete wound closure and re-epithelialization were achieved, with excellent functional and cosmetic outcomes, and no adverse events were observed in any patient. However, these studies did not include control groups to compare the results with other treatments [118,145,146].
Among all of these, only one in vitro study sprayed the cells in combination with a hydrogel, gellan gum. In this study, gellan gum produced a fluid gel with flexible viscoelastic properties that liquefies during spraying and self-structures post-spraying, showing no run-off when applied to a tilted surface. For clinical applications, this has the advantage of keeping the cells attached to the wound bed. Cells demonstrated high compatibility to the gellan gel, and good viability a week after the spraying process, but they showed a limited proliferation capacity in this gel, as cellular metabolic activity remained stable during the 7 days tested, in comparison to cells in culture medium, whose metabolic activity increased over time [13]. In contrast, for the rest of these studies [118,121,125,141,[144], [145], [146]], sprayed cells were suspended in culture medium, Hank's Balanced Salt Solution or Ringer's lactate solution, which could run off the wound [2,143]. In order to keep the cells attached to the wound bed, wounds were covered with dressings, dry gauze and bandages after the spraying process.
In 1993, Australian surgeon Dr. Fiona Wood developed an aerosol system to spray a layer of the patient's own skin stem cells onto the wounded area, called “spray-on skin”, consisting in taking a small biopsy from the patient's undamaged skin, suspending the skin cells in solution, and then spraying said solution over the burn wound. This permitted the coverage of an area 80 times the size of the biopsy [147,148]. Based on this, Avita Medical marketed a single-use, step-by-step kit, dubbed ReCell, which contains an enzyme solution to loosen and isolate the skin cells from the biopsy. Lastly, cells are suspended in a sodium lactate solution in a 10 mL syringe, and can either be dripped onto the wound (if the volume in the syringe is less than 2 mL) or sprayed through a spray nozzle attached to the syringe (if the volume in the syringe is greater than or equal to 2 mL). The cell suspension can be sprayed directly to partial-thickness wounds or over meshed autografts for full-thickness wounds. The ReCell non-cultured regenerative epidermal suspension includes keratinocytes, fibroblasts and melanocytes, a combination that may promote a better and faster healing outcome than one cell type alone. The FDA approved ReCell as the first spray-on skin treatment for burns in 2018 [122,147,[149], [150], [151]]. ReCell has been widely used for burns, but also for the treatment of other skin diseases and disorders such as vitiligo [152], piebaldism, leukoderma, treatment of scars, etc. [147]. Some studies using ReCell are summarized in . They report minimal cell breakdown during the spraying process, faster wound healing due to the reduced time of immediate post-harvesting application of the non-cultured cells, and a small harvested area for donor cells, with the downside of additional costs for the ReCell kits and a longer preparation time [2,122,153].
Professor Joerg C. Gerlach and colleagues employed a different enzymatic isolation technique from Wood et al. and ReCell to obtain the patient's own regenerative skin cells from a small biopsy, and in 2008 they developed a skin gun to spray the cells onto the wound [12,146]. The process takes about an hour and a half, and the electronically controlled pneumatic device has a 10 mL syringe where the cell solution is contained, which is pushed out of a 30G needle (with a 2.2 mL/min liquid flow) and into an air stream (3185 mL/min airflow), gently forming liquid droplets without injuring the cells [12]. The SkinGun was acquired by RenovaCare in 2013, and the patient's own skin stem cells solution received the name of ‘CellMist Solution’, which is placed in the SkinGun to be sprayed onto the patient's wound [154]. According to Esteban-Vives and Gerlach (2016), only a maximum wound area of 320 cm2 can be covered with the cells isolated using the ReCell kit, while their SkinGun's cell isolation method has no maximum wound coverage, and the patients treated with the SkinGun generally achieved complete re-epithelialization with good esthetic outcomes ( ) [12]. This device system requires further clinical evaluation and, at this time, it is an investigational system and is not available for general use or sale in the United States yet [154]. Although they are still currently seeking the FDA's approval, in August 2020 they did receive a conditional Investigational Device Exemption approval to start a clinical trial [155].
Both the ReCell syringe and the SkinGun only deliver the cells suspended in a lactate solution, which doesn't adhere to the wound by itself. Consequently, in order to avoid run-off, some studies have applied the cells harvested with the ReCell kit using a different spray device that allows them to co-deliver those cells with fibrin, like Johnstone et al. (2017), who used the Vivostat's Co-delivery System to co-deliver ReCell's cell suspension with Vivostat's fibrin to assist the anchoring of the viable keratinocytes onto the wound surface ( , ) [2]. Meanwhile, other cellular products, like the ones described next, include the use of fibrin to adhere the cells onto the wound.
KeraHeal® by Biosolution Co., Ltd. is a spray-type cultured epithelial autograft for burn wounds consisting of a suspension of autologous keratinocytes. A small biopsy is taken from the patient's undamaged skin and keratinocytes are isolated and cultured for their expansion for about two weeks. In order to prevent differentiation and to maintain a high take rate, once they reach 70–80% confluence, the pre-confluent keratinocytes are transferred into a vial at a cell density of 3·107 cells per 1 mL of Dulbecco's Modified Eagle's medium (DMEM) and are then sprayed over a wide meshed autograft that has just been applied to the wound bed. Right after the KeraHeal® suspension of cells has been sprayed, fibrin glue is often used to facilitate the attachment of epithelial cells to the wound [22,119,142,156]. KeraHeal® received product approval in 2006 by the Republic of Korea's Ministry of Food and Drug Safety [157]. shows two studies reporting positive results when using KeraHeal® to treat burn patients.
The downside of these autologous cell-spray methods is that the need for taking a biopsy, even if it is small, creates another wound and can produce more pain for the patient when they are already in such a delicate state. If the wounded area covers almost the entire body, it may even be difficult to find a suitable area for taking that small biopsy. These drawbacks can be avoided with the use of allogeneic cells, as long as they do not entail a risk of immune reaction and rejection.
Biosolution Co., Ltd. also offers KeraHeal®-Allo, based on allogeneic keratinocytes contained in syringes at a density of 2·107 cells per 1.5 mL of Poloxamer 407 and DMEM [158].
Although not commercialized, it is worth mentioning HP802-247 by Healthpoint Biotherapeutics, which was an investigational allogeneic spray-applied cell therapy using fibrin, used mostly to treat venous leg ulcers, and based on cryopreserved, growth-arrested fibroblasts and keratinocytes derived from neonatal foreskin. The fibrinogen solution was sprayed on the wound bed first, followed by the thrombin solution where the previously thawed cells were suspended. Allogeneic cells do not engraft, but according to in vitro studies, it was believed that HP802-247 released several cytokines and growth factors into the wound's micro-environment, which were expected to interact with the patient's own cells to encourage wound healing [26,159,160]. However, this product failed to demonstrate efficacy during phase 3 clinical trials. The main reason behind its failure is thought to be the batch to batch variability caused by changes in the phenotype of the cells, particularly for the keratinocytes, as a consequence of the age of the cell banks [161].
Therefore, there is a need for the development of new allogeneic products and therapies, which should carry out a thorough characterization and quality control of the cells before their application to patients.
The design, development and subsequent commercialization of a skin spray must be carried out in accordance with the legal and technical aspects defined by specific regulatory bodies in each jurisdiction. Based on whether the skin spray is made up of cells or not, its regulatory pathway to be marketed will be different. The acellular skin sprays are classified as medical devices [162,163]; and the cellular skin sprays, as advanced therapy medicinal products (ATMPs) [164]. Thus, the appropriate categorization of skin sprays is critical since it will define the specific requirements to be met and, therefore, the applicable rules governing their commercialization.
Acellular and cellular skin sprays must be authorized by competent authorities before appearing on the market. Their approval is based on the scientific evidence evaluation of the skin spray and on the quality data of manufacturing [165,166]. Both types of skin sprays must be manufactured under Good Manufacturing Practice (GMP) guidelines [166,167]. The data that should be provided in the authorization request for each type of skin spray will depend on its classification and its jurisdiction [164]. Thus, the regulatory framework of two of the main jurisdictions is described below: European Union (EU) and USA.
Regarding the legal requirements, an acellular skin spray for skin regeneration is considered as a medical device, because its activity should not be caused by chemical, pharmacological, immunological, or metabolic processes, or it would otherwise be considered as medicine [168].
Medical devices are classified according to their risk to the patient, based on their intended use, indications for use, the level of surgical invasion or body contact, structural characteristics, duration and activity [162]. Based on this classification, acellular cell sprays are considered as medical devices with the moderate risk being defined as class IIa (non-invasive) and IIb (invasive) devices under the European Medicines Agency (EMA) regulation in the EU [169] or class II (subject to general and special controls) under the FDA regulation in USA [170,171]. Nevertheless, if the medical device acts as a wound dressing, it must be classified as a class III due to the high risk of the device. In both jurisdictions it is necessary for the safety and efficacy data of an acellular skin spray to be submitted and evaluated by the health agency or a specific organization authorized for this purpose. The submission should include pre-clinical data based on in vitro and in vivo studies which determine the biocompatibility, local and systemic toxicology, activity, shelf life, microbiology, etc. of the medical devices [163,165]. These studies should be performed under Good Laboratory Practice (GLP). When the use of an acellular skin spray involves a high risk of illness or injury, clinical data should also be provided [172].
In the EU, for acellular skin sprays to be put into market as medical devices, they must fulfil the safety and effectiveness requirements, which are evaluated in a conformity assessment procedure by a notified body (NB). The NB evaluates the submitted technical documentation (scientific data and benefit-risk analysis) and audits the manufacturer's Quality Management System (QMS) [172]. This inspection is not required if the manufacturer has an International Organization for Standardization (ISO) 13485 certification [173]. Once the acellular skin spray is approved by the NB, it issues a declaration of conformity for manufacturers, allowing them to affix a CE marking to the medical devices, according to Regulation (EU) 2017/745 [174]. Acellular skin sprays that bear a CE mark can be then marketed in the EU and European Economic Area member states.
Access to the acellular skin sprays market in USA is regulated by the FDA, which requires that acellular skin sprays classified as Class II medical devices acquire clearance through the Premarket Notification 510(k) [175,176]. The application (administrative and scientific data) is reviewed by Accredited Persons and then, after the FDA receives this recommendation, the FDA must issue a final verdict. This pathway often does not require the provision of clinical data [163]. For acellular skin sprays considered as class III medical devices, a Premarket Approval (PMA) application is required, which is evaluated by FDA [177]. A PMA application must provide administrative, non-clinical laboratory studies and clinical data, in addition to a report that the manufacturer's quality control system has been audited. If the scientific evidence about safe and effective data are suitable enough, a marketing approval will be issued by the Center for Devices and Radiological Health of the FDA [171,178,179].
Regarding cellular skin sprays, they are considered medicinal products and represent an innovative strategy to repair, replace, restore, or regenerate wounds and skin injuries through their pharmacological, immunological, or metabolic activity [180]. The main characteristics of these types of products are that they must be made up of living cells (autologous, allogeneic or xenogeneic), and in addition, they might incorporate other components such as biomaterials, signaling molecules, non-viable cell derivatives, etc. [164,181].
Cellular skin sprays are subjected to different classifications, which are associated with the specific legislation where the medicinal product will be authorized. Thus, in the EU, cellular skin sprays are classified as tissue engineering products or combined advanced therapy medicinal products (incorporating a medical device), and therefore, they are considered ATMPs [182]. In USA, they are defined as human cells, tissues, and cellular and tissue-based products (HCT/Ps) [183]. The access to the market for the cellular skin sprays in both jurisdictions requires a prior marketing authorization (MA) by EMA and FDA, respectively [164,184]. The MA is based on the evaluation of the efficacy, safety, and quality data of the cellular skin spray. The request must include pre-clinical and clinical data similar to that described in the case of acellular skin sprays. In addition, data related to cell biological characteristics (viability, proliferation, migration, differentiation, identity, purity, potency, proliferative capacity, genomic stability, tumorigenicity, efficacy, and dosage) should be provided [166,185]. All this information must be compiled in a Common Technical Document (CTD).
In the EU, the procedure to request a MA for cellular skin sprays must be conducted under Regulation (EC) No. 1394/2007. This pathway involves the submission of a CTD, which is reviewed by the Committee for Advanced Therapies (CAT). Then, the CAT issues a draft opinion with its recommendation about the MA approval to the Committee for Medicinal Product for Human Use (CHMP). The CHMP is responsible for granting the final MA approval [186]. In some cases, the CHMP can issue a conditional MA and request additional post-authorization studies [164]. According to the USA regulatory framework, there are different regulatory pathways for the commercialization of cellular skin sprays, based on the risk of the product. If the risk is low or middle, they are classified as 361 HCT/Ps and marketed under Section 361 of the Public Health Service Act (PHSA) and under 21 Code of Federal Regulations (CFR) 1271 [183,187]. This pathway does not require any premarket approval but the administrative information, preclinical and clinical data should be provided [188]. However, if the risk is high, the cellular skin spray is classified as 351 HCT/Ps and regulated under Section 351 of the PHSA, and 21 CFR 1271, hence requiring a premarket review named Biologics License Application (BLA) [183,[187], [188], [189]]. In this regulatory pathway, data on the efficacy, safety, and quality of the cellular skin spray together with the administrative data of the sponsor must be submitted through a CTD to the Office of Tissues and Advanced Therapies (OTAT). When the evaluation is positive, the OTAT issues an approval for the marketing of the cellular skin spray [164].
Skin regeneration, especially in deep wounds, remains a challenge for many healthcare professionals. There are numerous and diverse strategies, but none of them provide a perfect solution. The use of sprays as a delivery system for hydrogels and cells allows the treatment of large wounded areas with much more ease and reduced time than other methods like traditional skin sheet grafts or bioengineered skin substitutes. There are some spray products for skin on the market, but cell spray grafting is mostly still under clinical evaluation.
Among the investigational and commercially available skin spray products, fibrin is the most widely used material, sometimes as a hydrogel alone, and other times as a carrier for cells. In general, cellular sprays for skin regeneration apply epidermal cells like keratinocytes and fibroblasts, but the incorporation of stem cells or growth factors could provide better stimulation for the healing of the skin. In the same way, the enrichment of fibrin hydrogels with other molecules, or the combined use of other materials present in the natural ECM (including components such as HA or collagen; or decellularized tissues, such as decellularized dermis) would most likely enhance the proliferation of cells at the wound bed matrix. Moreover, when formulating hydrogels to be used in skin sprays, it is necessary to carry out rheological studies to optimize the mechanical properties of the hydrogels. Viscosity is an important parameter to take into account, as hydrogels need to be able to pass through the spray nozzle without clogging, and then remain on the wound bed without running off. Furthermore, in the case of cellular sprays, hydrogels viscosity can affect cell viability. Since cells can be damaged when passing through the nozzle, an ideal hydrogel should have shear-thinning properties, having a high enough viscosity to maintain good mechanical properties, but reducing that viscosity when passing through the nozzle, and therefore reducing cellular damage. The viscoelastic properties are also important, as the materials sprayed on the skin need to be elastic and flexible to endure the body's movements. These parameters like viscosity and viscoelasticity can be adjusted by the combination of different materials or biopolymers at different concentrations, and thus the formulation of hydrogels using diverse materials that mimic the natural ECM of the skin and have satisfactory mechanical properties is an important factor for skin sprays.
When it comes to the translation of skin sprays to clinical practice, there are very few clinical trials studying cellular regenerative spray products, but many are acellular spray products using drugs like corticosteroids to relieve the irritation caused by some conditions like psoriasis. Therefore, it is important to develop more regenerative spray products for skin containing hydrogels and cells, and then translate them to clinical practice, which could benefit thousands of patients around the world suffering from burns, chronic wounds, and other skin conditions.
Finally, improved spraying devices should be developed. To date, only a small number of devices have been approved for commercialization and clinical use. Many preclinical studies utilize simple syringes with spray pumps, and they do not give much focus to the spraying parameters such as the pressure, distance, angle or volume, and therefore, these studies may hardly be reproducible, as these parameters can affect results significantly. New spraying devices should be able to regulate these parameters, especially the pressure (for example by adding a manometer or pressure gauge to the device) and volume. It is essential that new studies optimize these parameters for the device and the material used. Currently, most devices are designed to spray only one material at a time, with the exception of Baxter's double syringes for TISSEEL and ARTISS or the Vivostat's Co-Delivery system, but these are intended for the use of their own fibrin products. In case of wanting to deliver other types of hydrogels, it would be interesting to look at new devices that permit the spray delivery of two or more different materials or gels, simultaneously or subsequently. For example, in the case of hydrogels needing a gelling agent, it would be desirable if both materials could be sprayed simultaneously, so as to gel right as the material is applied on the wound (i.e. this is the case of the TISSEEL and ARTISS double syringes, spraying fibrinogen and thrombin to form fibrin, but it could also be used to spray other materials such as alginate and calcium). The use of diverse materials, such as different composite hydrogels sprayed in subsequent layers, could also be useful to simulate the different layers of the skin, and could consequently, greatly improve the healing of deep wounds.
This research was funded by Fundación Mutua Madrileña (FMM-AP17196-2019), the Consejería de Economía, Conocimiento, Empresas y Universidad de la Junta de Andalucía (ERDF funds, projects B-CTS-230-UGR18, PY18-2470, A-CTS-180-UGR20, SOMM17-6109, PYC20 RE 015 UGR and P18-FR-2465), and by the Ministry of Economy and Competitiveness, Instituto de Salud Carlos III, ERDF funds (DTS21/00098 and DTS19/00145).
Paula Pleguezuelos-Beltrán: Conceptualization, Investigation, Writing – Original Draft and Editing, Visualization; Patricia Gálvez-Martín: Writing – Original Draft, Review and Editing; Daniel Nieto-García: Writing – Review and Editing; Juan Antonio Marchal: Writing – Review and Editing, Supervision; Elena López-Ruiz: Conceptualization, Writing – Review and Editing, Supervision. All authors have read and approved the final manuscript.
The authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
Peer review under responsibility of KeAi Communications Co., Ltd.
Appendix ASupplementary data to this article can be found online at https://doi.org/10.1016/j.bioactmat.2022.02.023.
The following is the Supplementary data to this article:
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