**6. Regulatory considerations**

(mutTGFβ3) was generated by ablating its binding site for the latency‐associated TGFβ‐ binding protein (LTBP‐1) [78]. A localized intradermal transduction using a lentiviral vector expressing the mutTGFβ3 in a mouse skin wounding model was demonstrated to reduce reepithelialization density and fibroblast/myofibroblast trans‐differentiation within the wound area. Both of which reduced scar tissue formation (**Figure 2**). Using a noninvasive imaging system, the kinetics of luciferase gene expression was studied when delivered in an adenoviral vector (replication‐deficient adenovirus, Ad5). A peak of gene expression occurred at 7 days after delivery [79]. The esophageal cancer‐related gene‐4 (Ecrg4) delivering a viral‐ mediated gene was evaluated in a cutaneous wound healing model [80]. Both Ecrg4 mRNA and its protein product were localized to the epidermis, dermis, and hair follicles of healthy

Gene delivery using adenoviral vectors in tissue regeneration is hindered by a short duration of transgene expression. A fibrin scaffold was used to enhance delivery of the adenovirus to a wound site, precluding the need for high repeated doses [81]. An anti‐fibrotic interfering RNA (RNAi) delivery system using exogenous microRNA (miR)‐29B was proposed to modulate ECM remodeling following cutaneous injury. A collagen scaffold was used as the carrier of (miR)‐29B. The mRNA expressions of collagen type I and collagen type III were reduced up to 2 weeks after fibroblasts culture. *In vivo* evaluation in full‐thickness wounds treated with miR‐29B delivery revealed that collagen type III/I ratio and matrix metalloproteinase (MMP)‐ 8 to TIMP‐1 ratio were improved [82]. Porous (100 and 60 μm) and nonporous (n‐pore) hyaluronic acid‐MMP hydrogels with encapsulated reporter (pGFPluc) or proangiogenic (pVEGF) plasmids are used as a scaffold‐mediated gene delivery [83]. Alginate‐DNA gels were used to treat diabetic wounds, which provided sustained release of bioactive factors, such as neuropeptides and VEGF [13]. Silver nanoparticles (AgNPs) can be further augmented for gene delivery applications. The biofunctionalized stable AgNPs with good DNA‐binding ability for efficient transfection and minimal toxicity were developed [84]. Polyethylene glycol (PEG)‐ stabilized chitosan‐g‐polyacrylamide was used to modify AgNPs. To enhance the efficiency of gene transfection, the Arg‐Gly‐Asp‐Ser (RGDS) peptide was immobilized on the surface of AgNPs. The transfection efficiency of AgNPs increased significantly after immobilization of the RGDS peptide reaching up to 42 ± 4% and 30 ± 3% in HeLa and A549 cells, respectively. The transfection efficiency was significantly higher than 34 ± 3% and 23 ± 2%, respectively, with the

For treating diabetic patients with a threat of limb amputations, genes of various growth factors have been proposed in delivery systems. A simple nonviral gene delivery using minicircle plasmid DNA encoding VEGF was combined with an arginine‐grafted cationic dendrimer PAM‐RG4 [85]. Mouse ASCs were transfected with DNA plasmid encoding VEGF or green fluorescent protein (GFP) using biodegradable poly (β‐amino) esters (PBAE). Cells transfected with Lipofectamine™ 2000, a commercially available transfection reagent, were included as controls. ASCs transfected using PBAEs showed an enhanced transfection efficiency and 12– 15‐folds higher VEGF production compared with the controls (\**P* < 0.05) [86]. Keratinocyte

mouse skin.

**5.2. Nonviral vectors in gene delivery**

84 Wound Healing - New insights into Ancient Challenges

use of polyethylenimine (PEI, 25 kDa).

The major concerns of commercialization of drug/protein/cell/gene delivery wound dressings are the complicated registration process, specifically regulatory approval, protocol consider‐ ation, and clinical trial process. Among all the parameters of delivery wound dressings, the type and source of the materials (e.g., human and animal origin) are critical to the regulatory approval process. A product composed of two or more regulated components, that is, drug/ device, biologic/device, drug/biologic, or drug/device/biologic, that are physically, chemically, or otherwise combined or mixed and produced as a single entity is defined as a combination product [90]. The FDA (Food and Drug Administration, United States) regulation of a combination product (e.g., delivery system for wound healing) is mainly determined by the component with the primary mode of action. According to the classification of the product, the clinical trials (for premarket approval, PMA) must provide valid scientific evidence of safety and efficacy to support the indicated use of the wound healing delivery systems. Generally, preclinical studies contain toxicity studies and animal model evaluations. Delivery systems of drugs, bioactive proteins, cells, and genes in wound healing and nanomedicine should test their biocompatibility according to ISO 10993, including dermal irritation, dermal sensitization, cytotoxicity, acute systemic toxicity, hemocompatibility/hemolysis, pyrogenicity, mutagenicity studies, subchronic toxicity, chronic toxicity, and immunogenic potential [91]. Good clinical practices (GCPs) are the standards for designing, conducting, recording, and reporting clinical trials required for Class III medical devices.

For example, autologous stem cells are under clinical trial and are effective in ulcer healing and angiogenesis. However, translating delivery of stem cell application in *in vitro* and *in vivo* experiments from animal models to human clinical trials is still in its infancy. Preclinical studies suggest that cell delivery systems represent an effective and safe therapeutic strategy in the treatment of nonhealing wounds. More clinical studies on human subjects, including better data management of the patients and long‐term follow‐up of the patients' conditions, are necessary. Improved stem cell delivery vehicles in large‐scale human clinical trials may be promising for diabetics with foot ulcers. There are no serious complications or side effects, but its therapeutic mechanisms, effects, and standardization still require further research [92]. While delivery system‐based products offer increasingly important strategies for managing complex wounds, potential drawbacks include the risks of infectious agent transfer and immunological rejection. The manufacturing process, transport, and storage of delivery systems in wound healing are major cost implications; thus, their current clinical use remains limited [93]. Many current clinical trials are placing a high emphasis on addressing safety issues in all stem cell therapies, including stem cell delivery in wound healing [94]. The serious adverse effects of stem cell delivery are mainly immune response and tumorigenic potential. Delivery systems used in cell therapy encompass four main approaches, which are systemic administration, injection, topical, and local deliveries. Localized delivery of cells in wound healing is an optimal delivery approach for wound treatments [95]. Nonimmunogenic, nontoxic, biodegradable, and biocompatible biomaterials have been developed as carriers of stem cells that can protect cells and improve wound healing. However, clinical use of stem cells, for example, allogeneic EPCs, is currently inhibited by the risk of immunogenicity and tumorigenicity. To modulate the immune response, mesenchymal stromal cells or umbilical cord blood is already used in clinical trials, but definitive results are still pending. MSCs are known to be hypoimmunogenic [96]. Current challenges are standardized and quality‐ controlled cell therapy, the differentiation of MSCs to unwanted tissue, and potential tumori‐ genicity [94]. MSCs have been applied clinically for the treatment of diabetic wounds. Long *in vitro* expansion time and multiple handling procedures are barriers for its clinical application and increase the chances of infection [97]. Autologous induced pluripotent stem cells are nonimmunogenic and can be a promising cell source used in wound healing [98]. By compar‐ ison, clinical use of allogeneic cells is more complex and requires additional regulatory, legal, and safety hurdles to be overcome [99]. All things considered, the future prospects for the utilization of both autologous and allogeneic cells in cell delivery systems are bright. In the United States, there are three regulatory processes for the registration of wound healing delivery systems [100]. Only wound dressing with lower complexity and risk that is substan‐ tially equivalent to a marketed "predicate" device may be cleared through the 510(k) premarket notification process. In another words, those types of wound dressings are classified as Class I medical device. Clinical data are typically not needed for 510(k) clearance of Class II medical devices. Higher‐risk Class III medical devices typically require premarket approval (PMA). In summary, the regulatory processes are depending on multiple factors including the device's classification, the availability of a substantially equivalent predicate, and the level of risk. Before commercialization, investigational devices maybe clinically investigated within the USA through the investigational device exemption (IDE) process, which is a request to conduct clinical research on an investigational device with "significant" risk in the **United States**.

User fees are required with the submissions of 510(k) premarket notifications and PMA application in the **United States**. Recently, Health Canada released a consultation document that discusses the cost recovery (user fee) framework which shows the basis for accountability at Health Canada for the review process [101]. Essentially, the fees "guarantee" a certain level of service from Health Canada—for instance, specifying the target number of days in which Health Canada will process different types of applications. If the targets are not met, that is, if "performance" does not meet the established standard, the entity being charged the user fee will have their future fees reduced by a corresponding amount. Providing a framework for registration approval globally of delivery wound dressings would translate those delivery systems studied from the laboratory investigation stage to clinical use, which will benefit patients' quality of life.
