Acceleration of Diabetic Wound Healing with PHD2- and miR-210-Targeting Oligonucleotides.

In diabetes-associated chronic wounds, the normal response to hypoxia is impaired and many cellular processes involved in wound healing are hindered. Central to the hypoxia response is hypoxia-inducible factor-1α (HIF-1α), which activates multiple factors that enhance wound healing by promoting cellular motility and proliferation, new vessel formation, and re-epithelialization. Prolyl hydroxylase domain-containing protein 2 (PHD2) regulates HIF-1α activity by targeting it for degradation under normoxia. HIF-1α also upregulates microRNA miR-210, which in turn regulates proteins involved in cell cycle control, DNA repair, and mitochondrial respiration in ways that are antagonistic to wound repair. We have identified a highly potent short synthetic hairpin RNA (sshRNA) that inhibits expression of PHD2 and an antisense oligonucleotide (antimiR) that inhibits miR-210. Both oligonucleotides were chemically modified for improved biostability and to mitigate potential immunostimulatory effects. Using the sshRNA to silence PHD2 transcripts stabilizes HIF-1α and, in combination with the antimiR targeting miR-210, increases proliferation and migration of keratinocytes in vitro. To assess activity and delivery in an impaired wound healing model in diabetic mice, PHD2-targeting sshRNAs and miR-210 antimiRs both alone and in combination were formulated for local delivery to wounds using layer-by-layer (LbL) technology. LbL nanofabrication was applied to incorporate sshRNA into a thin polymer coating on a Tegaderm mesh. This coating gradually degrades under physiological conditions, releasing sshRNA and antimiR for sustained cellular uptake. Formulated treatments were applied directly to splinted full-thickness excisional wounds in db/db mice. Cellular uptake was confirmed using fluorescent sshRNA. Wounds treated with a single application of PHD2 sshRNA or antimiR-210 closed 4 days faster than untreated wounds, and wounds treated with both oligonucleotides closed on average 4.75 days faster. Markers for neovascularization and cell proliferation (CD31 and Ki67, respectively) were increased in the wound area following treatment, and vascular endothelial growth factor (VEGF) was increased in sshRNA-treated wounds. Our results suggest that silencing of PHD2 and miR-210 either together or separately by localized delivery of sshRNAs and antimiRs is a promising approach for the treatment of chronic wounds, with the potential for rapid clinical translation.

The Innate Immune System in Acute and Chronic Wounds.

Significance: This review article provides an overview of the critical roles of the innate immune system to wound healing. It explores aspects of dysregulation of individual innate immune elements known to compromise wound repair and promote nonhealing wounds. Understanding the key mechanisms whereby wound healing fails will provide seed concepts for the development of new therapeutic approaches. Recent Advances: Our understanding of the complex interactions of the innate immune system in wound healing has significantly improved, particularly in our understanding of the role of antimicrobials and peptides and the nature of the switch from inflammatory to reparative processes. This takes place against an emerging understanding of the relationship between human cells and commensal bacteria in the skin. Critical Issues: It is well established and accepted that early local inflammatory mediators in the wound bed function as an immunological vehicle to facilitate immune cell infiltration and microbial clearance upon injury to the skin barrier. Both impaired and excessive innate immune responses can promote nonhealing wounds. It appears that the switch from the inflammatory to the proliferative phase is tightly regulated and mediated, at least in part, by a change in macrophages. Defining the factors that initiate the switch in such macrophage phenotypes and functions is the subject of multiple investigations. Future Directions: The review highlights processes that may be useful targets for further investigation, particularly the switch from M1 to M2 macrophages that appears to be critical as dysregulation of this switch occurs during defective wound healing.

Psoriatic keratinocytes are resistant to tumor necrosis factor alpha's induction of mRNA for the NMDA-R2C subunit.

Psoriatic individuals demonstrate accelerated healing and the Koebner phenomenon, suggesting that psoriatic proliferation of keratinocytes is not inhibited appropriately after skin injury. Serial analysis of gene expression in TNFα-exposed keratinocytes shows the greatest alteration in expression of NMDA-R2C. Expression of the NMDA receptor is altered in diseased skin containing TNFα, and TNFα plays a prominent role in psoriasis. An abnormality in induction of NMDA-R2C by TNFα in psoriatic keratinocytes may explain their lack of growth inhibition. We compared the capacity of TNFα to induce expression of NMDA-R2C in normal and psoriatic (involved and uninvolved) keratinocytes in vitro. After 72 h of incubation with TNFα, normal keratinocytes demonstrated a significant induction of NMDA-R2C mRNA compared with control cultures, whereas psoriatic keratinocytes showed no induction. In an in vitro model of wounding (scratches on monolayers), TNFα inhibited migration/proliferation of keratinocytes only at the edge of NMDA-R2C expressing wounded monolayers of normal keratinocytes.

Hair regrowth following a Wnt- and follistatin containing treatment: safety and efficacy in a first-in-man phase 1 clinical trial.

Research has shown the importance of follistatin, Wnt 7a, and wound healing growth factors on the stimulation of bulge cells and inter-follicular stem cells to induce hair growth. We have studied the effects of a bioengineered, non-recombinant, human cell-derived formulation, termed Hair Stimulating Complex (HSC), containing these factors to assess its hair growth activity in male pattern baldness. HSC showed in vitro Wnt activity and contained follistatin, KGF, and VEGF. The clinical study was a double-blind, placebo-controlled, randomized single site trial and was designed to evaluate safety of the HSC product and assess efficacy in stimulating hair growth. All 26 subjects tolerated the single, intradermal injection of HSC procedures well, and no signs of an adverse reaction were reported. Histopathological evaluation of the treatment site biopsies taken at 22 and 52 weeks post-treatment revealed no abnormal morphology, hamartomas, or other pathological responses. Trichoscan image analysis of HSC-treated sites at 12 and 52 weeks showed significant improvements in hair growth over the placebo. At the initial 12-week evaluation period, HSC-treated sites demonstrated an increase in hair shaft thickness (6.3%±2.5% vs. -0.63%±2.1%; P=0.046), thickness density (12.8%±4.5% vs. -0.2%±2.9%; P=0.028), and terminal hair density (20.6±4.9% vs. 4.4±4.9%; P=0.029). At one year, a statistically significant increase in total hair count (P=0.032) continued to be seen. These results demonstrate that a single intradermal administration of HSC improved hair growth in subjects with androgenetic alopecia and is a clinical substantiation of previous preclinical research with Wnts, follistatin, and other growth factors associated with wound healing and regeneration.

Use of gelatin sponges in Mohs micrographic surgery defects and staged melanoma excisions: a novel approach to secondary wound healing.

BACKGROUND/AIMS: surgical closure or reconstruction is commonly used to treat wounds generated by Mohs micrographic surgeries (MMS) and staged melanoma excisions, which may result in contractures and scarring. The authors' objective was to determine the value of using gelatin sponges to promote secondary intention healing for surgical defects after MMS and staged melanoma excisions. METHODS: sixty-four surgeries from 54 predominantly elderly patients (median age=76 years) were treated with gelatin sponges to promote healing by secondary intention in this prospective investigation. Patients rated their satisfaction with outcomes on a scale of 1 (highly dissatisfied) to 5 (highly satisfied). RESULTS: in all patients, the wounds healed within four to 16 weeks (median=five weeks). Forty-five patients were highly satisfied with their results (mean score=4.9). CONCLUSION: healing by secondary intention using gelatin sponges was associated with improved hemostasis, excellent cosmesis and a high level of patient satisfaction.

Tissue-engineered skin substitutes in regenerative medicine.

Recent advance in cellular tissue-engineered skin constructs have refined the applications already commercially available, in particular, by the use of genetically modified cells to enhance their properties on the treatment of wounds and to ease the application of epidermis using sprayed keratinocytes. This approach lends itself to use of chimeric epidermis, cultured allogeneic cells, to provide short-term coverage, together with minimally cultured autologous cells for long-term repair. Experimental models of skin include pathological conditions, phenomena such as aging and organogenesis, as in the hair follicle grown from isolated cells in vitro. The recent development of induced pluripotent stem cells raises the possibility of realizing the dream of skin and even limb regeneration shown by animals such as the salamander.

Nonlinear modeling of venous leg ulcer healing rates.

BACKGROUND: The purpose of this manuscript was to determine whether the change in wound surface area over time could be described through nonlinear mathematics. METHODS: We studied 3,588 serial wound tracings of 338 venous leg ulcers (VLUs) that had been followed during a controlled, prospective, randomized trial of two topical wound treatments. RESULTS: A majority (72%) of VLUs exhibited surface area reduction via an exponential decay model, particularly during the early stages of healing. These results were consistent with the mechanics of wound contraction and epithelial cell proliferation, supported by the higher frequency at which exponential surface area reduction associated with full wound closure (35% of wounds that fit the exponential model healed vs. 21% of wounds that did not fit the exponential model completely healed during the study period, p = 0.018). Goodness-of-fit statistics suggested that much of the individual variation in healing could be described as nonlinear variation from the exponential model. CONCLUSION: We believe that parameter estimates from a mathematical model may provide a more accurate quantification of wound healing rates, and that similar models may someday reach routine use in comparing the efficacy of various treatments in routine practice and in product registration trials.

Skin tissue engineering.

The major applications of tissue-engineered skin substitutes are in promoting the healing of acute and chronic wounds. Several approaches have been taken by commercial companies to develop products to address these conditions. Skin substitutes include both acellular and cellular devices. While acellular skin substitutes act as a template for dermal formation, this discussion mainly covers cellular devices. In addressing therapeutic applications in tissue engineering generally, a valuable precursor is an understanding of the mechanism of the underlying pathology. While this is straightforward in many cases, it has not been available for wound healing. Investigation of the mode of action of the tissue-engineered skin substitutes has led to considerable insight into the mechanism of formation, maintenance and treatment of chronic wounds. Four aspects mediating healing are considered here for their mechanism of action: (i) colonization of the wound bed by live fibroblasts in the implant, (ii) the secretion of growth factors, (iii) provision of a suitable substrate for cell migration, particularly keratinocytes and immune cells, and (iv) modification of the immune system by secretion of neutrophil recruiting chemokines. An early event in acute wound healing is an influx of neutrophils that destroy planktonic bacteria. However, if the bacteria are able to form biofilm, they become resistant to neutrophil action and prevent reepithelialization. In this situation the wound becomes chronic. In chronic wounds, fibroblasts show a senescence-like phenotype with decreased secretion of neutrophil chemoattractants that make it more likely that biofilms become established. Treatment of the chronic wounds involves debridement to eliminate biofilm, and the use of antimicrobials. A role of skin substitutes is to provide non-senescent fibroblasts that attract and activate neutrophils to prevent biofilm re-establishment. The emphasis of the conclusion is the importance of preventing contaminating bacteria becoming established and forming biofilms.

Keratinocyte migration, proliferation, and differentiation in chronic ulcers from patients with diabetes and normal wounds.

Epithelialization of normal acute wounds occurs by an orderly series of events whereby keratinocytes migrate, proliferate, and differentiate to restore barrier function. The keratinocytes in the epidermis of chronic ulcers fail to execute this series of events. To better understand the epithelial dynamics of chronic ulcers, we used immunohistochemistry to evaluate proliferation, differentiation, adhesion, and migration in keratinocytes along the margin of chronic ulcers from patients with diabetes mellitus. We compared these features with keratinocytes from the migrating epithelial tongues of acute incisional and excisional wounds from normal volunteers. Keratinocytes at the chronic ulcer edge are highly proliferative (Ki67 proliferation marker), have an activated phenotype (K16), do not stain for keratins involved in epidermal differentiation (K10 and K2), and show a reduced expression of LM-3A32 (uncleaved, precursor of the alpha3 chain of laminin 5), a key molecule present on migrating epithelium. In contrast, keratinocytes in normal acute wound migrating epithelium do not express the proliferation marker Ki67 but do express K10, K2, and LM-3A32. A better understanding of molecular mechanisms involved in keratinocyte migration may lead to molecular targets for therapies for impaired wound healing.

Inhibition of keratinocyte migration by lipopolysaccharide.

A critical process in cutaneous wound healing is reepithelialization by keratinocytes that closes the breach in the epidermis. Chronic wounds fail to reepithelialize despite the presence of activated and proliferative keratinocytes around the wound perimeter. This type of wound is generally colonized to a greater or lesser extent by bacteria. This study examines the possibility that bacterial products might directly inhibit keratinocyte migration. Using conventional scratch assays, we observed a dose-dependent inhibition of keratinocyte migration by lipopolysaccharide (LPS) derived from either Pseudomonas aeruginosa or Escherichia coli. Although the P. aeruginosa preparation appeared to be slightly more inhibitory, both gave half-maximal inhibition at 0.5-0.6 ng/mL. Migration of fibroblasts was not inhibited. The result could not be attributed to a cytotoxic effect of the LPS. LPS inhibition of migration was relieved by neutralizing antibodies to toll-like receptors (TLR), 40% by anti-TLR2 and 75% by anti-TLR4. We conclude that keratinocyte migration is inhibited by bacterial products, detected through TLR4 and also through TLR2. Because chronic wounds always show some presence of bacteria, these findings provide a possible explanation for the lack of healing found in ulcers.

Commercial considerations in tissue engineering.

Tissue engineering is a field with immense promise. Using the example of an early tissue-engineered skin implant, Dermagraft, factors involved in the successful commercial development of devices of this type are explored. Tissue engineering has to strike a balance between tissue culture, which is a resource-intensive activity, and business considerations that are concerned with minimizing cost and maximizing customer convenience. Bioreactor design takes place in a highly regulated environment, so factors to be incorporated into the concept include not only tissue culture considerations but also matters related to asepsis, scaleup, automation and ease of use by the final customer. Dermagraft is an allogeneic tissue. Stasis preservation, in this case cryopreservation, is essential in allogeneic tissue engineering, allowing sterility testing, inventory control and, in the case of Dermagraft, a cellular stress that may be important for hormesis following implantation. Although the use of allogeneic cells provides advantages in manufacturing under suitable conditions, it raises the spectre of immunological rejection. Such rejection has not been experienced with Dermagraft. Possible reasons for this and the vision of further application of allogeneic tissues are important considerations in future tissue-engineered cellular devices. This review illustrates approaches that indicate some of the criteria that may provide a basis for further developments. Marketing is a further requirement for success, which entails understanding of the mechanism of action of the procedure, and is illustrated for Dermagraft. The success of a tissue-engineered product is dependent on many interacting operations, some discussed here, each of which must be performed simultaneously and well.

Morphological evidence for the role of suprabasal keratinocytes in wound reepithelialization.

The process by which wounds reepithelialize remains controversial. Two models have been proposed to describe reepithelialization: the "sliding" model and the "rolling" model. In the "sliding" model, basal keratinocytes are the principal cells responsible for migration and wound closure. In this model, basal and suprabasal keratinocytes remain strongly attached to leading edge basal keratinocytes and are then passively dragged along as a sheet. The "rolling" model postulates that basal keratinocytes remain strongly attached to the basement membrane zone while suprabasal keratinocytes at the wound margin are activated to roll into the wound site. The purpose of this study was to determine which populations of keratinocytes are actively involved in reepithelialization. We evaluated expression of keratins K14, K15, K10, K2e, and K16 as well as the proliferation marker Ki67 in the migrating tongue of normal human incisional 1-hour to 28-day wounds and normal human 3 mm diameter excisional 1- to 7-day wounds. Our results show dramatic changes in phenotype and protein expression of keratins K10, K2e, K14, K15, and K16 in suprabasal keratinocytes in response to injury. We conclude that this large population of suprabasal keratinocytes actively participates in wound closure.

Tissue-engineered skin substitutes.

The last two years have seen new tissue-engineered skin substitutes come onto the market and begin to resolve the various roles to which each is best suited. It is becoming evident that some of the very expensive cell-based products have cost-benefit advantage despite their high price and are valuable within the restricted applications for which they are intended. The use of skin substitutes for testing purposes has extended from epidermal keratinocytes to other integumentary epithelia and into preparations containing multiple cell types in which reactions resulting from paracrine interactions can be examined. Challenges remain in the application of gene therapy techniques to skin substitutes, both the control of transgene expression and in the selection of suitable genes to transfect. A coming challenge is the production of tissue-engineered products without the use of animal products other than human cells. A challenge that may be diminishing is the importance of acute rejection of allogeneic tissue-engineered skin substitutes.