Wound healing is a complex process that requires the intervention of cytoactive factors. The one-time application of soluble factors to a wound bed does not maintain a steady, sufficient concentration. Here we investigated the benefits of anchoring a factor in a wound bed via a tether to endogenous collagen. We used a collagen-mimetic peptide (CMP) as a pylon. The CMP binds to damaged but not intact collagen and thus localizes a pendant cytoactive factor in the regions of a wound bed that require intervention. As a model factor, we chose substance P, a peptide of the tachykinin family that promotes wound healing. Using splinted wounds in db/db mice, we found that the one-time application of a CMP-substance P conjugate enhances wound healing compared to unconjugated substance P and other controls. Specifically, all 16 wounds treated with the conjugate closed more thoroughly and, did so with extensive re-epithelialization and mitigated inflammatory activity. These data validate a simple and general strategy for re-engineering wound beds by the integration of beneficial cytoactive factors. Copyright © 2014 John Wiley & Sons, Ltd.

The persistence of bacterial biofilms in chronic wounds delays wound healing. Although Ga(3+) can inhibit or kill biofilms, precipitation as Ga(OH)3 has prevented its use as a topical wound treatment. The design of a microfilm construct comprising a polyelectrolyte film that releases noncytotoxic concentrations of Ga(3+) over 20 d and a dissolvable micrometer-thick film of polyvinylalcohol that enables facile transfer onto biomedically important surfaces is reported. By using infrared spectroscopy, it is shown that the density of free carboxylate/carboxylic acid and amine groups within the polyelectrolyte film regulates the capacity of the construct to be loaded with Ga(3+) and that the density of covalent cross-links introduced into the polyelectrolyte film (amide-bonds) controls the release rate of Ga(3+) . Following transfer onto the wound-contact surface of a biologic wound dressing, an optimized construct is demonstrated to release ≈0.7 μg cm(-2) d(-1) of Ga(3+) over 3 weeks, thus continuously replacing Ga(3+) lost to precipitation. The optimized construct inhibits formation of P. aeruginosa (two strains; ATCC 27853 and PA01) biofilms for up to 4 d and causes pre-existing biofilms to disperse. Overall, this study provides designs of polymeric constructs that permit facile modification of the wound-contacting surfaces of dressings and biomaterials to manage biofilms.

Accurate and rapid assessment of the healing status of a wound in a simple and noninvasive manner would enable clinicians to diagnose wounds in real time and promptly adjust treatments to hasten the resolution of nonhealing wounds. Histologic and biochemical characterization of biopsied wound tissue, which is currently the only reliable method for wound assessment, is invasive, complex to interpret, and slow. Here we demonstrate the use of Raman microspectroscopy coupled with multivariate spectral analysis as a simple, noninvasive method to biochemically characterize healing wounds in mice and to accurately identify different phases of healing of wounds at different time-points. Raman spectra were collected from "splinted" full thickness dermal wounds in mice at 4 time-points (0, 1, 5, and 7 days) corresponding to different phases of wound healing, as verified by histopathology. Spectra were deconvolved using multivariate factor analysis (MFA) into 3 "factor score spectra" (that act as spectral signatures for different stages of healing) that were successfully correlated with spectra of prominent pure wound bed constituents (i.e., collagen, lipids, fibrin, fibronectin, etc.) using non-negative least squares (NNLS) fitting. We show that the factor loadings (weights) of spectra that belonged to wounds at different time-points provide a quantitative measure of wound healing progress in terms of key parameters such as inflammation and granulation. Wounds at similar stages of healing were characterized by clusters of loading values and slowly healing wounds among them were successfully identified as "outliers". Overall, our results demonstrate that Raman spectroscopy can be used as a noninvasive technique to provide insight into the status of normally healing and slow-to-heal wounds and that it may find use as a complementary tool for real-time, in situ biochemical characterization in wound healing studies and clinical diagnosis.

Silver is a widely used antimicrobial agent, yet, when impregnated in macroscopic dressings, it stains wounds, can lead to tissue toxicity, and can inhibit healing. Recently, polymeric nanofilms containing silver nanoparticles were reported to exhibit antimicrobial activity at loadings and release rates of silver that are 100× lower than conventional dressings. Here, fabrication of composite microfilm constructs that provide a facile way to transfer the silver-loaded polymeric nanofilms onto wounds in vivo is reported. The construct is fabricated from a silver nanoparticle-loaded polymeric nanofilm that is laminated with a micrometer-thick-soluble film of polyvinylalcohol (PVA). When placed on a moist wound, the PVA dissolves, leaving the silver-loaded nanofilm immobilized on the wound-bed. In vitro, the immobilized nanofilms release <1 μg cm(-2) d(-1) of silver over 30 d from skin dermis and they kill 5 log10 CFUs of Staphylococcus aureus in 24 h. In mice, wounds inoculated with 10(5) CFU S. aureus presented up to 3 log10 less bacterial burden when treated with silver/nanofilms for 3 d, as compared to unmodified wounds. In uncontaminated wounds, silver/nanofilms allow normal and complete wound closure by re-epithelialization. Dissolvable microfilm constructs may overcome key limitations associated with current uses of silver in wound healing.

Silver is a commonly used topical antimicrobial. However, technologies to immobilize silver at the wound surface are lacking, while currently available silver-containing wound dressings release excess silver that can be cytotoxic and impair wound healing. We have shown that precise concentrations of silver at lower levels can be immobilized into a wound bed using a polyelectrolyte multilayer attachment technology. These silver nanoparticle-impregnated polyelectrolyte multilayers are noncytotoxic yet bactericidal in vitro, but their effect on wound healing in vivo was previously unknown. The purpose of this study was to determine the effect on wound healing of integrating silver nanoparticle/polyelectrolyte multilayers into the wound bed. A full-thickness, splinted, excisional murine wound healing model was employed in both phenotypically normal mice and spontaneously diabetic mice (healing impaired model). Gross image measurements showed an initial small lag in healing in the silver-treated wounds in diabetic mice, but no difference in time to complete wound closure in either normal or diabetic mice. Histological analysis showed modest differences between silver-treated and control groups on day 9, but no difference between groups at the time of wound closure. We conclude that silver nanoparticle/polyelectrolyte multilayers can be safely integrated into the wound beds of both normal and diabetic mice without delaying wound closure, and with transient histological effects. The results of this study suggest the feasibility of this technology for use as a platform to affect nanoscale wound engineering approaches to microbial prophylaxis or to augment wound healing.

Topical application of platelet-derived growth factor-BB (PDGF-BB) is considered to accelerate tissue repair of impaired chronic wounds. However, the vast literature is plagued with conflicting reports of its efficacy in animal models and this is often influenced by a wide array of experimental variables making it difficult to compare the results across the studies. To mitigate the confounding variables that influence the efficacy of topically applied PDGF-BB, we used a controlled full thickness splinted excisional wound model in db/db mice (type 2 diabetic mouse model) for our investigations. A carefully-defined silicone-splinted wound model, with reduced wound contraction, controlled splint and bandage maintenance, allowing for healing primarily by reepithelialization was employed. Two splinted 8 mm dorsal full thickness wounds were made in db/db mice. Wounds were topically treated once daily with either 3 µg PDGF-BB in 30 µl of 5% PEG-PBS vehicle or an equal volume of vehicle for 10 days. Body weights, wound contraction, wound closure, reepithelialization, collagen content, and wound bed inflammation were evaluated clinically and histopathologically. The bioactivity of PDGF-BB was confirmed by in vitro proliferation assay. PDGF-BB, although bioactive in vitro, failed to accelerate wound healing in vivo in the db/db mice using the splinted wound model. Considering that the predominant mechanism of wound healing in humans is by re-epithelialization, the most appropriate model for evaluating therapeutics is one that uses splints to prevent excessive wound contraction. Here, we report that PDGF-BB does not promote wound closure by re-epithelialization in a murine splinted wound model. Our results highlight that the effects of cytoactive factors reported in vivo ought to be carefully interpreted with critical consideration of the wound model used.

Chronic nonhealing skin wounds often contain bacterial biofilms that prevent normal wound healing and closure and present challenges to the use of conventional wound dressings. We investigated inhibition of Pseudomonas aeruginosa biofilm formation, a common pathogen of chronic skin wounds, on a commercially available biological wound dressing. Building on prior reports, we examined whether the amino acid tryptophan would inhibit P. aeruginosa biofilm formation on the three-dimensional surface of the biological dressing. Bacterial biomass and biofilm polysaccharides were quantified using crystal violet staining or an enzyme linked lectin, respectively. Bacterial cells and biofilm matrix adherent to the wound dressing were visualized through scanning electron microscopy. D-/L-tryptophan inhibited P. aeruginosa biofilm formation on the wound dressing in a dose dependent manner and was not directly cytotoxic to immortalized human keratinocytes although there was some reduction in cellular metabolism or enzymatic activity. More importantly, D-/L-tryptophan did not impair wound healing in a splinted skin wound murine model. Furthermore, wound closure was improved when D-/L-tryptophan treated wound dressing with P. aeruginosa biofilms were compared with untreated dressings. These findings indicate that tryptophan may prove useful for integration into wound dressings to inhibit biofilm formation and promote wound healing.

Re-epithelialization of wounds is a critical element of wound closure. Growth factors have been used in combination with conventional wound management to promote closure, but the method of delivery has been limited to the topical application of ointment formulations. Cytoactive factors delivered in this way have short resident times in wounds and have met with limited success. Here, we demonstrate that methods used to covalently immobilize proteins on synthetic materials can be extended to immobilize cytoactive factors such as the epidermal growth factor (EGF) onto the wound beds of genetically diabetic mice that exhibit impaired healing. Full-thickness splinted excisional wounds were created in diabetic (db/db) mice with a well-defined silicone splint to limit wound contracture. Wound surfaces were treated with a reducing agent to expose sulfhydryl groups and subsequently treated with EGF modified with a heterobifunctional crosslinker. This allowed for the covalent immobilization of the EGF to the wound surface. The conjugation chemistry was validated in vitro and in vivo. In a separate group of mice, wounds were topically treated twice daily with soluble EGF. The mice were evaluated over 11 days for wound closure. This covalent immobilization strategy resulted in EGF being retained on the wound surface for 2 days and significantly increased epithelial wound closure by 20% compared to wounds treated with topical EGF or topical vehicle. Covalent immobilization was not only therapeutically effective but also delivered a markedly reduced load of growth factor to the wound surface compared to topical application (when only 180 ng of EGF was immobilized onto the wound surface in comparison with 7200 ng of topically applied EGF over a period of 11 days). No adverse effects were observed in treated wounds. Results obtained provide proof of concept for the effectiveness of covalent immobilization in the treatment of dysregulated wounds. The covalent immobilization of cytoactive factors represents a potentially transformative approach to the management of difficult chronic wounds.