Bioengineered Human Skin: Working the Bugs Out
Authors: L Steinstraesser, S Al-Benna, M Kesting, F Jacobsen
Journal: Molecular Therapy (2009)

The bacterial contamination of wounds is an important global health-care issue that is bound to grow as the population ages. Bacterial wound infections increase morbidity and mortality and are of considerable socioeconomic impact. The risk of wound infection increases as disturbances in local conditions favor bacterial growth rather than host defense. This can lead to impaired wound healing, resulting on the one hand in rising treatment costs and on the other hand in a traumatic and potentially life-threatening condition for the patient.1Bowler PG Wound pathophysiology, infection and therapeutic options.Ann Med. 2002; 34: 419-427Crossref PubMed Scopus (192) Google Scholar The current clinical gold standard for treating wound infection is antibiotic therapy. However, the benefit of systemically delivered antibiotics to advance wound healing is still unclear.2Howell-Jones RS Wilson MJ Hill KE Howard AJ Price PE Thomas DW A review of the microbiology, antibiotic usage and resistance in chronic skin wounds.J Antimicrob Chemother. 2005; 55: 143-149Crossref PubMed Scopus (248) Google Scholar Moreover, the emergence of bacterial resistance to antibiotics is a growing threat, and this has increased the urgency of finding novel means of combating pathogenic assault. A growing number of studies indicate potential clinical applications of host defense peptides (HDPs) for skin antimicrobial therapy.3Steinstraesser L Koehler T Jacobsen F Daigeler A Goertz O Langer S et al.Host defense peptides in wound healing.Mol Med. 2008; 14: 528-537Crossref PubMed Scopus (125) Google Scholar,4Kaus A Jacobsen F Sorkin M Rittig A Voss B Daigeler A et al.Host defence peptides in human burns.Burns. 2008; 34: 32-40Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar,5Jacobsen F Mohammadi-Tabrisi A Hirsch T Mittler D Mygind PH Sonksen CP et al.Antimicrobial activity of the recombinant designer host defence peptide P-novispirin G10 in infected full-thickness wounds of porcine skin.J Antimicrob Chemother. 2007; 59: 493-498Crossref PubMed Scopus (33) Google Scholar,6Hirsch T von Peter S Dubin G Mittler D Jacobsen F Lehnhardt M et al.Adenoviral gene delivery to primary human cutaneous cells and burn wounds.Mol Med. 2006; 12: 199-207Crossref PubMed Scopus (30) Google Scholar,7Steinstraesser L Tippler B Mertens J Lamme E Homann HH Lehnhardt M et al.Inhibition of early steps in the lentiviral replication cycle by cathelicidin host defense peptides.Retrovirology. 2005; 2: 2Crossref PubMed Scopus (85) Google Scholar,8Jacobsen F Mittler D Hirsch T Gerhards A Lehnhardt M Voss B et al.Transient cutaneous adenoviral gene therapy with human host defense peptide hCAP-18/LL-37 is effective for the treatment of burn wound infections.Gene Ther. 2005; 12: 1494-1502Crossref PubMed Scopus (89) Google Scholar In this issue, Thomas-Virnig et al. report the potential for engineered keratinocytes that overexpress human HDPs to enhance wound healing by counteracting the growth of microbial pathogens.9Thomas-Virnig CL Centanni JM Johnston CE He L-K Schlosser SJ Van Winkle KF et al.Inhibition of multidrug-resistant Acinetobacter baumannii by nonviral expression of hCAP-18 in a bioengineered human skin tissue.Mol Ther. 2009; 17: 562-569Abstract Full Text Full Text PDF PubMed Scopus (30) Google ScholarHost Defense PeptidesParallel evolution of hosts and pathogens has led to a diversity of multifaceted survival mechanisms that are either encoded in the germ line (innate immunity) or acquired through adaptation to specific antigens (adaptive immunity). Among the numerous defense mechanisms, HDPs are important evolutionarily conserved elements of innate immunity. They are found in organisms as diverse as insects and humans. Salient examples include the cecropins (insects), magainins (amphibians), and defensins (mammals). They are generally small peptides (12–50 amino acids) that contain at least two positively charged (arginine or lysine) residues. Predominantly membrane active, these peptides exhibit a high positive net charge and can adopt amphipathic conformations. This amphipathic design, consisting of spatially separated hydrophobic and charged regions, permits intercalation of the peptide into microbial membranes. Their unique mechanism of action and ability (in many cases) to kill organisms make them attractive prototypes for antibacterial drug development. Recent developments highlight the importance of vertebrate epithelial cells as sites of antibiotic peptide production and suggest a critical role for this system in human health and disease. Several reviews on HDPs have already been published.3Steinstraesser L Koehler T Jacobsen F Daigeler A Goertz O Langer S et al.Host defense peptides in wound healing.Mol Med. 2008; 14: 528-537Crossref PubMed Scopus (125) Google Scholar,10Kaus A Jacobsen F Sorkin M Rittig A Voss B Daigeler A et al.Host defence peptides in human burns.Burns. 2008; 34: 32-40Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar,11Hirsch T Jacobsen F Steinau HU Steinstraesser L Host defense peptides and the new line of defence against multiresistant infections.Protein Pept Lett. 2008; 15: 238-243Crossref PubMed Scopus (38) Google ScholarThe importance of this innate system as a check on infection is evident when one considers that most bacteria have generation times of 20–30 minutes, whereas the mounting of a specific adaptive immune response may take days or weeks. Although many cationic peptides demonstrate direct antimicrobial activity against bacteria, fungi, eukaryotic parasites, and viruses,3Steinstraesser L Koehler T Jacobsen F Daigeler A Goertz O Langer S et al.Host defense peptides in wound healing.Mol Med. 2008; 14: 528-537Crossref PubMed Scopus (125) Google Scholar,7Steinstraesser L Tippler B Mertens J Lamme E Homann HH Lehnhardt M et al.Inhibition of early steps in the lentiviral replication cycle by cathelicidin host defense peptides.Retrovirology. 2005; 2: 2Crossref PubMed Scopus (85) Google Scholar,10Kaus A Jacobsen F Sorkin M Rittig A Voss B Daigeler A et al.Host defence peptides in human burns.Burns. 2008; 34: 32-40Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar,11Hirsch T Jacobsen F Steinau HU Steinstraesser L Host defense peptides and the new line of defence against multiresistant infections.Protein Pept Lett. 2008; 15: 238-243Crossref PubMed Scopus (38) Google Scholar,12Larrick JW Hirata M Balint RF Lee J Zhong J Wright SC Human CAP18: a novel antimicrobial lipopolysaccharide-binding protein.Infect Immun. 1995; 63: 1291-1297Crossref PubMed Google Scholar it has also been established that many also have a key modulatory role in the innate immune response and form an important link between the innate and adaptive immune responses.13Zasloff M Antimicrobial peptides of multicellular organisms.Nature. 2002; 415: 389-395Crossref PubMed Scopus (6644) Google Scholar Owing to their multiple functions, they are considered promising agents for new therapeutic approaches in infectious diseases and wound healing.3Steinstraesser L Koehler T Jacobsen F Daigeler A Goertz O Langer S et al.Host defense peptides in wound healing.Mol Med. 2008; 14: 528-537Crossref PubMed Scopus (125) Google Scholar,4Kaus A Jacobsen F Sorkin M Rittig A Voss B Daigeler A et al.Host defence peptides in human burns.Burns. 2008; 34: 32-40Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar,6Hirsch T von Peter S Dubin G Mittler D Jacobsen F Lehnhardt M et al.Adenoviral gene delivery to primary human cutaneous cells and burn wounds.Mol Med. 2006; 12: 199-207Crossref PubMed Scopus (30) Google Scholar,10Kaus A Jacobsen F Sorkin M Rittig A Voss B Daigeler A et al.Host defence peptides in human burns.Burns. 2008; 34: 32-40Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar,11Hirsch T Jacobsen F Steinau HU Steinstraesser L Host defense peptides and the new line of defence against multiresistant infections.Protein Pept Lett. 2008; 15: 238-243Crossref PubMed Scopus (38) Google Scholar,14Steinstraesser L Burghard O Nemzek J Fan MH Merry A Remick DI et al.Protegrin-1 increases bacterial clearance in sepsis but decreases survival.Crit Care Med. 2003; 31: 221-226Crossref PubMed Scopus (20) Google Scholar,15Steinstraesser L Tack BF Waring AJ Hong T Boo LM Fan MH et al.Activity of novispirin G10 against Pseudomonas aeruginosa in vitro and in infected burns.Antimicrob Agents Chemother. 2002; 46: 1837-1844Crossref PubMed Scopus (83) Google Scholar,16Nizet V Ohtake T Lauth X Trowbridge J Rudisill J Dorschner RA et al.Innate antimicrobial peptide protects the skin from invasive bacterial infection.Nature. 2001; 414: 454-457Crossref PubMed Scopus (1011) Google Scholar,17Steinstraesser L Oezdogan Y Wang SC Steinau HU Host defense peptides in burns.Burns. 2004; 30: 619-627Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar,18Steinstraesser L Ring A Bals R Steinau HU Langer S The human host defense peptide LL37/hCAP accelerates angiogenesis in PEGT/PBT biopolymers.Ann Plast Surg. 2006; 56: 93-98Crossref PubMed Scopus (45) Google ScholarThe emergence of bacterial resistance to antibiotics increased the urgency of exploring alternative means of combating pathogenic assault. HDPs are rapidly emerging as attractive candidates for antimicrobial treatment. Different therapies that effect wound repair have been proposed over the past few decades. Although HDP therapy was initially focused on antimicrobial activity, it can be applied equally well to the local, temporary treatment of acquired diseases, including impaired wound healing and tissue repair.Thomas-Virnig et al. elegantly investigated the potential therapeutic role of genetically engineered NIKS skin substitute tissue expressing hCAP-18 (NIKShCAP-18), which significantly counteracted bacterial infection by a multidrug-resistant nosocomial bacterial strain in a mouse model of infected third-degree cutaneous burn wounds.9Thomas-Virnig CL Centanni JM Johnston CE He L-K Schlosser SJ Van Winkle KF et al.Inhibition of multidrug-resistant Acinetobacter baumannii by nonviral expression of hCAP-18 in a bioengineered human skin tissue.Mol Ther. 2009; 17: 562-569Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Cathelicidins are a family of HDPs found in most mammalian species. They consist of an N-terminal cathelin domain, which is highly conserved throughout species, and a variable C-terminal peptide. This propeptide is activated extracellularly after proteolytic cleavage.The only known member of this family thus far is hCAP-18, an 18-kDa human cationic antimicrobial protein that is produced mainly by leukocytes and epithelial and mucosal cells. Its cationic C-terminal 37–amino acid domain, LL-37, displays broad antimicrobial activity mediated through direct interaction with and disruption of the microbial cell membrane. The enzyme responsible for cleavage of the proprotein in neutrophils is serine proteinase 3. In skin, the serine proteases kallikrein 5 and 7 were recently reported to mediate alternative processing of hCAP-18 generating several novel peptide fragments, suggesting that peptide profiles may differ among tissues and biological conditions.19Murakami M Ohtake T Dorschner RA Schittek B Garbe C Gallo RL Cathelicidin anti-microbial peptide expression in sweat, an innate defense system for the skin.J Invest Dermatol. 2002; 119: 1090-1095Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar This opens a potential new area of research, in that their functional profile may differ. Interestingly, both the proprotein, hCAP-18, and the mature processed form of LL-37 are constitutively produced in sweat.19Murakami M Ohtake T Dorschner RA Schittek B Garbe C Gallo RL Cathelicidin anti-microbial peptide expression in sweat, an innate defense system for the skin.J Invest Dermatol. 2002; 119: 1090-1095Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar The unprocessed proprotein does not have antimicrobial activity20Zaiou M Nizet V Gallo RL Antimicrobial and protease inhibitory functions of the human cathelicidin (hCAP18/LL-37) prosequence.J Invest Dermatol. 2003; 120: 810-816Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar until it is cleaved by proteinase 3.In addition to being antimicrobial, LL-37 is implicated in diverse biological processes, including chemotaxis, cytokine production, histamine release, and angiogenesis.18Steinstraesser L Ring A Bals R Steinau HU Langer S The human host defense peptide LL37/hCAP accelerates angiogenesis in PEGT/PBT biopolymers.Ann Plast Surg. 2006; 56: 93-98Crossref PubMed Scopus (45) Google Scholar,21Koczulla R von Degenfeld G Kupatt C Krotz F Zahler S Gloe T et al.An angiogenic role for the human peptide antibiotic LL-37/hCAP-18.J Clin Invest. 2003; 111: 1665-1672Crossref PubMed Scopus (721) Google Scholar,22Shaykhiev R Beisswenger C Kandler K Senske J Puchner A Damm T et al.Human endogenous antibiotic LL-37 stimulates airway epithelial cell proliferation and wound closure.Am J Physiol Lung Cell Mol Physiol. 2005; 289: L842-L848Crossref PubMed Scopus (223) Google Scholar Furthermore, hCAP-18/LL-37 is highly expressed during normal wound healing,23Dorschner RA Pestonjamasp VK Tamakuwala S Ohtake T Rudisill J Nizet V et al.Cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group A Streptococcus.J Invest Dermatol. 2001; 117: 91-97Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar,24Heilborn JD Nilsson MF Kratz G Weber G Sorensen O Borregaard N et al.The cathelicidin anti-microbial peptide LL-37 is involved in reepithelialization of human skin wounds and is lacking in chronic ulcer epithelium.J Invest Dermatol. 2003; 120: 379-389Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar displays growth factor–like properties enhancing the in vitro proliferation and migration of human keratinocytes, and is involved in re-epithelialization of wounds.24Heilborn JD Nilsson MF Kratz G Weber G Sorensen O Borregaard N et al.The cathelicidin anti-microbial peptide LL-37 is involved in reepithelialization of human skin wounds and is lacking in chronic ulcer epithelium.J Invest Dermatol. 2003; 120: 379-389Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar,25Heilborn JD Nilsson MF Jimenez CI Sandstedt B Borregaard N Tham E et al.Antimicrobial protein hCAP18/LL-37 is highly expressed in breast cancer and is a putative growth factor for epithelial cells.Int J Cancer. 2005; 114: 713-719Crossref PubMed Scopus (107) Google Scholar LL-37 is upregulated at the messenger RNA and protein levels upon exposure to bacteria or by sterile incision.26Kurosaka K Chen Q Yarovinsky F Oppenheim JJ Yang D Mouse cathelin-related antimicrobial peptide chemoattracts leukocytes using formyl peptide receptor-like 1/mouse formyl peptide receptor-like 2 as the receptor and acts as an immune adjuvant.J Immunol. 2005; 174: 6257-6265Crossref PubMed Scopus (180) Google Scholar Expression is also increased by various growth factors that are involved in wound healing, such as insulin-like growth factor. Consistent with this, LL-37 has been demonstrated to be involved in the re-epithelialization of skin wounds. Using a noninflammatory ex vivo wound healing model of organ-cultured human skin, upregulation of LL-37 expression has been demonstrated in the wound area, and antibodies specific to LL-37 have been shown to inhibit the re-epithelialization process in a concentration-dependent manner.27Territo MC Ganz T Selsted ME Lehrer R Monocyte-chemotactic activity of defensins from human neutrophils.J Clin Invest. 1989; 84: 2017-2020Crossref PubMed Scopus (399) Google Scholar Because LL-37 is probably not found at concentrations sufficient for antimicrobial activity,26Kurosaka K Chen Q Yarovinsky F Oppenheim JJ Yang D Mouse cathelin-related antimicrobial peptide chemoattracts leukocytes using formyl peptide receptor-like 1/mouse formyl peptide receptor-like 2 as the receptor and acts as an immune adjuvant.J Immunol. 2005; 174: 6257-6265Crossref PubMed Scopus (180) Google Scholar it is possible that one of the primary functions of LL-37 in the skin is to promote re-epithelialization. None of the clinically used antibiotics demonstrates these interesting features.As a result of the alarming increase of multidrug-resistant bacteria, the new experimental approach of Thomas-Virnig et al. with hCAP-18/LL-37 has considerable clinical relevance. The investigators demonstrated a two-log reduction in the growth of a clinical isolate of multidrug-resistant Acinetobacter baumannii. This well-conducted study provides strong evidence that the antimicrobial peptide hCAP-18/LL-37 may reduce the risk of infection from pathogenic bacteria, including multidrug-resistant nosocomial strains, and thereby actively enhance wound healing. Although this study did not show evidence of better wound healing in addition to reduced bacterial growth, as Thomas-Virnig et al. note, HDPs may also act to amplify the endogenous innate immune response to invading microbes. Moreover, there is evidence that HDPs such as hCAP-18/LL-37 exhibit wound healing properties distinct from their antimicrobial function, such as the stimulation of keratinocyte growth and/or migration and the stimulation of wound vascularization.3Steinstraesser L Koehler T Jacobsen F Daigeler A Goertz O Langer S et al.Host defense peptides in wound healing.Mol Med. 2008; 14: 528-537Crossref PubMed Scopus (125) Google Scholar,4Kaus A Jacobsen F Sorkin M Rittig A Voss B Daigeler A et al.Host defence peptides in human burns.Burns. 2008; 34: 32-40Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar,10Kaus A Jacobsen F Sorkin M Rittig A Voss B Daigeler A et al.Host defence peptides in human burns.Burns. 2008; 34: 32-40Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar,11Hirsch T Jacobsen F Steinau HU Steinstraesser L Host defense peptides and the new line of defence against multiresistant infections.Protein Pept Lett. 2008; 15: 238-243Crossref PubMed Scopus (38) Google Scholar,21Koczulla R von Degenfeld G Kupatt C Krotz F Zahler S Gloe T et al.An angiogenic role for the human peptide antibiotic LL-37/hCAP-18.J Clin Invest. 2003; 111: 1665-1672Crossref PubMed Scopus (721) Google Scholar,22Shaykhiev R Beisswenger C Kandler K Senske J Puchner A Damm T et al.Human endogenous antibiotic LL-37 stimulates airway epithelial cell proliferation and wound closure.Am J Physiol Lung Cell Mol Physiol. 2005; 289: L842-L848Crossref PubMed Scopus (223) Google Scholar,24Heilborn JD Nilsson MF Kratz G Weber G Sorensen O Borregaard N et al.The cathelicidin anti-microbial peptide LL-37 is involved in reepithelialization of human skin wounds and is lacking in chronic ulcer epithelium.J Invest Dermatol. 2003; 120: 379-389Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar,26Kurosaka K Chen Q Yarovinsky F Oppenheim JJ Yang D Mouse cathelin-related antimicrobial peptide chemoattracts leukocytes using formyl peptide receptor-like 1/mouse formyl peptide receptor-like 2 as the receptor and acts as an immune adjuvant.J Immunol. 2005; 174: 6257-6265Crossref PubMed Scopus (180) Google Scholar,27Territo MC Ganz T Selsted ME Lehrer R Monocyte-chemotactic activity of defensins from human neutrophils.J Clin Invest. 1989; 84: 2017-2020Crossref PubMed Scopus (399) Google Scholar,28Gallo RL Ono M Povsic T Page C Eriksson E Klagsbrun M et al.Syndecans, cell surface heparan sulfate proteoglycans, are induced by a proline-rich antimicrobial peptide from wounds.Proc Natl Acad Sci USA. 1994; 91: 11035-11039Crossref PubMed Scopus (325) Google Scholar,29Li J Post R Volk R Gao Y Li M Metais C et al.PR39, a peptide regulator of angiogenesis.Nat Med. 2000; 6: 49-55Crossref PubMed Scopus (336) Google ScholarThomas-Virnig and colleagues' study of enhanced production of an HDP using a genetically modified tissue substitute offers a feasible mechanism for cutaneous gene therapy to improve wound healing and may provide a major advantage in the ability to deliver a composite graft that aims to replicate the physiological function and anatomical architecture of skin. The skin is the site of many debilitating diseases for which effective therapy is currently unavailable. Gene therapy in the skin requires different gene transfer strategies depending on the therapeutic effect sought. Two major applications can be easily distinguished: (i) permanent correction of genetic disorders and (ii) transient treatment or improvement of pathological conditions.The skin is an attractive target for HDP therapy for many reasons. The predominant cells of the skin—fibroblasts and keratinocytes—are harvested easily, and protocols exist for their successful culture.30Terskikh VV Vasiliev AV Cultivation and transplantation of epidermal keratinocytes.Int Rev Cytol. 1999; 188: 41-72Crossref PubMed Google Scholar This situation not only enables testing of skin cells in vitro but also highlights their availability for use as vehicles in ex vivo protocols, because such cells can readily be transplanted back to a host. The superficial location of the skin makes it simple to monitor the behavior of a genetically modified skin area for any reactions and, if necessary, to remove it in case of an adverse reaction to the transgene product. The skin is also accessible to direct HDP transfer by many techniques, including injection and topical application, avoiding unnecessary systemic delivery.The skin allows (i) easy collection of the gene-targetable cells, (ii) efficient in vitro gene transfer (using viral vectors), (iii) expansion of the modified cell population (either selected/enriched or not), and (iv) grafting of genetically altered cells to the donor individual using the technique described by Thomas-Virnig et al. Diseases that could be treated using this approach can be divided into three major categories: (i) disorders that affect the skin and whose molecular defect has already been characterized, (ii) disorders that—whether or not they affect the skin—may improve when the skin is converted into a source of certain proteins, and (iii) disorders that do not affect the skin but may improve when the skin is converted into a metabolic sink.However, differences in important skin features, such as epidermal thickness, epidermal kinetics, and gene transfer efficiencies between animal and human tissue, have limited the use of HDP therapy. Thomas-Virnig et al. nicely circumvented this problem by using a human-skin substitute grafted onto immunodeficient mice (athymic nude mice). Their approach is particularly attractive because the regenerated human skin on these mice could preserve clinical, histological, molecular, and functional properties of the donor skin (either normal or diseased).For cutaneous HDP therapy aimed at the treatment of genetic skin disorders such as psoriasis, retention of pathological defects in vivo provides a valuable system for testing the efficacy and safety of strategies. It interesting to note that keratinocyte gene transfer has also been explored as a treatment modality for nondermatological conditions. Considerable progress has recently been made toward the treatment of systemic diseases caused by insufficient amounts of a protein in the circulation, using the epidermis as a "bioreactor" for producing therapeutic protein.The concept of delivery of HDPs by a skin substitute is a new development. Combinations of HDPs or their sequential use may be the future answer to accelerating wound healing processes, and with the advent of their delivery by a skin substitute, these HDPs may become increasingly important. Developments in matrix components and tissue engineering technology may offer promise for the future. The development of slow-release matrices may prolong the delivery of their potential antimicrobial and wound healing properties.Viral vectors are the original and most established technology for gene delivery. A wide range of applications have been developed, and many virus-mediated gene transfer models are successful. The production of viral vectors, however, is costly and time-consuming, transfection efficacy is variable, and the risk of local or systemic infections that may lead to fatal outcomes remains a concern. Thomas-Virnig and colleagues' nonviral approach using sustained delivery of hCAP-18 to the wound bed from temporarily applied human-skin-substitute tissue should also circumvent viral gene transfer issues such as the preexisting immunity exhibited by much of the human population toward adenoviruses. Other positive attributes of their approach include elimination of the risk of viral infection and avoidance of the higher cost of vector production.31Vogel JC Nonviral skin gene therapy.Hum Gene Ther. 2000; 11: 2253-2259Crossref PubMed Scopus (75) Google Scholar The transient nature of gene expression is also a benefit in wound healing applications. On the negative side, some nonviral gene transfer methods tend to be nonspecific and highly variable within the levels of gene expression reported.The Use of Skin Substitutes to Deliver HDPsThe problem then will be establishing which of the many HDPs identified as having potential therapeutic benefit should be the ones of choice. This question highlights the need for a database of HDP functions in wound healing to facilitate further study. The numerous therapeutic options for delivery of HDPs by a bioengineered skin substitute include anti-infection, anti-endotoxin, immunomodulation, chemotaxis, cell proliferation and differentiation, wound healing, angiogenesis, influencing initiation and polarization of adaptive immunity, induction of gene expression, and enhancement of protein secretion functions.Recent advances in cell biology have identified a variety of molecules—specifically, growth factors and their receptors—that are critically involved in directing cell functions during soft-tissue organogenesis. The study by Thomas-Virnig et al. indicates that delivery of HDPs via a skin substitute is a promising therapeutic tool for modifying this repair microenvironment. Skin with inherently poor or pathologically altered healing potential would thus benefit from a therapeutic bioengineered skin approach that could augment healing capacity while minimizing infection.A Paradigm ShiftHDP research is becoming an area of great importance. Delivery of HDPs by a bioengineered skin substitute has evolved from a purely experimental scientific endeavor to a clinically pertinent treatment for the skin. In wound healing, there remain challenges in the selection of optimal target cells, development of sequential therapeutic methods, and identification of factors that may be detrimental to the introduction of a genetically modified skin substitute.It is probable that the only way to answer many questions that still arise about therapeutic delivery of HDPs by a skin substitute will be through clinical trials. The following steps have been achieved/developed: (i) culture and targeting of human epidermal stem cells, (ii) surgical techniques that allow the grafting of large skin areas without scarring, and (iii) sustained transgene expression in vivo in preclinical models. Now is the time to validate these steps in a clinical context. Implementation of a phase I/II clinical trial of HDP therapy in selected individuals suffering from psoriasis may shed light on important issues, mainly, the persistence of transgene expression at therapeutic levels and host tolerance to the gene product. The focus in the development of HDPs for clinical applications has been on small HDPs containing the biologically active core of the endogenous molecules, thereby limiting related toxicity components, improving efficacy, and lowering the cost of goods. Only a modest number of HDPs have been tested in clinical-efficacy trials, and they demonstrated potential in both preclinical and clinical trials.24Heilborn JD Nilsson MF Kratz G Weber G Sorensen O Borregaard N et al.The cathelicidin anti-microbial peptide LL-37 is involved in reepithelialization of human skin wounds and is lacking in chronic ulcer epithelium.J Invest Dermatol. 2003; 120: 379-389Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar,25Heilborn JD Nilsson MF Jimenez CI Sandstedt B Borregaard N Tham E et al.Antimicrobial protein hCAP18/LL-37 is highly expressed in breast cancer and is a putative growth factor for epithelial cells.Int J Cancer. 2005; 114: 713-719Crossref PubMed Scopus (107) Google Scholar,26Kurosaka K Chen Q Yarovinsky F Oppenheim JJ Yang D Mouse cathelin-related antimicrobial peptide chemoattracts leukocytes using formyl peptide receptor-like 1/mouse formyl peptide receptor-like 2 as the receptor and acts as an immune adjuvant.J Immunol. 2005; 174: 6257-6265Crossref PubMed Scopus (180) Google Scholar,27Territo MC Ganz T Selsted ME Lehrer R Monocyte-chemotactic activity of defensins from human neutrophils.J Clin Invest. 1989; 84: 2017-2020Crossref PubMed Scopus (399) Google ScholarThomas-Virnig and co-workers' study indicates that delivery of HDPs via skin substitute is a promising therapeutic tool for modifying the repair microenvironment and may lead to a paradigm shift in the management of skin pathologies. Future basic and clinical research will tell if and when this new powerful "biological weapon" will become part of the health professionals' armamentarium. Further research of HDPs will improve our knowledge of their involvement in the recognition and neutralization of pathogens, which will support the development of new anti-infective therapeutic approaches. HDP immunomodulating functions will become clearer with additional research on structure–function analyses to elucidate their mechanisms of action. Most important, future research must take advantage of and build on the diverse nature of HDPs and adhere