Diabetes is a global health care issue. Three hundred-eighty-two million people were reported to have diabetes in 2013 (http://www.diabetesatlas.org). Additionally, the cost of diabetic foot ulcers to the American health care system was estimated to be $9–13 billion in addition to care for diabetes in 2013 [1]. Maggot debridement therapy (MDT) is a cost-effective, FDA-approved treatment for diabetic foot ulcers [2, 3]. MDT commonly involves the application of sterile Lucilia sericata larvae to a non-healing wound to promote healing and decrease infection. MDT has been applied successfully in more than 20 additional medical conditions [4, 5].

MDT promotes healing in part through digestion and mechanical removal of necrotic tissue. Debridement is a critical component of effective wound healing [4, 6]. Enzyme application and mechanical debridement have been studied in clinical trials, but challenges such as expense and potential damage to healthy tissue stunt the large-scale effectiveness of these treatment options [7]. In contrast, larvae leave behind healthy tissue. Larvae have been shown to ingest fluorescent bacteria in vitro [8] as well as raise the pH of the wound environment via excretions and secretions (ES), which results in inhibition of bacterial growth [9]. Most of the in vitro studies found that ES was more effective at inhibiting growth of gram positive than gram negative bacteria [10]. Further, in one small vivo study, sterile maggots were found to be more effective at inhibiting growth of gram positive bacteria in infected wounds [11]. Specific factors and fractions have been identified within ES that exhibit antibacterial activity in vitro [12]. For example, the insect defensin homologue lucifensin was detected in the gut and salivary glands of L. sericata larvae and identified in wound washings from MDT patients [13]. Lucifensin exhibited antibacterial activity against a panel of Gram positive bacteria [13, 14]. Some data suggests expression and secretion of antibacterial factors by larvae is not constitutive, but induced by the wound environment [15–17]. The antibacterial mechanisms of MDT are free from the limitations of antibiotic resistance frequently seen in the clinic. Indeed, maggot debridement therapy has been shown to be effective in treatment of MRSA in vitro as well as in clinical case studies [18].

It is clear from these studies that larvae significantly alter the wound environment during MDT. Maggot ES may also alter the local inflammatory response. For example, L. sericata ES modulate neutrophil migration and adhesion and alter expression of pattern recognition receptor levels [19]. ES also increased the secretion of anti-inflammatory cytokine IL-10, while inhibiting secretion of pro-inflammatory cytokines TNF-alpha and IL-12p40 [20].

In addition to the impact of MDT on the immune response in the wound, MDT also promotes wound healing through formation of granulation tissue [21, 22]. This could be a consequence of the physical action of the maggots in the wound, removal of dead tissue, change of wound pH and microbial killing [10]. In addition, there is some evidence that maggot ES could stimulate growth of human cells in the wound. ES was shown to stimulate fibroblast proliferation in culture [23] and hepatocyte growth factor (HGF) synthesis in 3T3 cells [24]. Further, increased HGF levels were measured in femoral vein blood of patient during MDT [24]. However, there is no evidence from randomized clinical trials that MDT shortens wound healing times [25]. This may reflect a limitation of the design of the trials [10] but highlights the need for further studies on the promotion of wound healing by maggots.

Studies have shown decreased concentrations of growth factors, including several isoforms of platelet-derived growth factor (PDGF), in chronic wounds when compared to acute wounds [26, 27]. This evidence precipitated investigation into topical recombinant growth factor treatment as a means to promote healing in chronic wounds. Several growth factors were investigated, however molecular stability limited their success despite the use of gels, micropheres, and other conjugates. Nevertheless, after achieving some pre-clinical and clinical success, human PDGF-BB became the first FDA-approved recombinant cytokine growth factor [28].

The role of PDGF in wound healing is well established [29]. PDGF is a cationic hetero or homo-dimer consisting of a combination of alpha and beta subunit chains containing multiple intra- and inter-chain disulfide bonds. The subunits are produced in a pro form by endothelial cells, fibroblasts, immune system cells, and others [23]. The pro subunits dimerize in the endoplasmic reticulum into homo or heterodimeric combinations. The pro dimers are further processed via N-terminal modification, remodeling, and cleavage to a mature dimer form [30]. The mature dimers are secreted, where they interact with the extra-cellular matrix (ECM) and cell surface PDGF receptors. Via activation of the PDGF receptor and subsequently PI3 kinase and mitogen-activated protein kinase (MAPK), PDGF stimulates cell survival, fibroblast proliferation and chemotaxis, actin reorganization, and production and secretion of other growth factors, ECM constituents, and metalloproteases [29]. Because of the extensive role of PDGF in wound healing, clinical trials have been done investigating the utility of a topical gel (Becaplermin) containing recombinant human PDGF-BB produced in Escherichia coli [31]. Immunostaining of wounds treated with PDGF-BB showed increased fibroblasts, increased collagen fibril formation, and healing (as measured by decreased wound size) [32]. In one study, ulcer surface area and time to complete healing were both reduced significantly in patients receiving topical PDGF-BB along with standard wound care, however, the authors purposefully selected large, severe ulcers for inclusion in the study [33]. Similarly, other trials reported increased healing and/or reduced time to wound closure [34–41]. However, some trials did not find that topical PDGF treatment significantly improved wound healing [42–44]. The mixed outcomes could reflect the complexity of the wound healing process that involves mutiple factors, which supports the need for a therapy that combines multiple mechanisms to promote wound healing.

Here, we present a novel concept in MDT technology that combines the established benefits of MDT with the potential power of engineered maggots to promote healing. Genetically modified larvae engineered to secrete selected human growth factors or antibacterial peptides effective against Gram positive and Gram negative bacteria could have the potential to synergistically improve wound healing and result in shorter hospital stays. Given the low cost of rearing maggots, the technology is likely to be cost-effective compared to dressing gels containing recombinant proteins. The objective of this study is to determine if L. sericata can be engineered to conditionally express and secrete human PDGF-BB. PDGF-BB was selected because PDGF-BB made in E. coli is active and has been approved for use in wound treatment. However, we view this study as proof-of-principle for the future development of engineered L. sericata strains that express a variety of growth factors and antimicrobial peptides.