Subcutaneous Anti-inflammatory Therapies to Prevent Burn Progression in a Swine Model of Contact Burn Injury.

INTRODUCTION: If left untreated, burn injuries can deepen or progress in depth within the first 72 hours after injury as a result of increased wound inflammation, subsequently worsening healing outcomes. This can be especially detrimental to warfighters who are constrained to resource-limited environments with delayed evacuation times to higher roles of care and more effective treatment. Preventing this burn progression at the point of injury has the potential to improve healing outcomes but requires a field-deployable therapy and delivery system. Subcutaneous therapies known to treat inflammation delivered local to the wound site may prove to be one such avenue for success. MATERIALS AND METHODS: Seven Yorkshire-cross swine received partial-thickness burn injuries using a previously established contact burn model. Each animal received one of the seven therapies: (1) saline, (2) heparin, (3) ibuprofen, (4) erythropoietin, (5) resolvin, (6) rapamycin, and (7) placental extract, all of which are either currently employed or are experimental in field use and indicated to treat inflammation. Treatments were delivered subcutaneously on the day of injury and 24 hours post-injury to simulate a prolonged field care scenario, before potential evacuation. Animals and wound development were observed for 28 days before euthanasia. Throughout the course of the study, wounds were observed macroscopically via non-invasive imaging. Histological analyses provided the critical metric of burn progression. Treatment success criteria were designated as the ability to prevent burn progression past 80% of the dermal depth in two of the three treated wounds, a clinically relevant metric of burn progression. RESULTS: It was determined that the applied model successfully created reproducible partial-thickness burn injuries in this porcine study. No significant differences with regard to lateral wound size or the rate of lateral wound closure were observed in any treatments. Several treatments including resolvin, rapamycin, ibuprofen, and erythropoietin successfully reduced burn progression to less than 80% of the dermal depth in two of the three wounds, 24 hours after injury. CONCLUSIONS: This report employs an established model of porcine contact burn injury in order to test the ability of local subcutaneous delivery of therapeutics to prevent burn progression at the point of injury, via what is believed to be the inhibition of inflammation. Several treatments successfully prevented burn progression to a full-thickness injury, potentially improving wound healing outcomes in a simulated battlefield scenario. Subcutaneously administered therapies combating burn-induced inflammation at the point of injury may serve as a field-deployable treatment modality to improve warfighter recovery and return to duty.

Coming to Consensus: What Defines Deep Partial Thickness Burn Injuries in Porcine Models?

Deep partial thickness burns are clinically prevalent and difficult to diagnose. In order to develop methods to assess burn depth and therapies to treat deep partial thickness burns, reliable, accurate animal models are needed. The variety of animal models in the literature and the lack of precise details reported for the experimental procedures make comparison of research between investigators challenging and ultimately affect translation to patients. They sought to compare deep partial thickness porcine burn models from five well-established laboratories. In doing so, they uncovered a lack of consistency in approaches to the evaluation of burn injury depth that was present within and among various models. They then used an iterative process to develop a scoring rubric with an educational component to facilitate burn injury depth evaluation that improved reliability of the scoring. Using the developed rubric to re-score the five burn models, they found that all models created a deep partial thickness injury and that agreement about specific characteristics identified on histological staining was improved. Finally, they present consensus statements on the evaluation and interpretation of the microanatomy of deep partial thickness burns in pigs.

Advancements in Regenerative Strategies Through the Continuum of Burn Care.

Burns are caused by several mechanisms including flame, scald, chemical, electrical, and ionizing and non-ionizing radiation. Approximately half a million burn cases are registered annually, of which 40 thousand patients are hospitalized and receive definitive treatment. Burn care is very resource intensive as the treatment regimens and length of hospitalization are substantial. Burn wounds are classified based on depth as superficial (first degree), partial-thickness (second degree), or full-thickness (third degree), which determines the treatment necessary for successful healing. The goal of burn wound care is to fully restore the barrier function of the tissue as quickly as possible while minimizing infection, scarring, and contracture. The aim of this review is to highlight how tissue engineering and regenerative medicine strategies are being used to address the unique challenges of burn wound healing and define the current gaps in care for both partial- and full-thickness burn injuries. This review will present the current standard of care (SOC) and provide information on various treatment options that have been tested pre-clinically or are currently in clinical trials. Due to the complexity of burn wound healing compared to other skin injuries, burn specific treatment regimens must be developed. Recently, tissue engineering and regenerative medicine strategies have been developed to improve skin regeneration that can restore normal skin physiology and limit adverse outcomes, such as infection, delayed re-epithelialization, and scarring. Our emphasis will be centered on how current clinical and pre-clinical research of pharmacological agents, biomaterials, and cellular-based therapies can be applied throughout the continuum of burn care by targeting the stages of wound healing: hemostasis, inflammation, cell proliferation, and matrix remodeling.

Biomaterials for the Delivery of Growth Factors and Other Therapeutic Agents in Tissue Engineering Approaches to Bone Regeneration.

Bone fracture followed by delayed or non-union typically requires bone graft intervention. Autologous bone grafts remain the clinical "gold standard". Recently, synthetic bone grafts such as Medtronic's Infuse Bone Graft have opened the possibility to pharmacological and tissue engineering strategies to bone repair following fracture. This clinically-available strategy uses an absorbable collagen sponge as a carrier material for recombinant human bone morphogenetic protein 2 (rhBMP-2) and a similar strategy has been employed by Stryker with BMP-7, also known as osteogenic protein-1 (OP-1). A key advantage to this approach is its "off-the-shelf" nature, but there are clear drawbacks to these products such as edema, inflammation, and ectopic bone growth. While there are clinical challenges associated with a lack of controlled release of rhBMP-2 and OP-1, these are among the first clinical examples to wed understanding of biological principles with biochemical production of proteins and pharmacological principles to promote tissue regeneration (known as regenerative pharmacology). After considering the clinical challenges with such synthetic bone grafts, this review considers the various biomaterial carriers under investigation to promote bone regeneration. This is followed by a survey of the literature where various pharmacological approaches and molecular targets are considered as future strategies to promote more rapid and mature bone regeneration. From the review, it should be clear that pharmacological understanding is a key aspect to developing these strategies.

Ciprofloxacin-loaded keratin hydrogels reduce infection and support healing in a porcine partial-thickness thermal burn.

Infection is a leading cause of morbidity and mortality in burn patients. Current therapies include silver-based creams and dressings, which display limited antimicrobial effectiveness and impair healing. The need exists for a topical, point-of-injury antibiotic treatment that provides sustained antimicrobial activity without impeding wound repair. Fitting this description are keratin-based hydrogels, which are fully biocompatible and support the slow-release of antibiotics. Here we develop a porcine model of an infected partial-thickness burn to test the effects of ciprofloxacin-loaded keratin hydrogels on infection and wound healing. Partial-thickness burns were inoculated with either Pseudomonas aeruginosa or Methicillin-resistant Staphylococcus aureus, resulting in infections that persisted for >2 weeks that exceeded 10(5) and 10(6) cfu per gram of tissue, respectively. Compared to silver sulfadiazine, ciprofloxacin-loaded keratin hydrogel treatment significantly reduced the amount of P. aeruginosa and S. aureus in the burn by >99% on days 4, 7, 11, and 15 postinjury. Further, burns treated with ciprofloxacin-loaded keratin hydrogels exhibited similar healing patterns as uninfected burns with regards to reepithelialization, macrophage recruitment, and collagen deposition and remodeling. The ability of keratin hydrogels to deliver antibiotics to fight infection and support healing of partial-thickness burns make them a strong candidate as a first-line burn therapy.

Keratin hydrogel carrier system for simultaneous delivery of exogenous growth factors and muscle progenitor cells.

Ideal material characteristics for tissue engineering or regenerative medicine approaches to volumetric muscle loss (VML) include the ability to deliver cells, growth factors, and molecules that support tissue formation from a system with a tunable degradation profile. Two different types of human hair-derived keratins were tested as options to fulfill these VML design requirements: (1) oxidatively extracted keratin (keratose) characterized by a lack of covalent crosslinking between cysteine residues, and (2) reductively extracted keratin (kerateine) characterized by disulfide crosslinks. Human skeletal muscle myoblasts cultured on coatings of both types of keratin had increased numbers of multinucleated cells compared to collagen or Matrigel(TM) and adhesion levels greater than collagen. Rheology showed elastic moduli from 10(2) to 10(5) Pa and viscous moduli from 10(1) to 10(4) Pa depending on gel concentration and keratin type. Kerateine and keratose showed differing rates of degradation due to the presence or absence of disulfide crosslinks, which likely contributed to observed differences in release profiles of several growth factors. In vivo testing in a subcutaneous mouse model showed that keratose hydrogels can be used to deliver mouse muscle progenitor cells and growth factors. Histological assessment showed minimal inflammatory responses and an increase in markers of muscle formation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 864-879, 2016.

Surface-mediated delivery of siRNA from fibrin hydrogels for knockdown of the BMP-2 binding antagonist noggin.

Antagonists and inhibitory molecules responsible for maintaining tissue homeostasis can present a significant barrier to healing when tissue engineering/regenerative medicine strategies are employed. One example of this situation is the up-regulation of antagonists such as noggin in response to increasing concentrations of bone morphogenetic protein-2 (BMP-2) present from endogenous bone repair processes or delivered exogenously from biomaterials (synthetic bone grafts). While recombinant human (rh)BMP-2 delivered from synthetic bone grafts has been shown to be an effective alternative to autografts and allografts, the supraphysiological doses of rhBMP-2 have led to clinically-adverse side effects. The high rhBMP-2 dosage may be required, in part, to overcome the presence of antagonists such as noggin. Small interfering RNA (siRNA) is an appealing approach to overcome this problem because it can knock-down antagonists or inhibitory molecules in a temporary manner. Here, we conducted fundamental studies on the delivery of siRNA from material surfaces as a means to knock-down antagonists like noggin. Non-viral cationic lipid (Lipofectamine)-siRNA complexes were delivered from a fibrin hydrogel surface to MC3T3-E1 preosteoblasts that were treated with a supraphysiological dose of rhBMP-2 to achieve noggin mRNA expression levels higher than cells naïve to rhBMP-2. Confocal microscopy and flow cytometry showed intracellular uptake of siRNA in over 98% of MC3T3-E1 cells after 48 h. Doses of 0.5 and 1 µg noggin siRNA were able to significantly reduce noggin mRNA to levels equivalent to those in MC3T3-E1 cells not exposed to rhBMP-2 with no effects on cell viability. STATEMENT OF SIGNIFICANCE: Small interfering RNA (siRNA) has been considered for treatment of diseases ranging from Alzheimer's to cancer. However, the ability to use siRNA in conjunction with biomaterials to direct tissue regeneration processes has received relatively little attention. Using the bone morphogenetic protein 2 antagonist, noggin, as a model, this research describes an approach to knock-down molecules that are inhibitory to desired regenerative pathways at the mRNA level via siRNA delivery from a hydrogel surface. Interactions between the material (fibrin) surface and polycation-siRNA complexes, release of the siRNA from the material surface, high levels of cellular uptake/internalization of siRNA, and significant knockdown of the targeting (noggin) mRNA are demonstrated. Broader future applications include those to nerve regeneration, cardiovascular tissue engineering, directing (stem) cell behavior, and mitigating inflammatory responses to materials.

Reduction of ectopic bone growth in critically-sized rat mandible defects by delivery of rhBMP-2 from kerateine biomaterials.

Absorbable collagen sponges (ACS) are used clinically as carriers of recombinant human bone morphogenetic protein 2 (rhBMP-2) to promote bone regeneration. ACS exhibit ectopic bone growth due to delivery of supraphysiological levels of rhBMP-2, which is particularly problematic in craniofacial bone injuries for both functional and esthetic reasons. We hypothesized that hydrogels from the reduced form of keratin proteins (kerateine) would serve as a suitable alternative to ACS carriers of rhBMP-2. The rationale for this hypothesis is that keratin biomaterials degrade slowly in vivo, have modifiable material properties, and have demonstrated capacity to deliver therapeutic agents. We investigated kerateine hydrogels and freeze-dried scaffolds as rhBMP-2 carriers in a critically-sized rat mandibular defect model. ACS, kerateine hydrogels, and kerateine scaffolds loaded with rhBMP-2 achieved bridging in animals by 8 weeks as indicated by micro-computed tomography. Kerateine scaffolds achieved statistically increased bone mineral density compared to ACS and kerateine hydrogels, with levels reaching those of native bone. Importantly, both kerateine hydrogels and kerateine scaffolds had significantly less ectopic bone growth than ACS sponges at both 8 and 16 weeks post-operatively. These studies demonstrate the suitability of keratins as rhBMP-2 carriers due to equal regenerative capacity with reduced ectopic growth compared to ACS.