The development of ventricular fibrillation as a complication of pericardiectomy in 16 dogs.

OBJECTIVE: To describe the clinical characteristics, perioperative protocols, and outcomes in dogs diagnosed with ventricular fibrillation (VF) while undergoing pericardiectomy. STUDY DESIGN: Retrospective, multi-institutional study. ANIMALS: Sixteen client-owned dogs. METHODS: Cases were accrued through a listserve request posted to 3 subspecialty veterinary societies. Dogs were included if they developed VF during a pericardiectomy performed through an open or thoracoscopic approach. Data collected included signalment, history and physical examination, surgical approach, histopathology, treatment, and outcome. RESULTS: Indications for pericardiectomy included idiopathic chylothorax (n = 7), neoplasia (4), idiopathic pericardial effusion (4), and foreign body granuloma (1). Surgical approaches included thoracoscopy (12), intercostal thoracotomy (3) and median sternotomy (1). Electrosurgical devices were used to complete at least part of the pericardiectomy in 15 of 16 dogs. Ventricular fibrillation appeared to be initiated during electrosurgical use in 8/15 dogs. However, in 5/15 dogs it was not obviously associated with electrosurgical use. In 3/16 dogs the timing of initiation of VF was unclear. In 7/16 dogs, cardiac arrhythmias were noted prior to the development of VF. Fourteen of 16 dogs died from intraoperative VF. CONCLUSION: In most dogs ventricular fibrillation was a fatal complication of pericardiectomy. Ventricular fibrillation might be associated with the use of electrosurgical devices and cardiac manipulation during pericardiectomy although a causal link could not be established from the data in this study. CLINICAL SIGNIFICANCE: Surgeons must be aware of the risk of VF during pericardial surgery. Electrosurgery might need to be used judiciously during pericardiectomy, particularly in dogs exhibiting cardiac arrythmias.

Dorsal offset rhinoplasty for treatment of stenotic nares in 34 brachycephalic dogs.

OBJECTIVE: To describe the technique, outcome, and owner satisfaction associated with dorsal offset rhinoplasty (DOR) to treat stenotic nares in brachycephalic dogs. STUDY DESIGN: Retrospective case series. ANIMALS: Thirty-four client-owned dogs. METHODS: Medical records of dogs treated with DOR at a veterinary teaching hospital over a 6-year period were identified. Dorsal offset rhinoplasty was defined as removal of a dorsal wedge of nasal planum from each naris with apposition of the rostral abaxial tissue to the caudal axial tissue, resulting in translocation of the alar cartilage in both median and dorsal planes. Immediate and postoperative complications were recorded. Owners were asked to report any complications with healing of the nares and to score their satisfaction with the appearance of the nares. RESULTS: Thirty-four dogs met the inclusion criteria. Twenty-nine (85%) dogs were examined a median of 402.5 days (range, 23-2042) postoperatively, with no major complications related to the rhinoplasty recorded. Eighteen owners responded a median of 701 days (range, 37-1622) postoperatively. One owner reported that self-trauma led to collapse of one naris. One owner reported collapse of both nares within 4 years; timing and cause were unknown. Sixteen of 17 responding owners reported that they were very satisfied with the outcome of the rhinoplasty. The owner of the dog with the collapsed naris was very unsatisfied. One owner did not provide a satisfaction score. CONCLUSION: Owners were generally highly satisfied with DOR, and complications were uncommon. CLINICAL SIGNIFICANCE: This report describes an alternate technique to treat stenotic nares.

Novel murine model for delayed wound healing using a biological wound dressing with Pseudomonas aeruginosa biofilms.

Bacterial biofilms impair healing in 60% of chronic skin wounds. Various animal models (mice, rats, rabbits, and pigs) have been developed to replicate biofilm infected wounds in vivo. We developed a sustained wound infection model by applying preformed Pseudomonas aeruginosa biofilms on a wound dressing to full-thickness murine skin wounds. We bathed a commercially available wound dressing in P. aeruginosa for 48 h, allowing a biofilm to establish on the dressing prior to application to the wound. Dressings were removed from the wounds after 3 days at which time the wound beds contained ∼108 bacterial cells per gram tissue. Significant numbers of P. aeruginosa persisted within the skin wounds for up to 21 days. Un-inoculated wounds reached closure between 9 and 12 days. In contrast, biofilm-inoculated wounds achieved closure between 18 and 21 days. Histologic analysis confirmed decreased re-epithelialization and collagen deposition, coupled with increased inflammation, in the biofilm-inoculated wounds compared to un-inoculated controls. This novel model of delayed healing and persistent infection of full-thickness murine skin wounds may provide a robust in vivo system in which to test novel treatments to prevent wound infection by bacterial biofilms.

A Carrel patch technique for renal transplantation in cats.

OBJECTIVE: To evaluate the feasibility of a Carrel patch method in feline renal transplantation. STUDY DESIGN: Descriptive case series. ANIMALS: Nine healthy donor cats and 9 client recipient cats with chronic renal failure. METHODS: Renal transplantation was performed in 9 cats with chronic renal failure after collection of a donor's left kidney with a Carrel patch technique. A patch of donor aortic wall was removed with either 2 or 1 renal artery (ies) (n = 1 and 8 cats, respectively) central to the patch, with a cuff of tissue (≤1 mm) protruding from the base of the vessels. The Carrel patch was implanted in recipient cats with an end-to-side artery-to-aorta anastomosis, in a simple-continuous pattern of 9-0 nylon. The renal vein and ureter were implanted as previously described. RESULTS: All donors and recipients survived surgery without vascular complication. CONCLUSION: The Carrel patch is a novel approach allowing the harvest of kidneys with multiple renal arteries. The technique also simplified the implant procedure, potentially decreasing the risks of bleeding and thrombosis.

Ureteral Papilla Implantation as a Technique for Neoureterocystostomy in Cats Undergoing Renal Transplantation: 30 Cases.

OBJECTIVE: To describe the clinical outcome of donor and recipient cats undergoing ureteral papilla harvest and implantation as a technique for neoureterocystostomy in clinical kidney transplant. STUDY DESIGN: Retrospective case series. ANIMALS: Donor (n=31) and recipient (n=30) cats that underwent kidney harvest and transplantation using ureteral papilla implantation technique for neoureterocystostomy. METHODS: Medical records for donor and recipient cats presented to the University of Wisconsin Veterinary Teaching Hospital from January 2003 to December 2014 were reviewed. Data recorded included complete blood count, serum chemistry panel, surgical technique, diagnostic imaging results, short- and long-term complications, and anesthetic survival. RESULTS: All 30 recipients recovered from anesthesia. Four died within 24 hours and 26 survived to hospital discharge. Serum creatinine was within the reference interval by 72 hours in 22/26 cats (85%). Complications related to the ureteral papilla implantation technique were seen in only 1 cat (3%). Uroabdomen diagnosed on day 3 ultimately resolved over the following 24 hours without surgical intervention. All 31 donor cats survived to discharge. Four donors (13%) experienced mild, transiently increased serum creatinine. CONCLUSION: Ureteral papilla implantation is a viable technique for neoureterocystostomy in cats undergoing kidney transplantation. Proposed benefits for the recipient include a less technically challenging anastomosis, decreased risk of ureteral obstruction at the anastomosis site, and reduced risk of leakage compared to previous reports. Benefits for recipients should be weighed against risks to donors, including a more complex ureteral harvest, increased surgical time, and potential injury or obstruction of the contralateral ureteral papilla.

Inhibition of Pseudomonas aeruginosa biofilm formation on wound dressings.

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.

Importance of defining experimental conditions in a mouse excisional wound model.

The murine dorsum dermal excisional wound model has been widely utilized with or without splint application. However, variations in experimental methods create challenges for direct comparison of results provided in the literature and for design of new wound healing studies. Here, we investigated the effects of wound location and size, number of wounds, type of adhesive used for splint fixation on wound healing using splinted or unsplinted dorsum excisional full thickness wound models. One or two 6- or 8-mm full thickness wounds were made with or without splinting in genetically diabetic but heterozygous mice (Dock7(m) + / + Lepr(db) ). Two different adhesives: tissue adhesive and an over the counter cyanoacrylate adhesive (OTCA) "Krazy glue" were used to fix splints. Wound contraction, wound closure, and histopathological parameters including reepithelialization, collagen deposition and inflammation were compared between groups. No significant effect of wound number (1 vs. 2), side (left vs. right and cranial vs. caudal) or size on wound healing was observed. The OTCA group had a significantly higher splint success compared to the tissue adhesive group that resulted in significantly higher reepithelialization and collagen deposition in the OTCA group. Understanding the outcomes and effects of the variables will help investigators choose appropriate experimental conditions for the study purpose and interpret data.

Outcome of Donor Cats After Unilateral Nephrectomy as Part of a Clinical Kidney Transplant Program.

OBJECTIVE: To compare 1) complications between 2 ureteral harvest techniques (ureteral papilla harvest [UPH] and ureteral transection [UT]); 2) to investigate the prevalence of kidney failure in a population of kidney donors; and 3) to evaluate owner satisfaction with commercially sourced cats adopted after kidney donation. STUDY DESIGN: Retrospective case series. ANIMALS: Cats (n = 72) that had unilateral nephrectomy for kidney donation. METHODS: Medical records were reviewed and information on short- and long-term complications and evidence of kidney failure was recorded. Clients were interviewed by telephone to ascertain their satisfaction with the adopted donor cats as pets. RESULTS: Seventy-two cats had unilateral nephrectomy. Forty-two owners were able to be contacted for survey data. Twenty-eight cats had complete medical records including serum BUN, creatinine, and urine specific gravity. For these 28 cats, mean age at nephrectomy was 1.9 years (median, 1.1 years; range, 0.5-9.3 years) and mean age at follow-up was 6.8 years (median, 5.1 years; range, 1.0-18.7 years). There was no difference in major or minor complication rates between UPH and UT techniques. Kidney failure occurred in 17.8% of cats. All owners were satisfied with the adopted donor cats, which were obtained from commercial facilities. CONCLUSIONS: UPH is a safe technique in cats being used for kidney donation. Commercially sourced cats make suitable pets after kidney donation. The prevalence of kidney failure in the donor population appears to be higher than that in the general population, but definitive conclusions cannot be made based on this study. Further, prospective study is needed to identify the true prevalence of kidney failure in cats after unilateral nephrectomy.

Raman spectroscopy enables noninvasive biochemical characterization and identification of the stage of healing of a wound.

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.

Tryptophan inhibits biofilm formation by Pseudomonas aeruginosa.

Biofilm formation by Pseudomonas aeruginosa has been implicated in the pathology of chronic wounds. Both the d and l isoforms of tryptophan inhibited P. aeruginosa biofilm formation on tissue culture plates, with an equimolar ratio of d and l isoforms producing the greatest inhibitory effect. Addition of d-/l-tryptophan to existing biofilms inhibited further biofilm growth and caused partial biofilm disassembly. Tryptophan significantly increased swimming motility, which may be responsible in part for diminished biofilm formation by P. aeruginosa.

Craniectomies for Dogs With Skull Multilobular Osteochondrosarcoma Using the Misonix Bone Scalpel: Cadaveric Evaluation and Retrospective Case Series.

To evaluate the Misonix bone scalpel (MBS) for craniotomies in dogs and describe clinical findings and surgical experience in 3 dogs with large multilobular osteochondrosarcoma (MLO) of the skull. Cadaver evaluation and retrospective case series. One canine cadaver; 3 client-owned dogs. Craniotomies of different sizes and at different locations were performed with MBS. Dural tear and bone discoloration were recorded. Clinical, imaging, and surgical findings of dogs diagnosed with MLO and where MBS was used for craniectomies were retrospectively included. Cadaveric evaluation identified MBS as an efficient tool for rapid craniectomies (>5minutes) albeit dural tears and some small foci of bone discoloration were observed. Craniectomies could be performed without complications in 3 dogs with MLO without dural tear or bone discoloration. .Excision was in complete in all cases. The short-term outcome was good, and the long-term outcome was fair to good. Piezoelectric bone surgery with the Misonix bone scalpel is an alternative technology to perform craniectomies in dogs. It was not associated with complications in 3 dogs diagnosed and surgically treated for MLO. Dural tears and suspected bone necrosis can occur. Great care should be taken when using CT to establish disease free surgical osteotomy.

Antibacterial efficacy of silver-impregnated polyelectrolyte multilayers immobilized on a biological dressing in a murine wound infection model.

OBJECTIVE: To investigate the antibacterial effect of augmenting a biological dressing with polymer films containing silver nanoparticles. BACKGROUND: Biological dressings, such as Biobrane, are commonly used for treating partial-thickness wounds and burn injuries. Biological dressings have several advantages over traditional wound dressings. However, as many as 19% of wounds treated with Biobrane become infected, and, once infected, the Biobrane must be removed and a traditional dressing approach should be employed. Silver is a commonly used antimicrobial in wound care products, but current technology uses cytotoxic concentrations of silver in these dressings. We have developed a novel and facile technology that allows immobilization of bioactive molecules on the surfaces of soft materials, demonstrated here by augmentation of Biobrane with nanoparticulate silver. Surfaces modified with nanometer-thick polyelectrolyte multilayers (PEMs) impregnated with silver nanoparticles have been shown previously to result in in vitro antibacterial activity against Staphylococcus epidermidis at loadings of silver that are noncytotoxic. METHODS: We demonstrated that silver-impregnated PEMs can be nondestructively immobilized onto the surface of Biobrane (Biobrane-Ag) and determined the in vitro antibacterial activity of Biobrane-Ag with Staphylococcus aureus. In this study, we used an in vivo wound infection model in mice induced by topical inoculation of S aureus onto full-thickness 6-mm diameter wounds. After 72 hours, bacterial quantification was performed. RESULTS: Wounds treated with Biobrane-Ag had significantly (P < 0.001) fewer colony-forming units than wounds treated with unmodified Biobrane (more than 4 log10 difference). CONCLUSIONS: The results of our study indicate that immobilizing silver-impregnated PEMs on the wound-contact surface of Biobrane significantly reduces bacterial bioburden in full-thickness murine skin wounds. Further research will investigate whether this construct can be considered for human use.

Peptides that anneal to natural collagen in vitro and ex vivo.

Collagen comprises » of the protein in humans and ¾ of the dry weight of human skin. Here, we implement recent discoveries about the structure and stability of the collagen triple helix to design new chemical modalities that anchor to natural collagen. The key components are collagen mimetic peptides (CMPs) that are incapable of self-assembly into homotrimeric triple helices, but are able to anneal spontaneously to natural collagen. We show that such CMPs containing 4-fluoroproline residues, in particular, bind tightly to mammalian collagen in vitro and to a mouse wound ex vivo. These synthetic peptides, coupled to dyes or growth factors, could herald a new era in assessing or treating wounds.

Retroperitoneal fibrosis as a postoperative complication following renal transplantation in cats.

OBJECTIVES: The aim of this report was to describe the clinical signs, diagnostic imaging findings, surgical management, histopathological findings, outcome and possible risk factors for cats that developed retroperitoneal fibrosis (RPF) following renal transplantation. METHODS: Medical records of cats that underwent renal transplantation and developed clinically significant RPF between 1995 and 2019 were reviewed. RESULTS: Eighty-one cats underwent 83 renal transplantations. Of these 81 cats, six developed clinically significant RPF. For all six cats, renal transplantation was performed using cold organ preservation solution and ureteral papilla implantation. Immunosuppression protocol included ciclosporin and prednisolone. All cats had at least one subtherapeutic trough ciclosporin level (<250 ng/ml) in the postoperative period. Cats presented with moderate-to-severe azotemia 39-210 days following renal transplantation. Abdominal ultrasonography and contrast pyelography revealed various degrees of hydroureter and hydronephrosis of the transplanted kidney. Surgical examination revealed a layer of dense fibrous tissue surrounding the transplanted kidney, ureter and bladder resulting in ureteral obstruction. Ureteral obstruction was managed by reimplantation of the proximal ureter or renal pelvis to the bladder. Histopathologic examination of the fibrous tissue and affected portion of the distal ureter revealed fibrous connective tissue with lymphoplasmacytic infiltration and perivascular inflammation suggestive of an autoimmune type reaction. Of the six cats, two died within 5 days after revision surgery, two developed signs consistent with recurrent partial ureteral obstruction (40 and 41 days after revision), one was euthanized 6 years later for an unrelated disease and one was lost to follow-up. CONCLUSIONS AND RELEVANCE: The incidence of RPF in this population of cats was relatively low (7%), but still represents a significant cause of morbidity and mortality. The cause of RPF remains unknown, although investigation into suboptimal immunosuppression as a potential cause for local rejection reaction is warranted.

Bifunctional Peptide that Anneals to Damaged Collagen and Clusters TGF-ß Receptors Enhances Wound Healing.

Transforming growth factor-ß (TGF-ß) plays important roles in wound healing. The activity of TGF-ß is initiated upon the binding of the growth factor to the extracellular domains of its receptors. We sought to facilitate the activation by clustering these extracellular domains. To do so, we used a known peptide that binds to TGF-ß receptors without diminishing their affinity for TGF-ß. We conjugated this peptide to a collagen-mimetic peptide that can anneal to the damaged collagen in a wound bed. We find that the conjugate enhances collagen deposition and wound closure in mice in a manner consistent with the clustering of TGF-ß receptors. This strategy provides a means to upregulate the TGF-ß signaling pathway without adding exogenous TGF-ß and could inspire means to treat severe wounds.

Polymeric Multilayers that contain Silver Nanoparticles can be Stamped onto Biological Tissues to Provide Antibacterial Activity.

We report the design of polyelectrolyte multilayers (PEMs) that can be prefabricated on an elastomeric stamp and mechanically transferred onto biomedically-relevant soft materials, including medical-grade silicone elastomers (E'~450-1500 kPa; E'-elastic modulus) and the dermis of cadaver-skin (E'~200-600 kPa). Whereas initial attempts to stamp PEMs formed from poly(allylamine hydrochloride) and poly(acrylic acid) resulted in minimal transfer onto soft materials, we report that integration of micrometer-sized beads into the PEMs (thicknesses of 6-160 nm) led to their quantitative transfer within 30 seconds of contact at a pressure of ~196 kPa. To demonstrate the utility of this approach, PEMs were impregnated with a range of loadings of silver-nanoparticles and stamped onto the dermis of human cadaver-skin (a wound-simulant) that was subsequently incubated with bacterial cultures. Skin-dermis stamped with PEMs that released 0.25±0.01 µg cm-2 of silver ions caused a 6 log10 reduction in colony forming units of Staphylococcus epidermidis and Pseudomonas aeruginosa within 12 h. Significantly, this level of silver release is below that which is cytotoxic to NIH 3T3 mouse fibroblast cells. Overall, this study describes a general and facile approach for the functionalization of biomaterial surfaces without subjecting them to potentially deleterious processing conditions.

Prospective comparison of cisterna chyli ablation to pericardectomy for treatment of spontaneously occurring idiopathic chylothorax in the dog.

OBJECTIVE: Prospective comparison of cisterna chyli ablation (CCA) or pericardectomy (PC) for chylothorax. STUDY DESIGN: Randomized prospective study. SUBJECT POPULATION: Dogs with idiopathic chylothorax (n= 23). METHODS: Dogs were treated by thoracic duct ligation (TDL) with either CCA (n = 12) or PC (n = 11). Long-term outcomes, intraoperative central venous pressures (CVPs) and pericardial histology were assessed. Dogs with persistent chylothorax were offered retreatment by the alternative procedure. RESULTS: Ten (83%) dogs treated by CCA-TDL and 6 (60%) treated by PC-TDL resolved their chylothorax. Retreatment in 4 dogs resulted in resolution in 2 dogs and 2 perioperative deaths. Four dogs developed nonchylous effusions; 2 of which resolved after initiating steroid therapy, 1 of which was unsuccessfully treated by PC, and 1 continues to be managed by thoracocentesis 6.5 years later. CVPs were normal in most dogs and unaffected by PC. On histology, pericardial tissues had extensive external surface fibrosis with mild inflammation. On follow-up (≤ 6.5 years), no recurrence of pleural effusions occurred after initial resolution. CONCLUSIONS: CCA-TDL appears to offer improved outcomes over historical results with TDL. Results with PC-TDL were more variable for unknown reasons. Venous pressure measurements did not support the hypothesis that venous hypertension was involved in chylothorax or response to therapy in these dogs.

Single paracostal approach to thoracic duct and cisterna chyli: experimental study and case series.

OBJECTIVE: To-determine the feasibility of a single paracostal abdominal approach for thoracic duct ligation (TDL) and cisterna chyli ablation (CCA) in dogs with chylothorax. STUDY DESIGN: Observational study and prospective case series. ANIMALS: Normal dogs (n = 5) and dogs with chylothorax (n = 8). METHODS: A single paracostal approach with transdiaphragmatic extension for TDL and CCA was developed experimentally (n = 5) and used in 8 clinical cases with subtotal pericardectomy (SPE) performed in 4 dogs. Surgery time, complications, hospitalization time, outcome, and follow-up of clinical cases were recorded. RESULTS: Exposure of relevant anatomy was excellent; vital lymphatic staining facilitated identification of lymphatic structures. In clinical cases, mean surgery time for TDL + CCA was 136 minutes. Mean hospitalization time was 3.1 days. Seven of 8 cases survived, with 1 dog dying of heart failure shortly after discharge. One dog required a second (left) paracostal approach to ligate 2 more lymphatic vessels. On follow-up (median, 7 months; range, 2-20 months), there was complete resolution of chylothorax in 6 dogs. CONCLUSIONS: A single paracostal approach provides excellent exposure of cisterna chyli, caudal thoracic duct, and intestinal lymphatics. This approach eliminates the need for repositioning during combined TDL + CCA procedures and avoids an intercostal thoracotomy.

Changing the Wound: Covalent Immobilization of the Epidermal Growth Factor.

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.