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Aim: Repair of peripheral nerves is recommended following transection. Systematic evaluation of longitudinal recovery in injury models is needed to improve patient management. Gompertz function provided straightforward interpretation and prediction of recovery outcomes. Materials & methods: Behavioural sciatic function index, measured 3 days post injury, and weekly for 12 weeks following full nerve transection and repair (n = 6) as well as crush injuries (n = 6). Results: Gompertz parametrization provided early classification between types of traumatic peripheral nerve injuries following surgical repair. Results distinguished injury nerves (A: p i: p

Delays in treatment of burn injuries can lead to significant morbidity, loss of function, and poor aesthetic appearance. Preventing conversion from partial- to full-thickness burns may help mitigate these sequelae. The pathophysiology of burn wound conversion remains unknown, but an overactive immune response is thought to be implicated. The purpose of this study was to determine whether downregulating the immune response via tacrolimus can decrease burn wound conversion.Assembly of the microfluidic hydrogels was achieved by embedding microfibers within a hydrogel scaffold composed of a gelatin-alginate blend. Tacrolimus stock solution for intraperitoneal injection was made by re-suspending powdered tacrolimus in DMSO at 10 mg/mL. 24 young (2-4 months) and 24 old (16 months) mice were given partial thickness burns. The treatment cohort received either tacrolimus ointment with a hydrogel dressing (6 young and 6 old) or an intraperitoneal injection of a tacrolimus solution (6 young and 6 old), while the control cohort only received either only the microcapillary hydrogel dressing or an intraperitoneal injection of saline. Mice were euthanized at day 3 after injury and skin samples were taken. Burn depth was evaluated using Vimentin immunostaining.In old mice, intraperitoneal injection of tacrolimus was able to significantly reduce burn wound depth compared to intraperitoneal injection of saline (p = 0.011). Similarly in old mice, topical hydrogel with tacrolimus was able to significantly reduce burn wound depth compared to hydrogel alone (p 0.001). Topical hydrogel with tacrolimus was able to mitigate the detrimental effects of older age on wound conversion, such that burn wounds of older mice treated with tacrolimus hydrogel dressing had similar burn depths as younger mice (p = 0.240).Utilizing a combination treatment of tacrolimus and microcapillary hydrogel is able to rescue the negative effects of aging and prevent partial- to full-thickness burn wound conversion. Hopefully these findings will encourage deeper investigation into the possible therapeutic advantages of utilizing immunosuppressive agents to decrease morbidity after burn injuries. Future research will need to specifically investigate IL-2 as an inhibitory target in the acute inflammatory cascade of burn injury.

Burn injury remains a serious cause of morbidity and mortality worldwide. Severity of burns is determined by the percentage of burned area compared to the body surface area, age of patient, and by the depth of skin and soft tissue involvement; these factors determine management as well as prospective outcomes. The pathophysiology of partial- to full-thickness burn conversion remains poorly understood and is associated with a worse overall prognosis. Recent studies have demonstrated that an altered inflammatory response may play a significant role in this conversion and therefore a reduction in early inflammation is crucial to ultimately decreasing burn severity and morbidity. We hypothesize that the application of a microcapillary gelatin-alginate hydrogel loaded with anti-TNF-α (infliximab) monoclonal antibodies to a partial-thickness burn will reduce inflammation within partially burned skin and prevent further progression to a full-thickness burn.Assembly of the microfluidic hydrogels is achieved by embedding microfibers within a hydrogel scaffold composed of a gelatin-alginate blend, which is then soaked in a solution containing anti-TNF-α antibodies for drug loading. 12 young (2-4 months) and 12 old (16 months) mice were given partial thickness burns. The treatment cohort received the anti-TNF-α infused hydrogel with an occlusive dressing and the control cohort only received an occlusive dressing. Mice were euthanized at post-burn day 3 and skin samples were taken. Burn depth was evaluated using Vimentin immunostaining.All mice in the treatment cohort demonstrated decreased conversion of burn from partial to full thickness injury (old = p0.01, young = p0.001) as compared to the control group. Old mice had greater depth of burn than young mice (p0.001). There were greater eosinophils in the treatment cohort for both young and old mice, but it did not reach statistical significance.The application of a novel microcapillary gelatin-alginate hydrogel infused with anti-TNF-α antibody to partial thickness burns in mice showed reduction in partial to full thickness burn secondary progression as compared to controls using this murine model; this promising finding might help decrease the high morbidity and mortality associated with burn injuries.

Introduction: Peripheral nerve injuries commonly result from trauma and can lead to devastating loss of sensory and motor function. A novel strategy to improve peripheral nerve regeneration is a chemical fusogen known as polyethylene glycol (PEG). Several animal studies have illustrated PEG’s potential to help prevent axon loss after peripheral nerve injury. However, the relative rate of success and potential complications of these studies have not been definitively shown in the literature. The purpose of this systematic review is to evaluate the literature regarding the success of PEG adjunct treatment after peripheral nerve injury in preclinical models. Materials and Methods: The MEDLINE database was queried using the PubMed search engine with the following keywords and phrases: “polyethylene glycol” OR “PEG” AND “nerve” AND “fusion”. All resulting articles were screened by two reviewers. Animal type, nerve type, injury type, type(s) of analyses, and overall superiority of outcomes were assessed. Results: One-hundred and seventy-nine articles were identified, and thirteen studies remained after the application of inclusion and exclusion criteria. Twelve of the thirteen studies utilized rats as the preclinical model, while one utilized a guinea pig. Superiority of peripheral nerve repair outcomes with adjunct PEG treatment compared to a control group was reported in eleven of thirteen studies. Conclusions: The majority of studies reported positive outcomes when using PEG; this indicates that PEG treatment has the potential to enhance peripheral nerve regeneration after injury. However, the results of some of these studies indicated several uncertainties that need to be addressed in future studies. These preclinical models may help guide clinicians regarding the use of PEG treatment in peripheral nerve repair.

Partial burn injury in older patients is associated with higher rates of morbidity, mortality, and conversion to full thickness burn (Finnerty et al., 2009; Pham et al., 2009). Both human and mouse models demonstrate an altered systemic immune response in older subjects, however less is known about the localized response (Jeschke et al., 2016; Farinas et al., 2018; Mohs et al., 2017). We hypothesized that a mouse model could demonstrate differences in the localized inflammatory response of the old.Six old (66 weeks) and young (8 weeks) mice received partial thickness thermal burns. Localized and systemic expression of nine chemokines (TNFalpha, MCP-1, MIP-2, S100A9, EGF, IL-10, RANTES, G-CSF, and EOTAXIN) were evaluated at day 3 after burn using Luminex analysis. Vimentin immunostaining was used to evaluate injury depth.Vimentin staining demonstrated increased burn depth in old mice (449±38μm) as compared to young (166±18μm) (p0.05). Both groups exhibited increased localized expression of EOTAXIN after burn (p0.05), however expression in old mice (83.6±6.1pg/ml) was lower than that of young (126.8±18.7pg/ml) (p0.05). Systemically, however, old mice had increased baseline EOTAXIN expression (1332.40±110.78pg/ml) compared to young (666.12±45.8pg/ml) (p0.005).EOTAXIN is one of the primary chemoattractants for selective eosinophilic recruitment and activation. While eosinophils are important for wound healing, a hyperactive eosinophilic response can result in tissue damage. We hypothesize that the increased baseline serum EOTAXIN in the old may prime their hyperactive response, and may contribute to their worse clinical outcomes. Long-term eosinophil activation requires further study, however our findings indicate a role for EOTAXIN and eosinophils in burn response.

Advanced age alters many physiological processes in the body, including both innate and adaptive immune responses, affecting burn wound healing. Previous findings in our lab led us to look more closely at eosinophil infiltration of burn tissues. We hypothesize that burn wounds within the older population present with an increased population of eosinophils than those in the younger population.A pilot study was performed utilizing samples collected from male and female patients 30-years-old and younger and 65-years-old and older. Samples were collected at day (PBD) 2-6 after burn. Deep partial-thickness burn tissues were collected during surgery, formalin-fixed paraffin embedded (FFPE), and assessed by HE to confirm deep partial-thickness injury. Immunohistochemistry (IHC) was then performed for Major Basic Protein (MBP) to identify eosinophils. Eosinophils/mm burn were calculated. Welch's Test was used to determine statistical significance of eosinophil measurements between young and old groups.Thirteen samples, were divided into two groups, Young (n=10) and Old (n=3). The mean and median age for Young was 23yo (Max 30yo; Min. 17yo). The mean age was 81yo and the median 84yo for the Old (Max. 93yo; Min. 67yo). Other demographics included race. It was found that the Young and Old groups had a mean of 0.171 Eos/mm and 0.910 Eos/mm, respectively, which was statistically significant (p=0.017).Older patients do present with increased eosinophil infiltration in the early stages of burn wound healing within our small sample set. Increased sample numbers will be required to confirm this discovery.

Porous, resorbable biomaterials can serve as temporary scaffolds that support cell infiltration, tissue formation, and remodeling of nonhealing skin wounds. Synthetic biomaterials are less expensive to manufacture than biologic dressings and can achieve a broader range of physiochemical properties, but opportunities remain to tailor these materials for ideal host immune and regenerative responses. Polyesters are a well-established class of synthetic biomaterials; however, acidic degradation products released by their hydrolysis can cause poorly controlled autocatalytic degradation. Here, we systemically explored reactive oxygen species (ROS)–degradable polythioketal (PTK) urethane (UR) foams with varied hydrophilicity for skin wound healing. The most hydrophilic PTK-UR variant, with seven ethylene glycol (EG7) repeats flanking each side of a thioketal bond, exhibited the highest ROS reactivity and promoted optimal tissue infiltration, extracellular matrix (ECM) deposition, and reepithelialization in porcine skin wounds. EG7 induced lower foreign body response, greater recruitment of regenerative immune cell populations, and resolution of type 1 inflammation compared to more hydrophobic PTK-UR scaffolds. Porcine wounds treated with EG7 PTK-UR foams had greater ECM production, vascularization, and resolution of proinflammatory immune cells compared to polyester UR foam–based NovoSorb Biodegradable Temporizing Matrix (BTM)–treated wounds and greater early vascular perfusion and similar wound resurfacing relative to clinical gold standard Integra Bilayer Wound Matrix (BWM). In a porcine ischemic flap excisional wound model, EG7 PTK-UR treatment led to higher wound healing scores driven by lower inflammation and higher reepithelialization compared to NovoSorb BTM. PTK-UR foams warrant further investigation as synthetic biomaterials for wound healing applications.

Although neurotization has the potential to improve sensory outcomes after autologous breast reconstruction, this technique remains controversial. There is debate regarding the clinical outcomes and the recipient nerve of choice. This histoanatomical study aims to quantitatively compare the sensory components of the recipient nerves involved in neurotization of the deep inferior epigastric perforator flap.Subjects undergoing bilateral autologous breast reconstruction were enrolled. Transected nerve specimens underwent immunohistochemical staining with antibodies against neurofilament 1 and choline acetyltransferase for total and motor neurons within the axons, respectively. Photomicrographs were captured, and axons were analyzed using ImageJ. Sensory axons were calculated as equal to the difference between the total and cholinergic axonal counts.Thirty-eight nerves from 19 subjects were included. The overall mean sensory axon count was 1246.3 (±1171.9) in the lateral cutaneous branch (LCB) of the fourth intercostal nerve and 1123.8 (±1213.0) in the anterior cutaneous branch (ACB) of the third intercostal nerve.The fourth LCB presented with an additional 10.9% sensory axonal count (P0.05). On average, sensory fibers constituted 36.7% and 31.7% of all fibers in the third ACBs and fourth LCBs, respectively.This study provides anatomic and histological evidence that the fourth LCB and third ACB contain comparable mean numbers of sensory axons. Both constitute adequate recipient nerves for coaptation in deep inferior epigastric perforator reinnervation to achieve optimal sensory return after breast reconstruction. The fourth LCB should be preferable when the third ACB remains intact to preserve any native breast flap sensation.

Impaired skin healing and progression into chronic wounds is a prevalent and growing medical problem. Porous, resorbable biomaterials can be used as temporary substrates placed into skin defects to support cell infiltration, neo-tissue formation, and remodeling of nonhealing wounds. Naturally-derived biomaterials have promising healing benefits, but their low mechanical properties and exuberant costs limit their performance and use. Synthetic materials can be affordably manufactured and tuned across a broader range of physiochemical properties, but opportunities remain for tailoring them for ideal host immune and regenerative responses. Polyesters are the most clinically-tested class of synthetic biomaterials, but their hydrolysis releases acidic degradation products that can cause autocatalytic degradation processes that are poorly controlled and are not tied to cellular or other biologic activities. Here, we systemically explored a series of ROS-degradable polythioketal (PTK) urethane (UR) foams with varied hydrophilicity as an alternative class of synthetic biomaterials for wound healing. It was found that the most hydrophilic PTK- UR variant, which had 7 ethylene glycol (EG7) repeats flanking each side of each thioketal bond, had the highest ROS reactivity of the PTK-URs tested. In an in vivo porcine excisional skin wound healing model, hydrophilic EG7 PTK-UR foams more effectively promoted tissue integration, ECM deposition, and re- epithelialization of full-thickness skin wound compared to more hydrophobic PTK-UR variants. Resolution of type 1 inflammation and lower foreign body response to scaffold remnants was also observed for EG7 versus more hydrophobic PTK-UR scaffolds. Finally, porcine wound healing studies showed that EG7 PTK-UR foams had similar wound healing response to a collagen-based clinical gold standard product, Integra Bilayer Wound Matrix (BWM), while outperforming polyester UR foam-based NovoSorb Biodegradable Temporizing Matrix (BTM) with respect to increased ECM production, vascularization, and biomaterial-associated immune phenotype. In sum, PTK-UR foams warrant further development toward a new class of synthetic biomaterial foams for skin wound healing applications.

Objective Primary repair of peripheral nerves is recommended following transection; however, patient management following repair is challenged by a lack of biomarkers to nerve regeneration. Previous studies have demonstrated that diffusion magnetic resonance imaging (MRI) may provide viable biomarkers of nerve regeneration in injury models; though, these methods have not been systematically evaluated in graded partial transections and repairs. Methods Ex vivo diffusion MRI was performed in fixed rat sciatic nerve samples 4 or 12 weeks following partial nerve transection and repair (25% cut = 12, 50% cut = 12 and 75% cut = 11), crush injuries (n = 12), and sham surgeries (n = 9). Behavioral testing and histologic evaluation were performed in the same animals and nerve samples for comparison. Results Diffusion tractography provided visual characterizations of nerve damage and recovery consistent with the expected degree of injury within each cohort. In addition, quantitative indices from diffusion MRI correlated with both histological and behavioral evaluations, the latter of indicated full recovery for sham and crush nerves and limited recovery in all partially transected/repaired nerves. Nerve recovery between 4 and 12 weeks was statistically significant in partial transections 50% and 75% depth cuts (p = 0.043 and p = 0.022) but not for 25% transections. Interpretation Our findings suggest that DTI can i) distinguish different degrees of partial nerve transection following surgical repair and ii) map spatially heterogeneous nerve recovery (e.g., due to collateral sprouting) from 4 to 12 weeks in partially transected nerves.

Nerve regeneration after injury must occur in a timely fashion to restore function. Unfortunately, current methods (e.g., electrophysiology) provide limited information following trauma, resulting in delayed management and suboptimal outcomes. Herein, we evaluated the ability of diffusion MRI to monitor nerve regeneration after injury/repair. Sprague-Dawley rats were divided into three treatment groups (sham = 21, crush = 23, cut/repair = 19) and ex vivo diffusion tensor imaging (DTI) and diffusion kurtosis imaging (DKI) was performed 1-12 weeks post-surgery. Behavioral data showed a distinction between crush and cut/repair nerves at 4 weeks. This was consistent with DTI, which found that thresholds based on the ratio of radial and axial diffusivities (RD/AD = 0.40 ± 0.02) and fractional anisotropy (FA = 0.53 ± 0.01) differentiated crush from cut/repair injuries. By the 12

BACKGROUND Nerve regeneration after an injury should occur in a timely fashion for function to be restored. Current methods cannot monitor regeneration prior to muscle reinnervation. Diffusion tensor imaging has been previously shown to provide quantitative indices after nerve recovery. The goal of this study was to validate the use of this technology following nerve injury via a series of rat sciatic nerve injury/repair studies. METHODS Sprague-Dawley rats were prospectively divided by procedure (sham, crush, or cut/repair) and time points (1, 2, 4, and 12 weeks after surgery). At the appropriate time point, each animal was euthanized and the sciatic nerve was harvested and fixed. Data were obtained using a 7-Tesla magnetic resonance imaging system. For validation, findings were compared to behavioral testing (foot fault asymmetry and sciatic function index) and cross-sectional axonal counting of toluidine blue-stained sections examined under light microscopy. RESULTS Sixty-three rats were divided into three treatment groups (sham, n = 21; crush, n = 23; and cut/repair, n = 19). Fractional anisotropy was able to differentiate between recovery following sham, crush, and cut/repair injuries as early as 2 weeks (p < 0.05), with more accurate differentiation thereafter. More importantly, the difference in anisotropy between distal and proximal regions recognized animals with successful and failed recoveries according to behavioral analysis, especially at 12 weeks. In addition, diffusion tension imaging-based tractography provided a visual representation of nerve continuity in all treatment groups. CONCLUSIONS Diffuse tensor imaging is an objective and noninvasive tool for monitoring nerve regeneration. Its use could facilitate earlier detection of failed repairs to potentially help improve outcomes.

Background: Epitendinous sutures not only join the 2 severed tendon edges but also supply strength and support to the repair. Multiple techniques have been described, but none of them include another thread of suture. This could potentially increase the strength of the repair without affecting gliding. Methods: Caprine tendons were harvested, transected, and sutured with 6-0 Prolene. Four groups were created: single thread running (SR), single thread locking (SL), double thread running (DR), and double thread locking (DL). An Instron 5542 was used to pull the repaired tendons apart, and the energy at the break was calculated (gf/mm). For gliding resistance, we harvested a human A2 pulley. A pre- and postrepair value was obtained, and a ratio was elaborated. A single-factor analysis of variance and independent sample t tests were performed. Results: The SR group had a mean energy at break of 9339.71 ± 1630.212 gf/mm; the SL group, 9629.96 ± 1476.45 gf/mm; and the DR group, 9600.221 ± 976.087 gf/mm, with no statistical significance. The DL group was significantly higher at 14 740.664 ± 2596.586 gf/mm ( P < .05). When comparing SR with DL for gliding, SR had less than half of the resistance than DL (0.018 ± 0.004 and 0.049 ± 0.015 N/mm, respectively), with statistical significance ( P < .05). Conclusion: Using a single suture thread for running epitendinous repair is no different than locking with a single thread or using an extra thread in a running fashion. Performing a double suture thread with a locking technique is significantly stronger than the previously mentioned repairs, with the disadvantage of more resistance at the pulley.

Peripheral nerve gap injuries continue to present a clinical challenge to today’s surgeons. One method of surgical repair, implantation of acellular allografts, has been developed with the aim of bridging the gap with a cadaveric graft following removal of its cellular components, thereby accelerating axonal regeneration and eliminating the need for immunosuppression in recipient patients. While decellularizing allografts reduces rates of graft rejection, the same chemical processing modifies the neural microenvironment, removing neutrotrophic factors and modifying the complex extracellular matrix. In this study we explore three common methods for producing acellular allografts. Extracellular matrix content remaining after processing was investigated, and was found to be highly dependent on the decellularization method. In addition, scanning electron micrographs were obtained to evaluate the structural effects of the decellularization methods. Though the content and structure of these processed allografts will contribute to their effectiveness as nerve gap repair candidates, we demonstrate that it also affects their capacity to be supplemented/pre-loaded with the prototypical neurotrophin, nerve growth factor (NGF), essential to neuronal regeneration. While all allografts had some capacity for retaining NGF in the first 24 hours, only Sondell-processed grafts retained NGF over the entire experimental period of 21 days. Future studies will include validating these processed and supplemented allografts as viable alternatives to traditional autograft nerve gap repair.

Purpose Given no definite consensus on the accepted autograft orientation during peripheral nerve injury repair, we compare outcomes between reverse and normally oriented autografts using an advanced magnetic resonance imaging technique, diffusion tensor imaging. Methods Thirty-six female Sprague-Dawley rats were divided into 3 groups: sham-left sciatic nerve isolation without injury, reverse autograft-10-mm cut left sciatic nerve segment reoriented 180° and used to coapt the proximal and distal stumps, or normally oriented autograft-10-mm cut nerve segment kept in its normal orientation for coaptation. Animals underwent sciatic functional index and foot fault behavior studies at 72 hours, and then weekly. At 6 weeks, axons proximal, within, and distal to the autograft were evaluated using diffusion tensor imaging and choline acetyltransferase motor staining for immunohistochemistry. Toluidine blue staining of 1-μm sections was used to assess axon count, density, and diameter. Bilateral gastrocnemius/soleus muscle weights were compared to obtain a net wet weight. Comparison of the groups was performed using Mann-Whiney U or Kruskal-Wallis H tests to determine significance. Results Diffusion tensor imaging findings including fractional anisotropy, radial diffusivity, and axial diffusivity were similar between reverse and normally oriented autografts. Diffusion tensor imaging tractography demonstrated proximodistal nerve regeneration in both autograft groups. Motor axon counts proximal, within, and distal to the autografts were similar. Likewise, axon count, density, and diameter were similar between the autograft groups. Muscle net weight at 6 weeks and behavioral outcomes (sciatic functional index and foot fault) at any tested time point were also similar between reverse and normally oriented autografts. Conclusions Diffusion tensor imaging may be a useful assessment tool for peripheral nerve regeneration. Reversing nerve autograft polarity did not demonstrate to have an influence on functional or regenerative outcomes.

Conduit-based nerve repairs are commonly used for small nerve gaps, whereas primary repair may be performed if there is no tension on nerve endings. We hypothesize that a conduit-based nerve coaptation device will improve nerve repair outcomes by avoiding sutures at the nerve repair site and utilizing the advantages of a conduit-based repair.The left sciatic nerves of female Sprague-Dawley rats were transected and repaired using a novel conduit-based device. The conduit-based device group was compared to a control group of rats that underwent a standard end-to-end microsurgical repair of the sciatic nerve. Animals underwent behavioral assessments at weekly intervals post-operatively using the sciatic functional index (SFI) test. Animals were sacrificed at four weeks to obtain motor axon counts from immunohistochemistry. A sub-group of animals were sacrificed immediately post repair to obtain MRI images.SFI scores were superior in rats which received conduit-based repairs compared to the control group. Motor axon counts distal to the injury in the device group at four weeks were statistically superior to the control group. MRI tractography was used to demonstrate repair of two nerves using the novel conduit device.A conduit-based nerve coaptation device avoids sutures at the nerve repair site and leads to improved outcomes in a rat model. Conduit-based nerve repair devices have the potential to standardize nerve repairs while improving outcomes.

Impaired wound healing that mimics chronic human skin pathologies is difficult to achieve in current animal models, hindering testing and development of new therapeutic biomaterials that promote wound healing. In this article, we describe a refinement and simplification of the porcine ischemic wound model that increases the size and number of experimental sites per animal. By comparing three flap geometries, we adopted a superior configuration (15 × 10 cm) that enabled testing of twenty 1 cm2 wounds in each animal: 8 total ischemic wounds within 4 bipedicle flaps and 12 nonischemic wounds. The ischemic wounds exhibited impaired skin perfusion for ∼1 week. To demonstrate the utility of the model for comparative testing of tissue regenerative biomaterials, we evaluated the healing process in wounds implanted with highly porous poly (thioketal) urethane (PTK-UR) scaffolds that were fabricated through reaction of reactive oxygen species (ROS)-cleavable PTK macrodiols with isocyanates. PTK-lysine triisocyanate (LTI) scaffolds degraded significantly in vitro under both oxidative and hydrolytic conditions whereas PTK-hexamethylene diisocyanate trimer (HDIt) scaffolds were resistant to hydrolytic breakdown and degraded exclusively through an ROS-dependent mechanism. Upon placement into porcine wounds, both types of PTK-UR materials fostered new tissue ingrowth over 10 days in both ischemic and nonischemic tissue. However, wound perfusion, tissue infiltration and the abundance of pro-regenerative, M2-polarized macrophages were markedly lower in ischemic wounds independent of scaffold type. The PTK-LTI implants significantly improved tissue infiltration and perfusion compared with analogous PTK-HDIt scaffolds in ischemic wounds. Both LTI and HDIt-based PTK-UR implants enhanced M2 macrophage activity, and these cells were selectively localized at the scaffold/tissue interface. In sum, this modified porcine wound-healing model decreased animal usage, simplified procedures, and permitted a more robust evaluation of tissue engineering materials in preclinical wound healing research. Deployment of the model for a relevant biomaterial comparison yielded results that support the use of the PTK-LTI over the PTK-HDIt scaffold formulation for future advanced therapeutic studies.

Purpose This study aims to compare engineered nerve conduits constructed from porcine-derived urinary bladder matrix (UBM) with the criterion-standard nerve autografts, for segmental loss peripheral nerve repairs. Methods Forty-eight Sprague-Dawley rats were divided into 2 groups. All underwent a 10-mm sciatic nerve gap injury. This was repaired using either (1) reverse autograft-the 10-mm cut segment was oriented 180 degrees and used to coapt the proximal and distal stumps or (2) UBM conduit-the 10-mm nerve gap was bridged with UBM conduit. Behavior assessments such as sciatic function index and foot fault asymmetry scores were performed weekly. At 3- or 6-week time endpoints, the repaired nerves and bilateral gastrocnemius/soleus muscles were harvested from each animal. Nerves were evaluated using immunohistochemistry for motor and sensory axon staining and with diffusion tensor imaging. The net wet muscle weights were calculated to assess the degree of muscle atrophy. Results The UBM group demonstrated significantly improved foot fault asymmetry scores at 2 and 4 weeks, whereas there was no difference in sciatic function index. The net muscle weights were similar between both groups. Motor axon counts proximal/inside/distal to the conduit/graft were similar between UBM conduits and reverse autografts, whereas sensory axon counts within and distal to the conduit were significantly higher than those of the autograft at 6 weeks. Sensory axonal regeneration seemed to be adherent to the inner surface of the UBM conduit, whereas it had a scattered appearance in autografts. Diffusion tensor imaging parameters between groups were similar. Conclusions Urinary bladder matrix conduits prove to be at least similar to nerve autografts for the repair of peripheral nerve injuries with a short gap. The matrix perhaps serves as a scaffold to augment sensory nerve growth. Clinical relevance In a clinical setting, UBM may eliminate the donor site morbidity and increased operative time associated with nerve autografting.

Nerve regeneration after injury must occur in a timely fashion to restore function. Unfortunately, current methods (e.g., electrophysiology) provide limited information following trauma, resulting in delayed management and suboptimal outcomes. Herein, we evaluated the ability of diffusion MRI to monitor nerve regeneration after injury/repair. Sprague-Dawley rats were divided into three treatment groups (sham = 21, crush = 23, cut/repair = 19) and ex vivo diffusion tensor imaging (DTI) and diffusion kurtosis imaging (DKI) was performed 1–12 weeks post-surgery. Behavioral data showed a distinction between crush and cut/repair nerves at 4 weeks. This was consistent with DTI, which found that thresholds based on the ratio of radial and axial diffusivities (RD/AD = 0.40 ± 0.02) and fractional anisotropy (FA = 0.53 ± 0.01) differentiated crush from cut/repair injuries. By the 12th week, cut/repair nerves whose behavioral data indicated a partial recovery were below the RD/AD threshold (and above the FA threshold), while nerves that did not recover were on the opposite side of each threshold. Additional morphometric analysis indicated that DTI-derived normalized scalar indices report on axon density (RD/AD: r = −0.54, p

Nerve regeneration after injury must occur in a timely fashion to restore function. Unfortunately, current methods (e.g., electrophysiology) provide limited information following trauma, resulting in delayed management and suboptimal outcomes. Herein, we evaluated the ability of diffusion MRI to monitor nerve regeneration after injury/repair. Sprague-Dawley rats were divided into three treatment groups (sham=21, crush=23, cut/repair=19) and ex vivo diffusion tensor imaging (DTI) and diffusion kurtosis imaging (DKI) was performed 1-12 weeks post-surgery. Behavioral data showed a distinction between crush and cut/repair nerves at 4 weeks. This was consistent with DTI, which found that thresholds based on the ratio of radial and axial diffusivities (RD/AD=0.40±0.02) and fractional anisotropy (FA=0.53±0.01) differentiated crush from cut/repair injuries. By the 12th week, cut/repair nerves whose behavioral data indicated a partial recovery were below the RD/AD threshold (and above the FA threshold), while nerves that did not recover were on the opposite side of each threshold. Additional morphometric analysis indicated that DTI-derived normalized scalar indices report on axon density (RD/AD: r=−0.54, p

Although the upper extremity is the most commonly injured part of the body, many studies have indicated that there is a lack of emergency hand coverage in the United States. In 2010, our laboratory evaluated on-call hand coverage in Tennessee (TN) and found that only 7% of hospitals had a hand surgeon on call for emergency cases at all times. In 2014, the Affordable Care Act (ACA) was implemented with the goal of increasing overall access to care and decreasing health care costs. Hand surgeons were surveyed on their attitudes toward the ACA, and the majority of surgeons surveyed disagreed or strongly disagree that the ACA would improve access to emergent hand surgery. This study aimed to determine if there has been an increase in emergency hand coverage in TN since the implementation of the ACA. A survey was administered to all hospitals in TN with both an emergency department and operating room to determine the percentage of TN hospitals offering elective hand surgery and on-call emergency hand coverage. With 94% of TN hospitals responding to the emergency department survey, we determined that there has been a 138% significant increase in the percentage of hospitals reporting 24/7 emergency hand coverage by a hand specialist since our last study in 2010. There has also been a significant increase in elective hand coverage in TN, although much smaller at 13% since 2010. This study suggests that there has been an overall increase in access to hand care in TN since the implementation of the ACA, most profoundly seen in the increase in hand specialists available for emergent cases.

The spatiotemporal analysis of the proteomic profile during human wound healing is a critical investigative step that can establish the complex interplay of molecular events that comprise the local response to burn injury. Partial-thickness wound samples with adjacent "normal" skin were collected from twenty-one patients with burn wounds and examined across a time spectrum ranging from the acute injury period at 3, 6, 11 days to the later hypertrophic scar period at 7 and 15 months. The techniques used for histology-directed tissue analyses highlighted inflammatory protein markers at the early time points after injury with diminished expression as burn wounds progressed into the proliferative phase. The datasets show the usefulness of MALDI MS and imaging mass spectrometry as discovery approaches to identify and map the cutaneous molecular sequence that is activated in response to the unique systemic inflammatory response following burn trauma. This information has the potential to define the unique factors that predispose human burn victims to disfiguring hypertrophic scar formation.

Treatment for mandibular fractures is commonly performed via open reduction fixation with mini titanium or resorbable plates and screws. The investigators hypothesized differences in maximum mechanical stress forces and deflection with each plating material; however, it was proposed that the experimental forces withstood by the resorbable system would be enough to withstand normal forces produced during mastication. The sample was composed of fresh cadaver mandibles that were harvested, fractured, and fixated with plates and screws. The predictor variable was fracture fixation and included a titanium plating or resorbable poly-L-lactide plating system. The primary outcome measure was maximum load withstood before plating failure measured in Newtons (N). Descriptive and bivariate statistics were computed, P value was set at .05. The sample was composed of six mandibles with and grouped by type of fixation modality, Titanium (T) (n = 3) and Resorbable (n = 3). There was a statistically significant correlation between the T group and increased maximum load (N) before failure as compared to the R group, (P = 0.023). The results suggest that fracture fixation with titanium plates and screws can withstand greater maximum forces before failure; however, the resorbable plating system withstood forces similar to/or greater than the maximum forces produced during normal mastication., Biomedical engineering; Dentistry; Materials science; Surgey; Resorbable plates; Titanium plates; Fracture; Fixation; Poly-l-lactic acid; Mandible.

BACKGROUND: Diffusion tensor tractography (DTT) has recently been shown to accurately detect nerve injury and regeneration. This study assesses whether 7-tesla (7T) DTT imaging is a viable modality to observe axonal outgrowth in a 4 cm rabbit sciatic nerve injury model fixed by a reverse autograft surgical technique. METHODS: Transection injury of unilateral sciatic nerve (4 cm long) was performed in 25 rabbits and repaired using a reverse autograft (RA) surgical technique. Analysis of the nerve autograft was performed at 3, 6, and 11 weeks postoperatively and compared to normal contralateral sciatic nerve, used as control group. High-resolution DTT from ex vivo sciatic nerves were obtained using 3D diffusion-weighted spin-echo acquisitions at 7-T. Total axons and motor and sensory axons were counted at defined lengths along the graft. RESULTS: At 11 weeks, histologically, the total axon count of the RA group was equivalent to the contralateral uninjured nerve control group. Similarly, by qualitative DTT visualization, the 11-week RA group showed increased fiber tracts compared to the 3 and 6 weeks counterparts. Upon immunohistochemical evaluation, 11-week motor axon counts did not significantly differ between reverse autograft and control; but significantly decreased sensory axon counts remained. Nerves explanted at 3 weeks and 6 weeks showed decreased motor and sensory axon counts. DISCUSSION: 7-T DTT is an effective imaging modality that may be used qualitatively to visualize axonal outgrowth and regeneration. This has implications for the development of technology that non-invasively monitors peripheral nerve regeneration in a variety of clinical settings.

Polyethylene glycol (PEG) is a cell membrane fusogen (de StGroth et al., 1980; Hoehn et al., 1978 [2]; Pontecorvo et al., 1975 [3]). In clinical application PEG allows some axonal fusion in peripheral nerve repairs, resulting in retention of some mobility and sensation during the regeneration process and reducing muscular atrophy. Several manuscripts exist concerning in vivo models of PEG fusion, while there is a distinct lack of in vitro studies. This study optimizes an experimental in vitro PEG fusion procedure in B35 rat neuroblastoma cells, through alteration of PEG and dimethyl sulfoxide (DMSO) concentrations and utilizes flow cytometry for assessment. The optimized procedure was then tested on B104-1-1 cells, a mouse neuroblastoma line expressing P2X7R, to validate our in vitro procedure/model against previous in vivo testing of P2X7R modulators, Brilliant Blue FCF (FCF) and bzATP. In brief, two cell populations were differentially stained, mixed, pre-treated (if applicable) and PEG-fused. Initial optimization generated the highest fusion rates with 70% PEG + 0% DMSO. Subsequent testing of the model with FCF and bzATP proved that assessment over time is required when evaluating potential modulators of PEG fusion. Our conclusion, at 72 h post PEG fusion, is that the optimized procedure is suitable for initial candidate testing of modulators of PEG fusion. In vitro experiments with FCF and bzATP corroborated previous in vivo findings and confirmed that DMSO is not required to aid PEG fusion in the in vitro model. The development of this cellular based in vitro model will be invaluable as a substitute for initial assessment of chemical modulators of PEG fusion.

Herein we present a simple, reproducible and versatile approach for in situ protein digestion and identification on formalin-fixed and paraffin-embedded (FFPE) tissues. This adaptation is based on the use of an enzyme delivery platform (hydrogel discs) that can be positioned on the surface of a tissue section. By simultaneous deposition of multiple hydrogels over select regions of interest within the same tissue section, multiple peptide extracts can be obtained from discrete histological areas. After enzymatic digestion, the hydrogel extracts are submitted for LC-MS/MS analysis followed by database inquiry for protein identification. Further, imaging mass spectrometry (IMS) is used to reveal the spatial distribution of the identified peptides within a serial tissue section. Optimization was achieved using cutaneous tissue from surgically excised pressure ulcers that were subdivided into two prime regions of interest: the wound bed and the adjacent dermal area. The robust display of tryptic peptides within these spectral analyses of histologically defined tissue regions suggests that LC-MS/MS in combination with IMS can serve as useful exploratory tools.

The filling of wound cavities with new tissue is a challenge. We previously reported on the physical properties and wound healing kinetics of prefabricated, gas-blown polyurethane (PUR) scaffolds in rat and porcine excisional wounds. To address the capability of this material to fill complex wound cavities, this study examined the in vitro and in vivo reparative characteristics of injected PUR scaffolds employing a sucrose porogen. Using the porcine excisional wound model, we compared reparative outcomes to both preformed and injected scaffolds as well as untreated wounds at 9, 13, and 30 days after scaffold placement. Both injected and preformed scaffolds delayed wound contraction by 19% at 9 days and 12% at 13 days compared to nontreated wounds. This stenting effect proved transient since both formulations degraded by day 30. Both types of scaffolds significantly inhibited the undesirable alignment of collagen and fibroblasts through day 13. Injected scaffolds were highly compatible with sentinel cellular events of normal wound repair cell proliferation, apoptosis, and blood vessel density. The present study provides further evidence that either injected or preformed PUR scaffolds facilitate wound healing, support tissue infiltration and matrix production, delay wound contraction, and reduce scarring in a clinically relevant animal model, which underscores their potential utility as a void-filling platform for large cutaneous defects. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1679-1690, 2016.

Individuals in the geriatric age range are more prone than younger individuals to convert their partial thickness thermal burns into full thickness injuries. We hypothesized that this often observed clinical phenomenon is strongly related to differential local injury responses mediated by the immune system.Skin samples from areas with partial thickness thermal burns were obtained during routine excision and grafting procedures between post burn days 2-6. Tissue samples were grouped by age ranges with young patients defined as30 years of age or aged patients defined as65. Formalin fixed samples were used to confirm depth of burn injury and companion sections were homogenized for multiplex analysis using a Luminex platform. Immunohistochemical staining was used to quantify total macrophage numbers as well as the M1 and M2 subpopulations.Our analysis includes samples derived from 11 young subjects (mean age=23) and 3 aged subjects (mean age=79.2). Our initial survey of analytes examined 31 cytokines/chemokines. Twelve were excluded from consideration as they were present in concentrations either above or below the optimal detection range. Two analytes emerged as candidate molecules with significant differences between the young and the aged patient responses to burn injury. EGF levels were on average 21.69pg/ml in young vs 14.87pg/ml in aged (p=0.032). RANTES/CCL5 levels were on average 14.86pg/ml in young vs 4.26pg/ml in aged (p=0.026). Elevated macrophage numbers were present within wounds of younger patients compared to the old (p0.01), with a higher concentration of the M1 type in the elderly (p0.05).Our study has identified at least 2 well known cytokines, CCL5 (RANTES) and EGF, which are differentially regulated in response to burn injury by young versus aged burn victims. Evidence suggests that a proinflammatory environment can explain the high conversion rate from partial to full thickness burns. Our data suggest the need for future studies at the point of injury (cutaneous targets) that may be modulated by post burn release of cytokines/chemokines.

Background The management of peripheral nerve injuries remains a large challenge for plastic surgeons. With the inability to fuse axonal endings, results after microsurgical nerve repair have been inconsistent. Our current nerve repair strategies rely upon the slow and lengthy process of axonal regeneration (~1 mm/d). Polyethylene glycol (PEG) has been investigated as a potential axonal fusion agent; however, the percentage of axonal fusion has been inconsistent. The purpose of this study was to identify a PEG delivery device to standardize outcomes after attempted axonal fusion with PEG. Materials and Methods We used a rat sciatic nerve injury model in which we completely transected and repaired the left sciatic nerve to evaluate the efficacy of PEG fusion over a span of 12 weeks. In addition, we evaluated the effectiveness of a delivery device's ability to optimize results after PEG fusion. Results We found that PEG rapidly (within minutes) restores axonal continuity as assessed by electrophysiology, fluorescent retrograde tracer, and diffusion tensor imaging. Immunohistochemical analysis shows that motor axon counts are significantly increased at 1 week, 4 weeks, and 12 weeks postoperatively in PEG-treated animals. Furthermore, PEG restored behavioral functions up to 50% compared with animals that received the criterion standard epineurial repair (control animals). Conclusions The ability of PEG to rapidly restore nerve function after neurotmesis could have vast implications on the clinical management of traumatic injuries to peripheral nerves.

Polyethylene glycol (PEG) has been shown to restore axonal continuity after peripheral nerve transection in animal models. We hypothesized that PEG can also restore axonal continuity in the central nervous system. In this current experiment, coronal sectioning of the brains of Sprague-Dawley rats was performed after animal sacrifice. 3Brain high-resolution microelectrode arrays (MEA) were used to measure mean firing rate (MFR) and peak amplitude across the corpus callosum of the ex-vivo brain slices. The corpus callosum was subsequently transected and repeated measurements were performed. The cut ends of the corpus callosum were still apposite at this time. A PEG solution was applied to the injury site and repeated measurements were performed. MEA measurements showed that PEG was capable of restoring electrophysiology signaling after transection of central nerves. Before injury, the average MFRs at the ipsilateral, midline, and contralateral corpus callosum were 0.76, 0.66, and 0.65 spikes/second, respectively, and the average peak amplitudes were 69.79, 58.68, and 49.60 μV, respectively. After injury, the average MFRs were 0.71, 0.14, and 0.25 spikes/second, respectively and peak amplitudes were 52.11, 8.98, and 16.09 μV, respectively. After application of PEG, there were spikes in MFR and peak amplitude at the injury site and contralaterally. The average MFRs were 0.75, 0.55, and 0.47 spikes/second at the ipsilateral, midline, and contralateral corpus callosum, respectively and peak amplitudes were 59.44, 45.33, 40.02 μV, respectively. There were statistically differences in the average MFRs and peak amplitudes between the midline and non-midline corpus callosum groups (P < 0.01, P < 0.05). These findings suggest that PEG restores axonal conduction between severed central nerves, potentially representing axonal fusion.

Imaging mass spectrometry (IMS) was employed for the analysis of frozen skin biopsies to investigate the differences between stage IV pressure ulcers that remain stalled, stagnant, and unhealed versus those exhibiting clinical and histological signs of improvement. Our data reveal a rich diversity of proteins that are dynamically modulated, and we selectively highlight a family of calcium binding proteins (S-100 molecules) including calcyclin (S100-A6), calgranulins A (S100-A8) and B (S100-A9), and calgizzarin (S100-A11). IMS allowed us to target three discrete regions of interest: the wound bed, adjacent dermis, and hypertrophic epidermis. Plots derived using unsupervised principal component analysis of the global protein signatures within these three spatial niches indicate that these data from wound signatures have potential as a prognostic tool since they appear to delineate wounds that are favorably responding to therapeutic interventions versus those that remain stagnant or intractable in their healing status. Our discovery-based approach with IMS augments current knowledge of the molecular signatures within pressure ulcers while providing a rationale for a focused examination of the role of calcium modulators within the context of impaired wound healing.

Restoration of neuronal functions by outgrowths regenerating at ∼1 mm/day from the proximal stumps of severed peripheral nerves takes many weeks or months, if it occurs at all, especially after ablation of nerve segments. Distal segments of severed axons typically degenerate in 1-3 days. This study shows that Wallerian degeneration can be prevented or retarded, and lost behavioral function can be restored, following ablation of 0.5-1-cm segments of rat sciatic nerves in host animals. This is achieved by using 0.8-1.1-cm microsutured donor allografts treated with bioengineered solutions varying in ionic and polyethylene glycol (PEG) concentrations (modified PEG-fusion procedure), being careful not to stretch any portion of donor or host sciatic nerves. The data show that PEG fusion permanently restores axonal continuity within minutes, as initially assessed by action potential conduction and intracellular diffusion of dye. Behavioral functions mediated by the sciatic nerve are largely restored within 2-4 weeks, as measured by the sciatic functional index. Increased restoration of sciatic behavioral functions after ablating 0.5-1-cm segments is associated with greater numbers of viable myelinated axons within and distal to PEG-fused allografts. Many such viable myelinated axons are almost certainly spared from Wallerian degeneration by PEG fusion. PEG fusion of donor allografts may produce a paradigm shift in the treatment of peripheral nerve injuries.

The authors' earlier retrospective report of surgical complications after abdominal contouring surgery provided evidence that post-bariatric surgery patients are at increased risk of developing wound complications compared with a normal population. This prospective pilot study was designed as a comparative analysis of both surgical and wound healing characteristics between massive weight loss and normal patients who present for abdominal contouring surgery.Excisional wounds were created and polytetrafluoroethylene tubing was inserted during the preoperative period for later harvesting in patients undergoing abdominal contouring following Roux-en-Y gastric bypass for weight loss (n = 16) or abdominoplasty (n = 17). Wound fluids were sequentially collected from drains and subjected to matrix metalloproteinase (MMP) analysis. Standard postsurgical complications were documented.Surgical complications were more common in weight loss patients (47 percent) than in control patients (25 percent). MMP analyses showed that MMP-9 levels remained significantly elevated at postoperative day 4 in patients who subsequently experienced complications in either the weight loss group (p = 0.02) or the control group (p = 0.03). Other parameters showed no significant differences between massive weight loss patients and controls.Although many markers were examined, the ratio of MMP-9 to albumin was the only predictor of postsurgical complications in any group. This lends further support to growing evidence that MMP-9 may be a useful biomarker of postsurgical complications. This pilot work showed no causal factors that explain the higher rates of postsurgical complications in the post-bariatric surgery patient population.Risk, II.

Currently, determination of burn depth and healing outcomes has been limited to subjective assessment or a single modality, e.g., laser Doppler imaging. Such measures have proven less than ideal. Recent developments in other non-contact technologies such as optical coherence tomography (OCT) and pulse speckle imaging (PSI) offer the promise that an intelligent fusion of information across these modalities can improve visualization of burn regions thereby increasing the sensitivity of the diagnosis. In this work, we combined OCT and PSI images to classify the degree of burn (superficial, partial-thickness and full-thickness burns). Algorithms were developed to integrate and visualize skin structure (with and without burns) from the two modalities. We have completed the proposed initiatives by employing a porcine burn model and compiled results that attest to the utility of our proposed dual-modal fusion approach. Computer-derived data indicating the varying burn depths were validated through immunohistochemical analysis performed on burned skin tissue. The combined performance of OCT and PSI modalities provided an overall ROC-AUC=0.87 (significant at p

Peripheral nerve repair using nerve grafts has been investigated for several decades using traditional techniques such as histology, immunohistochemistry, and electron microscopy. Recent advances in mass spectrometry techniques have made it possible to study the proteomes of complex tissues, including extracellular matrix rich tissues similar to peripheral nerves. The present study comparatively assessed three previously described processing methods for generating acellular nerve grafts by mass spectrometry. Acellular nerve grafts were additionally examined by F-actin staining and nuclear staining for debris clearance. Application of newer techniques allowed us to detect and highlight differences among the 3 treatments. Isolated proteins were separated by mass on polyacrylamide gels serving 2 purposes. This further illustrated that these treatments differ from one another and it allowed for selective protein extractions within specific bands/molecular weights. This approach resulted in small pools of proteins that could then be analyzed by mass spectrometry for content. In total, 543 proteins were identified, many of which corroborate previous findings for these processing methods. The remaining proteins are novel discoveries that expand the field. With this pilot study, we have proven that mass spectrometry techniques complement and add value to peripheral nerve repair studies.

Small interfering RNA (siRNA) delivered from reactive oxygen species-degradable tissue engineering scaffolds promotes diabetic wound healing in rats. Porous poly(thioketal-urethane) scaffolds implanted in diabetic wounds locally deliver siRNA that inhibits the expression of prolyl hydroxylase domain protein 2, thereby increasing the expression of progrowth genes and increasing vasculature, proliferating cells, and tissue development in diabetic wounds.

Polyethylene glycol (PEG) has long been used as a membrane fusogen, but recently it has been adopted as a technique for peripheral nerve repair. Vertebrate models using PEG fusion have shown improved outcomes when PEG is applied during repair of severed peripheral nerves. The cellular mechanism of PEG fusion in the peripheral nerve repair model has not previously been assessed via flow cytometry. PEG fusion was assessed in this experiment by dying B35 rat neuroblastoma cells with different color fluorescent labels. The different color cells were combined and PEG was applied in concentrations of 50%, 75% and 100%. The amount of cell fusion was assessed via flow cytometry as the percentage of double positive cells. Results showed increasing fusion and decreasing viability with increasing concentrations of PEG.

The spatiotemporal analysis of the proteomic profile during human wound healing is a critical investigative step that can establish the complex interplay of molecular events that comprise the local response to burn injury. Partial-thickness wound samples with adjacent "normal" skin were collected from twenty-one patients with burn wounds and examined across a time spectrum ranging from the acute injury period at 3, 6, 11 days to the later hypertrophic scar period at 7 and 15 months. The techniques used for histology-directed tissue analyses highlighted inflammatory protein markers at the early time points after injury with diminished expression as burn wounds progressed into the proliferative phase. The datasets show the usefulness of MALDI MS and imaging mass spectrometry as discovery approaches to identify and map the cutaneous molecular sequence that is activated in response to the unique systemic inflammatory response following burn trauma. This information has the potential to define the unique factors that predispose human burn victims to disfiguring hypertrophic scar formation.

Approximately 12% of operations for traumatic neuropathy are for patients with segmental nerve loss, and less than 50% of these injuries obtain meaningful functional recovery. Polyethylene glycol (PEG) therapy has been shown to improve functional outcomes after nerve severance, and we hypothesized this therapy could also benefit nerve autografting.We used a segmental rat sciatic nerve injury model in which we repaired a 0.5-cm defect with an autograft using microsurgery. We treated experimental animals with solutions containing methylene blue (MB) and PEG; control animals did not receive PEG. We recorded compound action potentials (CAPs) before nerve transection, after solution therapy, and at 72 h postoperatively. The animals underwent behavioral testing at 24 and 72 h postoperatively. After we euthanized the animals, we fixed the nerves, sectioned and immunostained them to allow for quantitative morphometric analysis.The introduction of hydrophilic polymers greatly improved morphological and functional recovery of rat sciatic axons at 1-3 d after nerve autografting. Polyethylene glycol therapy restored CAPs in all animals, and CAPs were still present 72 h postoperatively. No CAPS were detectable in control animals. Foot Fault asymmetry scores and sciatic functional index scores were significantly improved for PEG therapy group at all time points (P0.05 and P0.001; P0.001 and P0.01). Sensory and motor axon counts were increased distally in nerves treated with PEG compared with control (P = 0.019 and P = 0.003).Polyethylene glycol therapy improves early physiologic function, behavioral outcomes, and distal axonal density after nerve autografting.

The pathogenesis of impaired healing within pressure ulcers remains poorly characterized and rarely examined. We describe the results of a pilot study that applies matrix-assisted laser desorption/ionization imaging mass spectrometry technology for direct tissue analysis to evaluate proteomic signatures ranging from 2 to 20 kDa and phospholipids from 300-1,200 Da in focal regions within the wound microenvironment. Distinguishing molecular differences were apparent between upper vs. lower regions of ulcers and further contrasted against adjacent dermis and epidermal margins using protein profiles, ion density maps, principal component analysis and significant analysis of microarrays. Several proteins previously uncharacterized in pressure ulcers, the α-defensins (human neutrophil peptide [HNP]-1, -2, -3), are potential markers indicating whether the wound status is improving or being prolonged in a deleterious, chronic state. Thymosin β4 appears to be a favorable protein marker showing higher relative levels in adjacent dermis and maturing areas of the wound bed. Lipidomic examination revealed the presence of major lipid classes: glycerophosphocholines, glycerophosphoglycerols, glycerophosphoinositols, and triacylglycerols. Our pilot data examined from either a global perspective using proteomic or lipidomic signatures or as individual distributions reveal that imaging mass spectrometry technology can be effectively used for discovery and spatial mapping of molecular disturbances within the microenvironment of chronic wounds.

Investigations of the human skin proteome by classical analytical procedures have not addressed spatial molecular distributions in whole skin biopsies. The aim of this study was to develop methods for detection of protein signatures and their spatial disposition in human skin using advanced molecular imaging technology based on mass spectrometry technologies. This technology allows for the generation of protein images at specific molecular weight values without the use of antibody while maintaining tissue architecture. Two experimental approaches were employed: MALDI-MS profiling, where mass spectra were taken from discrete locations based on histology, and MALDI-IMS imaging, where complete molecular images were obtained at various MW values. In addition, proteins were identified by in situ tryptic digestion, sequence analysis of the fragment peptides, and protein database searching. We have detected patterns of protein differences that exist between epidermis and dermis as well as subtle regional differences between the papillary and reticular dermis. Furthermore, we were able to detect proteins that are constitutive features of human skin as well as those associated with unique markers of individual variability.

Extracellular functions of the endoplasmic reticulum chaperone protein calreticulin (CRT) are emerging. Here we show novel roles for exogenous CRT in both cutaneous wound healing and diverse processes associated with repair. Compared with platelet-derived growth factor-BB-treated controls, topical application of CRT to porcine excisional wounds enhanced the rate of wound re-epithelialization. In both normal and steroid-impaired pigs, CRT increased granulation tissue formation. Immunohistochemical analyses of the wounds 5 and 10 days after injury revealed marked up-regulation of transforming growth factor-beta3 (a key regulator of wound healing), a threefold increase in macrophage influx, and an increase in the cellular proliferation of basal keratinocytes of the new epidermis and of cells of the neodermis. In vitro studies confirmed that CRT induced a greater than twofold increase in the cellular proliferation of primary human keratinocytes, fibroblasts, and microvascular endothelial cells (with 100 pg/ml, 100 ng/ml, and 1.0 pg/ml, respectively). Moreover, using a scratch plate assay, CRT maximally induced the cellular migration of keratinocytes and fibroblasts (with 10 pg/ml and 1 ng/ml, respectively). In addition, CRT induced concentration-dependent migration of keratinocytes, fibroblasts macrophages, and monocytes in chamber assays. These in vitro bioactivities provide mechanistic support for the positive biological effects of CRT observed on both the epidermis and dermis of wounds in vivo, underscoring a significant role for CRT in the repair of cutaneous wounds.

The role of the EGF superfamily of growth factors and their receptors during wound healing continues to unfold. The aim of the study was to determine the effect of overexpression of erbB3 in wound healing and whether topical application of its various ligands enhances repair. Partial thickness porcine wounds were transfected with recombinant adenoviral particles containing erbB3 receptor gene or a vehicle β-galactosidase gene (Lac Z). Highly similar ligands: Epidermal growth factor (EGF), Epiregulin (EPR), Heparin-binding epidermal growth factor (HB-EGF), Heregulin (HER) were topically applied to wounds boosted with erbB3 receptors. Enhanced wound healing parameters were assessed 5 days after initial treatment for reepithelization, dermal depth, macrophage infiltration, proliferating cells, angiogenesis, and apoptotic cells. Treatment with EPR and HB-EGF promoted greater resurfacing of the epidermal layer than EGF, HER, erbB3 only or vehicle treated wounds. Neodermal formation as measured by dermal or granulation depth was significantly different in the EPR and HB-EGF treated wounds compared to EGF, HER, erbB3 only or vehicle. Numerous hallmarks of enhanced wound maturity (fewer infiltrating macrophages, significant reductions in neovascularization, fibroblastic proliferation) were noted in EPR- and HB-EGF-treated wounds transfected with erbB3 as compared to vehicle controls. Only erbB3 wounds treated with these particular ligands showed significant increases in apoptotic cells. Our results indicate that wounds treated with this erbB3 receptor form supplied with topical application of EPR and HB-EGF exhibit synergistic and accelerating reparative effects on porcine partial thickness wounds as compared to vehicle. These in vivo findings suggest the complexity and evidence of synergism between the erbB receptor and its various ligands on wound healing. (This research work was funded by the NIH GM40437.)

Background Hydrophilic polymers have been shown to improve physiologic recovery following repair of transected nerves with microsutures. Our study was designed to combine hydrophilic polymer therapy with nerve tubes (NT) to enhance polymer delivery to the site of nerve injury. Methods Using a rat sciatic nerve injury model, a single transection injury was repaired in an end-to-end fashion with NT+polyethylene glycol (PEG) to NT alone. Compound action potentials (CAPs) were recorded before nerve transection and after repair. Behavioral testing was performed for 5 weeks. Results PEG therapy restored CAPS in all, but one, animals, while no CAPS were recorded in animals not receiving PEG. Behavioral nerve function was measured using the standardized functional assessment technique and foot fault asymmetry scores (FF). FF scores were improved for the PEG therapy groups on postoperative days 7, 14, and 21. However, after expected eventual axonal outgrowth, the benefit was less noticeable at days 28 and 35. Immunohistochemistry of the distal axon segments showed an increase number of sensory and motor axons in the NT+PEG group as compared to NT alone. Conclusion These data suggest that PEG delivery via a conduit may provide a simple, effective way to fuse severed axons and regain early nerve function. For proximal nerve injuries in large animals, recovery of axonal continuity could dramatically improve outcomes, even if fusion only occurs in a small percentage of axons.

Diagnosis and management of peripheral nerve injury is complicated by the inability to assess microstructural features of injured nerve fibers via clinical examination and electrophysiology. Diffusion tensor imaging (DTI) has been shown to accurately detect nerve injury and regeneration in crush models of peripheral nerve injury, but no prior studies have been conducted on nerve transection, a surgical emergency that can lead to permanent weakness or paralysis. Acute sciatic nerve injuries were performed microsurgically to produce multiple grades of nerve transection in rats that were harvested 1 hour after surgery. High-resolution diffusion tensor images from ex vivo sciatic nerves were obtained using diffusion-weighted spin-echo acquisitions at 4.7 T. Fractional anisotropy was significantly reduced at the injury sites of transected rats compared with sham rats. Additionally, minor eigenvalues and radial diffusivity were profoundly elevated at all injury sites and were negatively correlated to the degree of injury. Diffusion tensor tractography showed discontinuities at all injury sites and significantly reduced continuous tract counts. These findings demonstrate that high-resolution DTI is a promising tool for acute diagnosis and grading of traumatic peripheral nerve injuries.