Luke R. Burnett

The Effect of Daily Caffeine Use on Cerebral Blood Flow: How Much Caffeine Can We Tolerate?

Caffeine is a commonly used neurostimulant that also produces cerebral vasoconstriction by antagonizing adenosine receptors. Chronic caffeine use results in an adaptation of the vascular adenosine receptor system presumably to compensate for the vasoconstrictive effects of caffeine. We investigated the effects of caffeine on cerebral blood flow (CBF) in increasing levels of chronic caffeine use. Low (mean = 45 mg/day), moderate (mean = 405 mg/day), and high (mean = 950 mg/day) caffeine users underwent quantitative perfusion magnetic resonance imaging on four separate occasions: twice in a caffeine abstinent state (abstained state) and twice in a caffeinated state following their normal caffeine use (native state). In each state, there were two drug conditions: participants received either caffeine (250 mg) or placebo. Gray matter CBF was tested with repeated‐measures analysis of variance using caffeine use as a between‐subjects factor, and correlational analyses were conducted between CBF and caffeine use. Caffeine reduced CBF by an average of 27% across both caffeine states. In the abstained placebo condition, moderate and high users had similarly greater CBF than low users; but in the native placebo condition, the high users had a trend towards less CBF than the low and moderate users. Our results suggest a limited ability of the cerebrovascular adenosine system to compensate for high amounts of daily caffeine use. Hum Brain Mapp 2009.

Regenerative Medicine as Applied to General Surgery

The present review illustrates the state of the art of regenerative medicine (RM) as applied to surgical diseases and demonstrates that this field has the potential to address some of the unmet needs in surgery. RM is a multidisciplinary field whose purpose is to regenerate in vivo or ex vivo human cells, tissues, or organs to restore or establish normal function through exploitation of the potential to regenerate, which is intrinsic to human cells, tissues, and organs. RM uses cells and/or specially designed biomaterials to reach its goals and RM-based therapies are already in use in several clinical trials in most fields of surgery. The main challenges for investigators are threefold: Creation of an appropriate microenvironment ex vivo that is able to sustain cell physiology and function in order to generate the desired cells or body parts; identification and appropriate manipulation of cells that have the potential to generate parenchymal, stromal and vascular components on demand, both in vivo and ex vivo; and production of smart materials that are able to drive cell fate.

Superior colliculus lesions preferentially disrupt multisensory orientation.

The general involvement of the superior colliculus (SC) in orientation behavior and the striking parallels between the multisensory responses of SC neurons and overt orientation behaviors have led to assumptions that these neural and behavioral changes are directly linked. However, deactivation of two areas of cortex which also contain multisensory neurons, the anterior ectosylvian sulcus and rostral lateral suprasylvian sulcus have been shown to eliminate multisensory orientation behaviors, suggesting that this behavior may not involve the SC. To determine whether the SC contributes to this behavior, cats were tested in a multisensory (i.e. visual-auditory) orientation task before and after excitotoxic lesions of the SC. For unilateral SC lesions, modality-specific (i.e. visual or auditory) orientation behaviors had returned to pre-lesion levels after several weeks of recovery. In contrast, the enhancements and depressions in behavior normally seen with multisensory stimuli were severely compromised in the contralesional hemifield. No recovery of these behaviors was observed within the 6 month testing period. Immunohistochemical labeling of the SC revealed a preferential loss of parvalbumin-immunoreactive pyramidal neurons in the intermediate layers, a presumptive multisensory population that targets premotor areas of the brainstem and spinal cord. These results highlight the importance of the SC for multisensory behaviors, and suggest that the multisensory orientation deficits produced by cortical lesions are a result of the loss of cortical influences on multisensory SC neurons.

Hemostatic properties and the role of cell receptor recognition in human hair keratin protein hydrogels

Driven by new discoveries in stem-cell biology and regenerative medicine, there is broad interest in biomaterials that go beyond basic interactions with cells and tissues to actively direct and sustain cellular behavior. Keratin biomaterials have the potential to achieve these goals but have been inadequately described in terms of composition, structure, and cell-instructive characteristics. In this manuscript we describe and characterize a keratin-based biomaterial, demonstrate self-assembly of cross-linked hydrogels, investigate a cell-specific interaction that is dependent on the hydrogel structure and mediated by specific biomaterial-receptor interactions, and show one potential medical application that relies on receptor binding - the ability to achieve hemostasis in a lethal liver injury model. Keratin biomaterials represent a significant advance in biotechnology as they combine the compatibility of natural materials with the chemical flexibility of synthetic materials. These characteristics allow for a system that can be formulated into several varieties of cell-instructive biomaterials with potential uses in tissue engineering, regenerative medicine, drug and cell delivery, and trauma.

Novel keratin (KeraStat™) and polyurethane (Nanosan®-Sorb) biomaterials are hemostatic in a porcine lethal extremity hemorrhage model

Traumatic injury is the leading cause of death in people aged 44 or less in the US. It is also estimated that 82% of deaths from battlefield hemorrhage may be survivable with better treatment options. In this study, two biomaterial hemostats having disparate mechanisms were evaluated in a large animal lethal hemorrhage model and compared to a commercial product and standard cotton gauze. We hypothesized that the biomaterial with a biologically active mechanism, as opposed to a mechanical mechanism, would be the most effective in this model. Using a published study protocol, the femoral artery in swine was punctured and treated. KeraStat™ (KeraNetics) and Nanosan®-Sorb (SNS Nano) hemostats were compared to a commercial chitosan dressing (second generation Hemcon®) and cotton gauze. Both KeraStat and Nanosan increased survival, significantly increased mean arterial pressure (MAP), and significantly decreased shock index compared to both controls. The Hemcon dressing was no different than gauze. Platelet adhesion assays suggested that the KeraStat mechanism of action involves β1 integrin mediated platelet adhesion while Nanosan-Sorb operates similar to one reported mechanism for Hemcon, absorbing fluid and concentrating clotting components. The Nanosan also swelled considerably and created pressure within the wound site even after direct pressure was removed.

Excitotoxic lesions of the superior colliculus preferentially impact multisensory neurons and multisensory integration

The superior colliculus (SC) plays an important role in integrating visual, auditory and somatosensory information, and in guiding the orientation of the eyes, ears and head. Previously we have shown that cats with unilateral SC lesions showed a preferential loss of multisensory orientation behaviors for stimuli contralateral to the lesion. Surprisingly, this behavioral loss was seen even under circumstances where the SC lesion was far from complete. To assess the physiological changes induced by these lesions, we employed single unit electrophysiological methods to record from individual neurons in both the intact and damaged SC following behavioral testing in two animals. In the damaged SC of these animals, multisensory neurons were preferentially reduced in incidence, comprising less than 25% of the sensory-responsive population (as compared with 49% on the control side). In those multisensory neurons that remained following the lesion, receptive fields were nearly twofold larger, and less than 25% showed normal patterns of multisensory integration, with those that did being found in areas outside of the lesion. These results strongly suggest that the multisensory behavioral deficits seen following SC lesions are the combined result of a loss of multisensory neurons and a loss of multisensory integration in those neurons that remain.

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

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

Development and Characterization of a 3D Printed, Keratin-Based Hydrogel

Keratin, a naturally-derived polymer derived from human hair, is physiologically biodegradable, provides adequate cell support, and can self-assemble or be crosslinked to form hydrogels. Nevertheless, it has had limited use in tissue engineering and has been mainly used as casted scaffolds for drug or growth factor delivery applications. Here, we present and assess a novel method for the printed, sequential production of 3D keratin scaffolds. Using a riboflavin-SPS-hydroquinone (initiator–catalyst–inhibitor) photosensitive solution we produced 3D keratin constructs via UV crosslinking in a lithography-based 3D printer. The hydrogels obtained have adequate printing resolution and result in compressive and dynamic mechanical properties, uptake and swelling capacities, cytotoxicity, and microstructural characteristics that are comparable or superior to those of casted keratin scaffolds previously reported. The novel keratin-based printing resin and printing methodology presented have the potential to impact future research by providing an avenue to rapidly and reproducibly manufacture patient-specific hydrogels for tissue engineering and regenerative medicine applications.

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

Infection is a leading cause of morbidity and mortality in burn patients. Current therapies include silver‐based creams and dressings, which display limited antimicrobial effectiveness and impair healing. The need exists for a topical, point‐of‐injury antibiotic treatment that provides sustained antimicrobial activity without impeding wound repair. Fitting this description are keratin‐based hydrogels, which are fully biocompatible and support the slow‐release of antibiotics. Here we develop a porcine model of an infected partial‐thickness burn to test the effects of ciprofloxacin‐loaded keratin hydrogels on infection and wound healing. Partial‐thickness burns were inoculated with either Pseudomonas aeruginosa or Methicillin‐resistant Staphylococcus aureus, resulting in infections that persisted for >2 weeks that exceeded 105 and 106 cfu per gram of tissue, respectively. Compared to silver sulfadiazine, ciprofloxacin‐loaded keratin hydrogel treatment significantly reduced the amount of P. aeruginosa and S. aureus in the burn by >99% on days 4, 7, 11, and 15 postinjury. Further, burns treated with ciprofloxacin‐loaded keratin hydrogels exhibited similar healing patterns as uninfected burns with regards to reepithelialization, macrophage recruitment, and collagen deposition and remodeling. The ability of keratin hydrogels to deliver antibiotics to fight infection and support healing of partial‐thickness burns make them a strong candidate as a first‐line burn therapy.

Vasoactive properties of keratin-derived compounds.

Please cite this paper as: Nunez, Trach, Burnett, Handa, Dyke, Callahan, and Smith (2011). Vasoactive Properties of Keratin‐Derived Compounds. Microcirculation18(8), 663–669.