Robert W. Hitchcock

Intracellular calcium transients evoked by pulsed infrared radiation in neonatal cardiomyocytes

Non‐technical summary  We have investigated the mechanisms underlying the response of cells to pulsed infrared radiation (IR, ∼1862 nm) using the neonatal rat ventricular cardiomyocyte as a model. Fluorescence monitoring of the intracellular free calcium (Ca2+) demonstrated that infrared irradiation induced rapid (millisecond time scale) intracellular Ca2+ transients in the cells. The results showed that the Ca2+ transients were sufficient to elicit contractile responses from the cardiomyocytes and could be ‘paced’ or entrained to the pulsing frequency of the IR. Pharmacological results strongly implicate mitochondria as the primary intracellular organelles contributing to the IR‐evoked Ca2+ cycling.

Skeletal myogenesis on elastomeric substrates: Implications for tissue engineering

Studies geared towards understanding the interaction between skeletal muscle and biomaterials may provide useful information for the development of various emerging technologies, ranging from novel delivery vehicles for genetically modified cells to fully functional skeletal muscle tissue. To determine the utility of elastomeric materials as substrates for such applications, we asked whether skeletal myogenesis would be supported on a commercially available polyurethane, Tecoflex SG-80A. G8 skeletal myoblasts were cultured on Tecoflex two-dimensional solid thin films fabricated by a spin-casting method. Myoblasts attached, proliferated, displayed migratory activity and differentiated into multinucleated myotubes which expressed myosin heavy chain on solid thin films indicating that Tecoflex SG-80A was permissive for skeletal myogenesis. Porous three-dimensional (3-D) cell scaffolds were fabricated in a variety of shapes, thicknesses, and porosities by an immersion precipitation method, and where subsequently characterized with microscopic and mechanical methods. Mechanical analysis revealed that the constructs were elastomeric, recovering their original length following 100% elongation. The 3-D substrates were seeded with muscle precursors to determine if muscle differentiation could be obtained within the porous network of the fabricated constructs. Following several weeks in culture, histological studies revealed the presence of multinucleated myotubes within the elastomeric material. In addition, immunohistochemical analysis indicated that the myotubes expressed the myosin heavy chain protein suggesting that the myotubes had reached a state of terminal differentiation. Together the results of the study suggest that it is indeed feasible to engineer bioartificial systems consisting of skeletal muscle cultivated on a 3-D elastomeric substrate.

Comparison of human fibroblast ECM-related gene expression on elastic three-dimensional substrates relative to two-dimensional films of the same material.

Three-dimensional elastic substrates were fabricated from a commercially available polyurethane with an internal porosity of approximately 70% and elastic modulus of 27.4+/-2.76 KPa and examined for suitability in vocal fold tissue engineering. Using immunohistochemistry, biomechanical testing, and RT-PCR; we examined human fibroblast viability, distribution and extracellular matrix related gene expression within substrates for periods up to 4 weeks. We found that cells were capable of colonizing the entire volume of a 5mm wide x 3mm deep x 20mm long substrate at high viability. Histological cross-sections showed extensive extracellular matrix deposited around the cells and throughout the pore structure of the substrates, which consisted of fibronectin and type I collagen. Cell seeded substrates displayed a significantly higher elastic modulus than unseeded controls similar to native tissue. The transfer of cell growth from two-dimensional to three-dimensional culture resulted in changes in ECM-related gene expression consistent with decreasing cell migration and increasing tissue formation. We found that fibroblasts cultured in three-dimensional substrates expressed significantly higher levels of mRNA for elastin and fibromodulin, while expressing significantly lower levels of mRNA for MMP-1 and hyaluronidase relative to two-dimensional substrates of the same material. The results suggest that three-dimensionally porous, Tecoflex-derived elastic biomaterials may be suitable substrates for engineering vocal fold tissue.

Electrical stimulation directs engineered cardiac tissue to an age-matched native phenotype

Quantifying structural features of native myocardium in engineered tissue is essential for creating functional tissue that can serve as a surrogate for in vitro testing or the eventual replacement of diseased or injured myocardium. We applied three-dimensional confocal imaging and image analysis to quantitatively describe the features of native and engineered cardiac tissue. Quantitative analysis methods were developed and applied to test the hypothesis that environmental cues direct engineered tissue toward a phenotype resembling that of age-matched native myocardium. The analytical approach was applied to engineered cardiac tissue with and without the application of electrical stimulation as well as to age-matched and adult native tissue. Individual myocytes were segmented from confocal image stacks and assigned a coordinate system from which measures of cell geometry and connexin-43 spatial distribution were calculated. The data were collected from 9 nonstimulated and 12 electrically stimulated engineered tissue constructs and 5 postnatal day 12 and 7 adult hearts. The myocyte volume fraction was nearly double in stimulated engineered tissue compared to nonstimulated engineered tissue (0.34 ± 0.14 vs 0.18 ± 0.06) but less than half of the native postnatal day 12 (0.90 ± 0.06) and adult (0.91 ± 0.04) myocardium. The myocytes under electrical stimulation were more elongated compared to nonstimulated myocytes and exhibited similar lengths, widths, and heights as in age-matched myocardium. Furthermore, the percentage of connexin-43-positive membrane staining was similar in the electrically stimulated, postnatal day 12, and adult myocytes, whereas it was significantly lower in the nonstimulated myocytes. Connexin-43 was found to be primarily located at cell ends for adult myocytes and irregularly but densely clustered over the membranes of nonstimulated, stimulated, and postnatal day 12 myocytes. These findings support our hypothesis and reveal that the application of environmental cues produces tissue with structural features more representative of age-matched native myocardium than adult myocardium. We suggest that the presented approach can be applied to quantitatively characterize developmental processes and mechanisms in engineered tissue.

Early weight bearing after lower extremity fractures in adults.

Abstract Weight‐bearing protocols should optimize fracture healing while avoiding fracture displacement or implant failure. Biomechanical and animal studies indicate that early loading is beneficial, but high‐quality clinical studies comparing weight‐bearing protocols after lower extremity fractures are not universally available. For certain fracture patterns, well‐designed trials suggest that patients with normal protective sensation can safely bear weight sooner than most protocols permit. Several randomized, controlled trials of surgically treated ankle fractures have shown no difference in outcomes between immediate and delayed (≥6 weeks) weight bearing. Retrospective series have reported low complication rates with immediate weight bearing following intramedullary nailing of femoral shaft fractures and following surgical management of femoral neck and intertrochanteric femur fractures in elderly patients. For other fracture patterns, particularly periarticular fractures, the evidence in favor of early weight bearing is less compelling. Most surgeons recommend a period of protected weight bearing for patients with calcaneal, tibial plafond, tibial plateau, and acetabular fractures. Further studies are warranted to better define optimal postoperative weight‐bearing protocols.

Towards Modeling of Cardiac Micro-Structure With Catheter-Based Confocal Microscopy: A Novel Approach for Dye Delivery and Tissue Characterization

This work presents a methodology for modeling of cardiac tissue micro-structure. The approach is based on catheter-based confocal imaging systems, which are emerging as tools for diagnosis in various clinical disciplines. A limitation of these systems is that a fluorescent marker must be available in sufficient concentration in the imaged region. We introduce a novel method for the local delivery of fluorescent markers to cardiac tissue based on a hydro-gel carrier brought into contact with the tissue surface. The method was tested with living rabbit cardiac tissue and applied to acquire three-dimensional image stacks with a standard inverted confocal microscope and two-dimensional images with a catheter-based confocal microscope. We processed these image stacks to obtain spatial models and quantitative data on tissue microstructure. Volumes of atrial and ventricular myocytes were 4901plusmn1713 and 10299plusmn3598 mum3 (meanplusmnsd), respectively. Atrial and ventricular myocyte volume fractions were 72.4plusmn4.7% and 79.7plusmn2.9% (mean plusmn sd), respectively. Atrial and ventricular myocyte density was 165571plusmn55836 and 86957plusmn32280 cells/mm3 (mean plusmn sd), respectively. These statistical data and spatial descriptions of tissue microstructure provide important input for modeling studies of cardiac tissue function. We propose that the described methodology can also be used to characterize diseased tissue and allows for personalized modeling of cardiac tissue.

Development of a wireless intra-vaginal transducer for monitoring intra-abdominal pressure in women

Pelvic floor disorders (PFD) affect one of every four women in the United States. Elevated intra-abdominal pressure (IAP) during daily activity or strenuous physical activity has been identified as a risk factor in the prevalence of PFD. However, the relationship between IAP and physical activity is poorly understood and oftentimes activity restrictions are prescribed by physicians without clinical evidence linking various activities to elevated IAP. There are currently no pressure transducers capable of monitoring IAP non-invasively out of a clinical environment. To overcome this shortcoming, a novel intra-vaginal pressure transducer (IVT) was developed to continuously monitor IAP. Improvements were made to the first generation IVT by incorporating wireless capability to enhance the device’s mobility while creating a more robust IAP monitoring system. To ensure the changes maintained the functionality of the original device design, comparison testing with standard clinical pressure transducers in both bench top and clinical settings was conducted. The wireless device was found to have high linearity, robust signal transmission, and dynamic response that outperforms the clinical standard rectal transducer and is similar to the original first generation non-wireless design. The wireless IVT presented here is a mobile wireless device capable of measuring, storing and transmitting IAP data during various physical activities.

Intra-abdominal pressures during activity in women using an intra-vaginal pressure transducer

Abstract Strenuous physical activity has been linked to pelvic floor disorders in women. Using a novel wireless intra-vaginal pressure transducer, intra-abdominal pressure was measured during diverse activities in a laboratory. Fifty-seven women performed a prescribed protocol using the intra-vaginal pressure transducer. We calculated maximal, area under the curve and first moment of the area intra-abdominal pressure for each activity. Planned comparisons of pressure were made between levels of walking and cycling and between activities with reported high pressure in the literature. Findings indicate variability in intra-abdominal pressure amongst individuals doing the same activity, especially in activities that required regulation of effort. There were statistically significant differences in maximal pressure between levels of walking, cycling and high pressure activities. Results for area under the curve and first moment of the area were not always consistent with maximal pressure. Coughing had the highest maximal pressure, but had lower area under the curve and first moment of the area compared to most activities. Our data reflect novel findings of maximal, area under the curve and first moment of the area measures of intra-abdominal pressure, which may have clinical relevance for how physical activity relates to pelvic floor dysfunction.

Engineered channels enhance cellular density in perfused scaffolds.

Scaffold-based tissue engineering provides cells with an engineered matrix to enhance and direct cell attachment, proliferation and differentiation. One critical limitation to current tissue engineering approaches is the inability to create densely populated constructs thicker than a few 100 μm. We hypothesized that development of porous, channeled scaffolds would increase cell density and uniformity of their spatial distribution through scaffold channel perfusion. Patterned polyurethane sheets were fabricated using a sprayed phase separation technique and laminated together to form 1.5 mm thick channeled scaffolds. Hydraulic permeability testing confirmed the presence of functional channels throughout the multilaminate construct. A continuous flow bioreactor was used to perfuse the construct with medium during the culture period. Cross-sectional cell densities and spatial uniformities were measured in channeled and nonchanneled scaffolds under different seeding and culture conditions. Channeled scaffolds were found to have higher densities of human mesenchymal stem cells than nonchanneled samples. Perfused scaffolds had more uniform spatial distribution of cells within the scaffold compared to statically cultured scaffolds. In conclusion, we have shown the channeled scaffolds to be a promising approach toward creating thick tissue-engineered constructs.

Three-Dimensional Modeling and Quantitative Analysis of Gap Junction Distributions in Cardiac Tissue

Gap junctions play a fundamental role in intercellular communication in cardiac tissue. Various types of heart disease including hypertrophy and ischemia are associated with alterations of the spatial arrangement of gap junctions. Previous studies applied two-dimensional optical and electron-microscopy to visualize gap junction arrangements. In normal cardiomyocytes, gap junctions were primarily found at cell ends, but can be found also in more central regions. In this study, we extended these approaches toward three-dimensional reconstruction of gap junction distributions based on high-resolution scanning confocal microscopy and image processing. We developed methods for quantitative characterization of gap junction distributions based on analysis of intensity profiles along the principal axes of myocytes. The analyses characterized gap junction polarization at cell ends and higher-order statistical image moments of intensity profiles. The methodology was tested in rat ventricular myocardium. Our analysis yielded novel quantitative data on gap junction distributions. In particular, the analysis demonstrated that the distributions exhibit significant variability with respect to polarization, skewness, and kurtosis. We suggest that this methodology provides a quantitative alternative to current approaches based on visual inspection, with applications in particular in characterization of engineered and diseased myocardium. Furthermore, we propose that these data provide improved input for computational modeling of cardiac conduction.