Biomedical subjects

Robert G Dennis

Publications and source records attributed to Robert G Dennis.

At least 19 recordsLinked to original sources

PubMed2007

Incisional herniation induces decreased abdominal wall compliance via oblique muscle atrophy and fibrosis.

OBJECTIVE: The purpose of this study is to measure abdominal wall myopathic histologic and mechanical changes during incisional herniation and its effect on incisional hernia repairs. SUMMARY BACKGROUND DATA: Unloaded skeletal muscles undergo characteristic atrophic changes, including change in fiber type composition, decreased cross-sectional area, and pathologic fibrosis. We hypothesize that these atrophic changes decrease muscle elastic properties and may contribute to the high laparotomy wound failure rate observed following incisional hernia repair. METHODS: A rat model of chronic incisional hernia formation was used. Failing midline laparotomy incisions developed into incisional hernias. Controls were uninjured and sham laparotomy (healed) groups. Internal oblique muscles were harvested for fiber typing, measurement of cross-sectional area, collagen deposition, and mechanical analysis. Mesh hernia repairs were performed on a second group of rats with chronic incisional hernias or acute anterior abdominal wall myofascial defects. RESULTS: The hernia group developed lateral abdominal wall shortening and oblique muscle atrophy. This was associated with a change in the distribution of oblique muscle fiber types, decreased cross-sectional area, and pathologic fibrosis consistent with myopathic disuse atrophy. These muscles exhibited significant decreased extensibility and increased stiffness. The healed (sham) laparotomy group expressed an intermediate phenotype between the uninjured and hernia groups. Recurrent hernia formation was most frequent in the chronic hernia model, and hernia repairs mechanically disrupted at a lower force compared with nonherniated abdominal walls. CONCLUSIONS: The internal oblique muscles of the abdominal wall express a pattern of changes consistent with those seen in chronically unloaded skeletal muscles. The internal oblique muscles become fibrotic during herniation, reducing abdominal wall compliance and increasing the transfer of load forces to the midline wound at the time of hernia repair.

Abdominal Muscles↗

PubMed2006

Regional variation of tibialis anterior tendon mechanics is lost following denervation.

Denervation or inactivity is known to decrease the mass and alter the phenotype of muscle. The mechanical response of tendon to inactivity that has been determined experimentally differs from what is reported by patients. We investigated the hypothesis that this difference was the result of artifacts of the testing process and did not represent what occurred in vivo. To test this hypothesis, a novel approach was used to determine the mechanical properties of the tibialis anterior (TA) tendon by optically measuring the end-to-end mechanical strains as well as the local strains at specific regions of excised TA tendon units. When the end-to-end strain of normal TA tendon is determined, stress-strain response curves show considerably more extensibility than when strain is measured across only the midsection of the tendon (mid-tendon). The strain experienced by the region close to the muscle (muscle tendon) is five times greater than the strain in either the mid-tendon or near the bone (bone-tendon). Five weeks of denervation decreased muscle mass by 67%; increased tendon mass by 10%; and changed the entire shape of the nonlinear response curve, including a loss in regional variation in strain, a 3.9-fold increase in end-to-end tangent modulus, and a 70% reduction in the toe region, as a result of a drastic reduction of the extensibility in the muscle-tendon region. The stress-strain response in the mid-tendon region of a normal TA tendon is therefore not indicative of its overall ability to deform in vivo as it transmits forces from muscle to bone.

Animals↗

PubMed2006

Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation.

In vitro studies have used protein markers to distinguish between myogenic cells isolated from fast and slow skeletal muscles. The protein markers provide some support for the hypothesis that satellite cells from fast and slow muscles are different, but the data are equivocal. To test this hypothesis directly, three-dimensional skeletal muscle constructs were engineered from myogenic cells isolated from fast tibialis anterior (TA) and slow soleus (SOL) muscles of rats and functionality was tested. Time to peak twitch tension (TPT) and half relaxation time (RT(1/2)) were approximately 30% slower in constructs from the SOL. The slower contraction and relaxation times for the SOL constructs resulted in left shift of the force-frequency curve compared with those from the TA. Western blot analysis showed a 60% greater quantity of fast myosin heavy chain in the TA constructs. 14 days of chronic low-frequency electrical stimulation resulted in a 15% slower TPT and a 14% slower RT(1/2), but no change in absolute force production in the TA constructs. In SOL constructs, slow electrical stimulation resulted in an 80% increase in absolute force production with no change in TPT or RT(1/2). The addition of cyclosporine A did not prevent the increase in force in SOL constructs after chronic low-frequency electrical stimulation, suggesting that calcineurin is not responsible for the increase in force. We conclude that myogenic cells associated with a slow muscle are imprinted to produce muscle that contracts and relaxes slowly and that calcineurin activity cannot explain the response to a slow pattern of electrical stimulation.

Animals↗

PubMed2006

Mesh incisional herniorrhaphy increases abdominal wall elastic properties: a mechanism for decreased hernia recurrences in comparison with suture repair.

BACKGROUND: An improved understanding of load-bearing soft tissue repair suggests that the mechanism for the improved outcomes after alloplastic incisional herniorrhaphy involves more than simple tissue replacement or material strength. We test the hypothesis that postrepair abdominal wall elastic properties are most predictive of successful abdominal wall reconstruction. METHODS: A rodent model of chronic incisional hernia formation was used. Midline incisional hernias were repaired primarily with suture (n = 24) or polypropylene mesh (n = 24). Rodents were sacrificed at serial postoperative time points over 60 days. Intact abdominal wall strips were cut perpendicular to the wound for tensiometric analysis. Biopsies of wound provisional matrix were obtained for biochemical analysis. RESULTS: Recurrent incisional hernia formation was significantly decreased in the mesh-repair group, compared with the suture-repair group (5/24 vs 14/24, P = .02). Mesh-repaired abdominal walls demonstrated significantly more elongation (P < .01) and less stiffness (P < .01). Toughness was equal between wounds, although the suture-repaired wounds had increased recovery of tensile strength (P < .01). There were no significant differences in collagen deposition after postoperative day 7. CONCLUSIONS: Mesh incisional herniorrhaphy increases abdominal wall elastic properties as measured by increased elongation and reduced stiffness. Increased abdominal wall elasticity after incisional hernia repair in turn results in lower recurrence rates.

Abdominal Wall↗

PubMed2006

Tissue-engineered axially vascularized contractile skeletal muscle.

BACKGROUND: As tissue-engineered muscle constructs increase in scale, their size is limited by the need for a vascular supply. In this work, the authors demonstrate a method of producing three-dimensional contractile skeletal muscles in vivo by incorporating an axial vascular pedicle. METHODS: Primary myoblast cultures were generated from adult F344 rat soleus muscle. The cells were suspended in a fibrinogen hydrogel contained within cylindrical silicone chambers, and situated around the femoral vessels in isogeneic adult recipient rats. The constructs were allowed to incubate in vivo for 3 weeks, at which point they were explanted and subjected to isometric force measurements and histologic evaluation. RESULTS: The resulting three-dimensional engineered skeletal muscle constructs produced longitudinal contractile force when electrically stimulated. Length-tension, force-voltage, and force-frequency relationships were similar to those found in developing skeletal muscle. Desmin staining demonstrated that individual myoblasts had undergone fusion to form multinucleated myotubes. Von Willebrand staining showed that the local environment within the chamber was richly angiogenic, and capillaries had grown into and throughout the constructs from the femoral artery and vein. CONCLUSIONS: Three-dimensional, vascularized skeletal muscle can be engineered in vivo. The resulting tissues have histologic and functional properties consistent with native skeletal muscle.

Animals↗

PubMed2006

Electrical stimulation of denervated muscles of rats maintains mass and force, but not recovery following grafting.

PURPOSE: Denervated skeletal muscles lack contractile activity and subsequently lose mass and force generation. Prolonged periods of denervation prior to nerve-implant grafting limit the recovery of mass and force. We hypothesized that electrical stimulation during a period of denervation that maintains mass and force above the levels of denervated muscles enhances the recovery of mass and force following nerve-implant grafting. METHODS: The extensor digitorum longus (EDL) muscles of anesthetized rats were denervated, and a stimulator was implanted. Following 4 or 7 months of denervation, with or without electrical stimulation, the EDL muscles were removed, evaluated in vitro for mass and contractile properties, and then nerve-implant grafted into syngeneic rats. Unoperated, contralateral muscles were also evaluated and grafted. RESULTS: The hypothesis was not supported by the experimental data. Compared with values for 4- or 7-month denervated muscles, the stimulated-denervated muscles maintained higher mass and force, less prolonged time-to-peak tensions and half-relaxation times, and higher excitability. Nevertheless, the recovery of mass and force following grafting was not improved. CONCLUSION: The factors within long-term denervated muscles that hinder recovery following grafting appear to be related primarily to factors associated with the duration of denervation and not to the level of atrophy and weakness prior to grafting.

Animals↗

PubMed2005

Comparison of gene expression of 2-mo denervated, 2-mo stimulated-denervated, and control rat skeletal muscles.

Loss of innervation in skeletal muscles leads to degeneration, atrophy, and loss of force. These dramatic changes are reflected in modifications of the mRNA expression of a large number of genes. Our goal was to clarify the broad spectrum of molecular events associated with long-term denervation of skeletal muscles. A microarray study compared gene expression profiles of 2-mo denervated and control extensor digitorum longus (EDL) muscles from 6-mo-old rats. The study identified 121 genes with increased and 7 genes with decreased mRNA expression. The expression of 107 of these genes had not been identified previously as changed after denervation. Many of the genes identified were genes that are highly expressed in skeletal muscles during embryonic development, downregulated in adults, and upregulated after denervation of muscle fibers. Electrical stimulation of denervated muscles preserved muscle mass and maximal force at levels similar to those in the control muscles. To understand the processes underlying the effect of electrical stimulation on denervated skeletal muscles, mRNA and protein expression of a number of genes, identified by the microarray study, was compared. The hypothesis was that loss of nerve action potentials and muscle contractions after denervation play the major roles in upregulation of gene expression in skeletal muscles. With electrical stimulation of denervated muscles, the expression levels for these genes were significantly downregulated, consistent with the hypothesis that loss of action potentials and/or contractions contribute to the alterations in gene expression in denervated skeletal muscles.

Animals↗

PubMed2005

Development of a three-dimensional physiological model of the internal anal sphincter bioengineered in vitro from isolated smooth muscle cells.

Fecal incontinence affects people of all ages and social backgrounds and can have devastating psychological and economic consequences. This disorder is largely attributed to decreased mechanical efficiency of the internal anal sphincter (IAS), yet little is known about the pathophysiological mechanisms responsible for the malfunction of sphincteric smooth muscle at the cellular level. The object of this study was to develop a three-dimensional (3-D) physiological model of the IAS bioengineered in vitro from isolated smooth muscle cells. Smooth muscle cells isolated from the IAS of rabbits were seeded in culture on top of a loose fibrin gel, where they migrated and self-assembled in circumferential alignment. As the cells proliferated, the fibrin gel contracted around a 5-mm-diameter SYLGARD mold, resulting in a 3-D cylindrical ring of sphincteric tissue. We found that 1) the bioengineered IAS rings generated a spontaneous basal tone, 2) stimulation with 8-bromo-cAMP (8-Br-cAMP) caused a sustained decrease in the basal tone (relaxation) that was calcium-independent, 3) upon stimulation with ACh, bioengineered IAS rings showed a calcium- and concentration-dependent peak contraction at 30 s that was sustained for 4 min, 4) addition of 8-Br-cAMP induced rapid relaxation of ACh-induced contraction and force generation of IAS rings, and 5) bioengineered sphincter rings show striking functional differences when compared with bioengineered rings made from isolated colonic smooth muscle cells. This is the first report of a 3-D in vitro model of a gastrointestinal smooth muscle IAS. Bioengineered IAS rings demonstrate physiological functionality and may be used in the elucidation of the mechanisms causing sphincter malfunction.

Acetylcholine↗

PubMed2005

Progressive fascial wound failure impairs subsequent abdominal wall repairs: a new animal model of incisional hernia formation.

BACKGROUND: Fascial wound failure alters the phenotype of the abdominal wall. This study introduces a novel animal model of progressive failure of the ventral abdominal wall fascia, which generates large incisional hernias. MATERIAL AND METHODS: A mechanistic model of incisional hernia was compared with a model of acute myofascial defect hernia repair. Using biological tissue repair markers, tensiometric measurements and recurrent hernia rate, we measured the mechanism by which incisional hernias regenerate abdominal wall structure and function after mesh and suture herniorrhaphy. RESULTS: Recurrent incisional hernia formation was significantly increased after repairs of the hernia model, compared with the myofascial defect model (6/16 vs 0/16, P < .05). In the hernia model, there were significant decreases in the recovery of wound strength, energy, and extensibility before mechanical disruption, compared with the myofascial defect model. Unexpectedly, excision of fascial hernia wound edges did not significantly improve tissue repair outcomes in the hernia model group. CONCLUSIONS: Clinically accurate animal modeling can recreate the wound pathology expressed in mature incisional hernias. Progressive fascial wound failure decreases the fidelity of subsequent incisional hernia repair, compared with identically sized acute abdominal wall defect repairs. The mechanism appears to include decreased fascial wound strength and decreased tissue compliance after herniorrhaphy.

Abdominal Muscles↗

PubMed2005

Electrical stimulation attenuates denervation and age-related atrophy in extensor digitorum longus muscles of old rats.

Skeletal muscles of old rats and elderly humans lose muscle mass and maximum force. Denervation is a major cause of age-related muscle atrophy and weakness, because denervated fibers do not contract, and undergo atrophy. At any age, surgical denervation causes even more dramatic muscle atrophy and loss in force than aging does. Electrical stimulation that generates tetanic contractions of denervated muscles reduces the denervation-induced declines. We investigated whether a stimulation protocol that maintains mass and force of denervated extensor digitorum longus muscles of adult rats would also maintain these properties in denervated muscles of old rats during a 2-month period of age-induced declines in these properties. Contractile activity generated by the electrical stimulation eliminated age-related losses in muscle mass and reduced the deficit in force by 50%. These data provide support for the hypothesis that during aging, lack of contractile activity in fibers contributes to muscle atrophy and weakness.

Aging↗

PubMed2005

In vivo conditioning of tissue-engineered heart muscle improves contractile performance.

The ability to engineer cardiac tissue in vitro is limited by the absence of a vasculature. In this study we describe an in vivo model which allows neovascularization of engineered cardiac tissue. Three-dimensional cardiac tissue, termed "cardioids," was engineered in vitro from the spontaneous delamination of a confluent monolayer of cardiac cells. Cardioids were sutured onto a support framework and then implanted in a subcutaneous pocket in syngeneic recipient rats. Three weeks after implantation, cardioids were recovered for in vitro force testing and histological evaluation. Staining for hematoxylin and eosin demonstrated the presence of viable cells within explanted cardioids. Immunostaining with von Willebrand factor showed the presence of vascularization. Electron micrographs revealed the presence of large amounts of aligned contractile proteins and a high degree of intercellular connectivity. The peak active force increased from an average value of 57 microN for control cardioids to 447 microN for explanted cardioids. There was also a significant increase in the specific force. There was a significant decrease in the time to peak tension and half relaxation time. Explanted cardioids could be electrically paced at frequencies of 1-5 Hz. Explanted cardioids exhibited a sigmoidal response to calcium and positive chronotropy in response to epinephrine. As the field of cardiac tissue engineering progresses, it becomes desirable to engineer larger diameter tissue equivalents and to induce angiogenesis within tissue constructs. This study describes a relatively simple in vivo model, which promotes the neovascularization of tissue-engineered heart muscle and subsequent improvement in contractile performance.

Animals↗

PubMed2005

Distribution of rest periods between electrically generated contractions in denervated muscles of rats.

Stimulation protocols for denervated muscles distribute the generated contractions either within treatment sessions followed by hours of rest, or repeated 24 h per day with each contraction followed by a constant interval of rest. Our purpose was to directly compare the effects of the same number of identically generated contractions having different temporal daily distributions. For 5 weeks in denervated extensor digitorum longus muscles of rats, between 100 and 800 contractions were generated daily, distributed either within worksets that alternated periods of activity and rest, or separated by constant intervals of rest. Most of the tested protocols maintained muscle mass and maximum force near values of innervated controls. Although 100 contractions daily generated at constant intervals were sufficient to maintain mass and force, 100 contractions during a 4-h treatment session followed by 20 h of rest were not sufficient, and mass and force were not different from values of denervated muscles.

Analysis of Variance↗

PubMed2004

Self-organization of rat cardiac cells into contractile 3-D cardiac tissue.

The mammalian heart is not known to regenerate following injury. Therefore, there is great interest in developing viable tissue-based models for cardiac assist. Recent years have brought numerous advances in the development of scaffold-based models of cardiac tissue, but a self-organizing model has yet to be described. Here, we report the development of an in vitro cardiac tissue without scaffolding materials in the contractile region. Using an optimal concentration of the adhesion molecule laminin, a confluent layer of neonatal rat cardiomyogenic cells can be induced to self-organize into a cylindrical construct, resembling a papillary muscle, which we have termed a cardioid. Like endogenous heart tissue, cardioids contract spontaneously and can be electrically paced between 1 and 5 Hz indefinitely without fatigue. These engineered cardiac tissues also show an increased rate of spontaneous contraction (chronotropy), increased rate of relaxation (lusitropy), and increased force production (inotropy) in response to epinephrine. Cardioids have a developmental protein phenotype that expresses both alpha- and beta-tropomyosin, very low levels of SERCA2a, and very little of the mature isoform of cardiac troponin T.

Animals↗

PubMed2004

A swimming robot actuated by living muscle tissue.

Biomechatronics is the integration of biological components with artificial devices, in which the biological component confers a significant functional capability to the system, and the artificial component provides specific cellular and tissue interfaces that promote the maintenance and functional adaptation of the biological component. Based upon functional performance, muscle is potentially an excellent mechanical actuator, but the larger challenge of developing muscle-actuated, biomechatronic devices poses many scientific and engineering challenges. As a demonstratory proof of concept, we designed, built, and characterized a swimming robot actuated by two explanted frog semitendinosus muscles and controlled by an embedded microcontroller. Using open loop stimulation protocols, the robot performed basic swimming maneuvers such as starting, stopping, turning (turning radius ~400 mm) and straight-line swimming (max speed >1/3 body lengths/second). A broad spectrum antibiotic/antimycotic ringer solution surrounded the muscle actuators for long term maintenance, ex vivo. The robot swam for a total of 4 hours over a 42 hour lifespan (10% duty cycle) before its velocity degraded below 75% of its maximum. The development of functional biomechatronic prototypes with integrated musculoskeletal tissues is the first critical step toward the long term objective of controllable, adaptive and robust biomechatronic robots and prostheses.

Journal Article↗

PubMed2004

Rapid formation of functional muscle in vitro using fibrin gels.

The transition of a muscle cell from a differentiated myotube into an adult myofiber is largely unstudied. This is primarily due to the difficulty of isolating specific developmental stimuli in vivo and the inability to maintain viable myotubes in culture for sufficient lengths of time. To address these limitations, a novel method for rapidly generating three-dimensional engineered muscles using fibrin gel casting has been developed. Myoblasts were seeded and differentiated on top of a fibrin gel. Cell-mediated contraction of the gel around artificial anchors placed 12 mm apart culminates 10 days after plating in a tubular structure of small myotubes (10-microm diameter) surrounded by a fibrin gel matrix. These tissues can be connected to a force transducer and electrically stimulated between parallel platinum electrodes to monitor physiological function. Three weeks after plating, the three-dimensional engineered muscle generated a maximum twitch force of 329 +/- 26.3 microN and a maximal tetanic force of 805.8 +/- 55 microN. The engineered muscles demonstrated normal physiological function including length-tension and force-frequency relationships. Treatment with IGF-I resulted in a 50% increase in force production, demonstrating that these muscles responded to hormonal interventions. Although the force production was maximal at 3 wk, constructs can be maintained in culture for up to 6 wk with no intervention. We conclude that fibrin-based gels provide a novel method to engineer three-dimensional functional muscle tissue and that these tissues may be used to model the development of skeletal muscle in vitro.

Animals↗

PubMed2004

Number of contractions to maintain mass and force of a denervated rat muscle.

Within 5 weeks, denervated extensor digitorum longus (EDL) muscles of rats lose 66% of mass, 91% of force, and 76% of fiber cross-sectional area (CSA). We previously determined the parameters of electrical stimulation for denervated rat EDL muscles to generate tetanic contractions sufficient to maintain mass and force close to control values. Using these parameters, we tested the hypothesis that a range exists for number of contractions per day, below and above which values for mass, maximum force, and fiber CSA are lower than values for innervated control muscles. For 5 weeks, denervated EDL muscles were stimulated to generate between 25 and 5000 contractions daily with contractions separated by constant intervals of rest, repeated 24 h per day. Force was not maintained with fewer than 200 or more than 800 contractions daily, whereas mass and fiber CSA were not maintained with fewer than 50 contractions daily. Protocols of stimulation that maintain muscle mass and force during prolonged periods of denervation may minimize problems clinically associated with denervation atrophy.

Animals↗

PubMed2004

The tensile properties of alginate hydrogels.

Alginate hydrogels are currently being employed and explored for a broad range of medical applications including cell encapsulation, drug delivery, and tissue engineering. In these capacities, knowledge of the mechanical and material properties of the hydrogels and the properties that govern and influence them is necessary to adequately design and effectively use these systems. Although much is known about the mechanical properties of alginate in compression and shear, little is known about the tensile characteristics. Thus, an extensive tensile assessment of alginate hydrogels was completed as a function of alginate type, formulation, gelling conditions, incubation, and strain rate. In general, the initial tensile behavior and properties of alginate hydrogels were highly dependent on the choice of the alginate polymer and how it was processed. Specifically, high guluronic acid containing alginate polymers yielded stronger, more ductile hydrogels than high mannuronic acid containing alginates. The ultimate stress, ultimate strain, and tensile modulus were decreased by increased phosphate concentrations, solution reconstitution with phosphate buffered saline instead of culture media, and peptide modification. Incubation of hydrogels for at least 7 days diminished many of the initial tensile property differences associated with formulation and gelling conditions. Overall, by controlling the specific alginate polymer and processing methods, a wide range of tensile properties are available from these hydrogels.

Alginates↗

PubMed2004

Contractile skeletal muscle tissue-engineered on an acellular scaffold.

For the reconstructive surgeon, tissue-engineered skeletal muscle may offer reduced donor-site morbidity and an unlimited supply of tissue. Using an acellularized mouse extensor digitorum longus muscle as a scaffold, the authors produced engineered skeletal muscle capable of generating longitudinal force. Eight extensor digitorum longus muscles from adult mice were made acellular using a protocol developed in the authors' laboratory. The acellular muscles were then placed in a bath of 20% fetal bovine serum in Dulbecco's modified Eagle's medium and 100 U/ml penicillin for 1 week at room temperature. C2C12 myoblasts were injected into the acellular muscle matrix using a 26-gauge needle and a 100-microl syringe. The resulting constructs were placed in growth medium for 1 week at 37 degrees C under 5% carbon dioxide, with media changes every 48 hours. The constructs were then placed in differentiation medium for 1 week, with media changes every 48 hours. Isometric contractile force testing of the constructs demonstrated production of longitudinal contractile force on electrical stimulation. A length-tension, or Starling, relationship was observed. Light and electron microscopy studies demonstrated recapitulation of some of the normal histologic features of developing skeletal muscle.

Animals↗