Raphael C. Lee

Mechanisms and dynamics of mechanical strengthening in ligament-equivalent fibroblast-populated collagen matrices

We have measured the dynamics of extracellular matrix consolidation and strengthening by human dermal fibroblasts in hydrated collagen gels. Constraining matrix consolidation between two porous polyethylene posts held rigidly apart set up the mechanical stress which led to the formation of uniaxially oriented fibroblast-populated collagen matrices with a histology resembling a ligament. We measured the mechanical stiffness and tensile strength of these ligament equivalents (LEs) as a function of age at biweekly intervals up to 12 weeks in culture using a mechanical spectrometer customized for performing experiments under physiologic conditions. The LE load-strain curve changed as a function of LE age, increasing in stiffness and exhibiting less plastic-like behavior. At 12 weeks, LEs had acquired up to 30 times the breaking strength of 1-week-old LEs. Matrix strengthening occurred primarily through the formation of BAPN-sensitive, lysyl oxidase catalyzed crosslinks. Sulfated glycosaminoglycan (GAG) content increased monotonically with LE age, reaching levels that are characteristic of ligaments. Cells in the LEs actively incorporated [3H]proline and [35S]sulfate into the extracellular matrix. Over the first three weeks, DNA content increased rapidly but thereafter remained constant. This data represent the first documentation of strengthening kinetics for cell-assembled biopolymer gels and the results suggest that this LE tissue may be a valuable model for studying the cellular processes responsible for tissue growth, repair, and remodeling.

Electrical injury mechanisms: electrical breakdown of cell membranes.

Electric fields are capable of damaging cells through both thermal and nonthermal mechanisms. While joule heating is generally recognized to mediate tissue injury in electrical trauma, the possible role of electrical breakdown of cell membranes has not been thoroughly considered. Evidence is presented suggestive that in many instances of electrical trauma the local electric field is of sufficient magnitude to cause electrical breakdown of cell membranes and cell lysis. In theory, large cells such as muscle and nerve cells are more vulnerable to electrical breakdown. To illustrate the significance of cell size and orientation, a geometrically simple model of an elongated cell is analyzed.

Oscillatory compressional behavior of articular cartilage and its associated electromechanical properties.

The compressive stiffness of articular cartilage was examined in oscillatory confined compression over a wide frequency range including high frequencies relevant to impact loading. Nonlinear behavior was found when the imposed sinusoidal compression amplitude exceeded a threshold value that depended on frequency. Linear behavior was attained only by suitable control of the compression amplitude. This was enabled by real time Fourier analysis of data which provided an accurate assessment of the extent of nonlinearity. For linear viscoelastic behavior, a stiffness could be defined in the usual sense. The dependence of the stiffness on ionic strength and proteoglycan content showed that electrostatic forces between matrix charge groups contribute significantly to cartilages compressive stiffness over the 0.001 to 20 Hz frequency range. Sinusoidal streaming potentials were also generated by oscillatory compression. A theory relating the streaming potential field to the fluid velocity field is derived and used to interpret the data. The observed magnitude of the streaming potential suggests that interstitial fluid flow is significant to cartilage behavior over the entire frequency range. The use of simultaneous streaming potential and stiffness data with an appropriate theory appears to be an important tool for assessing the relative contribution of fluid flow, intrinsic matrix viscoelasticity, or other molecular mechanisms to energy dissipation in cartilage. This method is applicable in general to hydrated, charged polymers.

Injury by electrical forces: Pathophysiology, Manifestations, and therapy

The pathogenesis and pathophysiologic features of electrical injury are more complex than once thought. The relative contributions of thermal and pure electrical damage depend on the duration of electric current passage, the orientation of the cells in the current path, their location, and other factors. If the contact time is brief, nonthermal mechanisms of cell damage will be most important and the damage is relatively restricted to the cell membrane. When contact time is much longer, however, heat damage predominates and the whole cell is affected directly. These parameters also determine the anatomic tissue distribution of injury. Damage by Joule heating is not known to be dependent on cell size, whereas larger cells are more vulnerable to membrane breakdown by electroporation. Cells do survive transient plasma membrane rupture under appropriate circumstances or if therapy is instituted quickly. If membrane permeabilization is the primary cellular pathologic condition, then injured tissue may be salvageable and the challenge for the future is to identify a technique to reseal the damaged membranes promptly. Present standards of care for electrical injury require a fully staffed and well-equipped intensive care unit, available operating suites, and the availability of the full range of medical specialists. Major teaching hospitals with burn centers may be the ideal setting for the treatment of an electrical trauma victim. After the initial resuscitation, efforts are directed primarily towards preventing additional tissue loss mediated through the compartment syndrome, compressive neuropathies, or the presence of necrotic tissue. Renal and cardiac failure caused by the release of intracellular muscle contents into the circulation must be prevented. Attention can then be directed towards maximizing tissue salvage and preventing late skeletal and neuromuscular complications. Reconstructive procedures that transfer healthy tissue from a distance are necessary to optimize the functional value of the remaining tissue. Finally, unless the patient is rehabilitated psychologically, the real benefit from other sophisticated care will not be fully realized. These goals are important throughout the acute care of the patient. In the future, new guidelines for treating electrical trauma will be based on a clearer understanding of the relevant pathophysiologic features. These strategies will rely on improved diagnostic imaging and on reversing the fundamental problem of cell membrane damage. Moreover, complex biochemical and organ system pathophysiologic interactions will require careful management. If successful, research efforts presently underway should improve the prognosis of victims after electrical trauma.

Intralesional Verapamil Injection for the Treatment of Peyronie’s Disease

Peyronies disease remains a therapeutic dilemma for the practicing urologist. Multiple nonoperative therapies have been offered with variable suboptimal response rates. Calcium channel blockers have been shown to alter the metabolism of fibroblasts, resulting in decreased extracellular matrix secretion of collagen as well as increased collagenase activity. In this nonrandomized dose-escalating format study 14 men received biweekly injections of verapamil into the Peyronies plaques for 6 months. Subjectively, there was significant improvement in plaque-associated penile narrowing (100%) and curvature (42%). Objectively, a decreased plaque volume of greater than 50% was noted in 30% of the subjects. Plaque softening was noted in all patients, while 83% noticed that plaque-related changes in erectile function had arrested or improved. There was no toxicity nor did symptoms recur when improvement was noted. This preliminary study suggests that intralesional calcium antagonist (verapamil) therapy offers an economical and sensible nonoperative approach to the treatment of Peyronies disease.

The mechanisms of action of vacuum assisted closure: More to learn

Division of Plastic and Reconstructive Surgery, Brigham and Women’s Hospital , Boston, MA; Division of Plastic and Reconstructive Surgery, University of Pittsburg School of Medicine , Pittsburg, PA; Department of Surgery, Yale University School of Medicine , New Haven, CT; Department of Surgery, University of Chicago Hospitals , Chicago, IL; Limb Center, Georgetown University Medical Center, Washington, DC; Department of Surgery, Stanford University Medical Center, Stanford, CA; Division of Plastic Surgery, Department of Surgery, Penn State University Milton S. Hershey Medical Center, Hershey, PA

Rhabdomyolysis Due to Pulsed Electric Fields

High-voltage electrical trauma frequently results in extensive and scattered destruction of skeletal muscle along the current path. The damage is commonly believed to be mediated by heating. Recent experimental and theoretical evidence suggests, however, that the rhabdomyolysis and secondary myoglobin release that occur also can result from electroporation, a purely non-thermal mechanism. Based on the results of a computer simulation of a typical high-voltage electric shock, we have postulated that electroporation contributes substantially to skeletal muscle damage and could be the primary mechanism of damage in some cases of electrical injury. In this study, we determined the threshold field strength and exposure duration required to produce rhabdomyolysis by the electroporation mechanism. The change in the electrical impedance of intact skeletal muscle tissue following the application of short-duration, high-intensity electric field pulses is used as an indicator of membrane damage. Our experiments show that a decrease in impedance magnitude occurs following electric field pulses that exceed threshold values of 60 V/cm magnitude and 1-ms duration. The field strength, pulse duration, and number of pulses are factors that determine the extent of damage. The effect does not depend on excitation-contraction coupling. Electron micrographs confirm structural defects created in the membranes by the applied electric field pulses, and these represent the first clear demonstration of rhabdomyolysis in intact muscle due to electroporation. These results provide compelling evidence in support of our postulate.

Calcium antagonists retard extracellular matrix production in connective tissue equivalent.

Hypertrophic and keloid scars are characterized by the overproduction of extracellular matrix collagen and proteoglycans. Because cellular secretion of macromolecules is known to be a calcium-dependent process, we have examined the effect of calcium antagonists on the rate of incorporation of [3H]proline into protein and Na235SO4 into sulfated glycosaminoglycans. Experiments were carried out in vitro using uniaxially oriented mammalian fibroblast-populated collagen matrices which exhibit many histoarchitectural features of scar matrix. We observed that nifedipine and verapamil in sufficient concentrations inhibited the incorporation of proline into extracellular matrix protein. The concentration range studied was 1 to 100 microM. Cobalt chloride (200 microM) had a similar effect on proline incorporation, but enhanced the incorporation of sulfate into extracellular matrix glycosaminoglycans. We conclude that cellular calcium metabolism appears to regulate extracellular matrix production and that hypertrophic disorders of wound healing may respond to therapy with calcium antagonist drugs.

Significance of cell size and tissue structure in electrical trauma

High-voltage electrical trauma frequently leads to extensive and selective destruction of muscle and nerve tissue. In this paper, the mechanism of plasma membrane disruption due to the large transmembrane potentials imposed during electrical trauma is used to explain the particular susceptibility of muscle and nerve cells to damage. It is proposed that this vulnerability is partially due to the relatively large size of these cells. A distributed-parameter electric cable model of an elongated cell is used to examine the alteration of the transmembrane potential caused by a 60 Hz electric field applied parallel to the long axis of the cell. The maximum predicted transmembrane potential occurs at the ends of the cell and is strongly cell-size dependent. Theories are discussed which illustrate how this could explain the predisposition of skeletal muscle to cell membrane breakdown and rupture. The predicted effect of either close-neighboring cells in a tissue or cell contact with cortical bone is even greater induced transmembrane potentials and increased probability of rupture. This is the first hypothesis which explains the clinically-observed pattern of tissue damage resulting from electrical trauma.

Kinetics of the chondrocyte biosynthetic response to compressive load and release

To gain insight regarding the rate at which cartilage tissue can sense and respond to a dynamic mechanical stimulus, we have examined the time-course of changes in biosynthetic activity following both the application and release of a static compressive stress. Cartilage harvested from the reserve zone of calf epiphyseal plate was subjected to unconfined static compressive stresses of 0, 0.25 and 0.5 MPa. Incorporation of [35S]sulfate and [3H]proline was measured during loading periods of less than 1 to 26 h and after preloading periods of 0.5, 2 or 12 h. During loading, total incorporation decreased to steady levels with time constants estimated to be 0.25-4 h (proline) and 1-5 h (sulfate). Proline incorporation exceeded control levels for 3 h after release of a 2 or 12 h preload. Sulfate incorporation remained depressed for at least 4 h after release of a 12 h preload and remained at control levels following release of 0.5 and 2 h preloads. We conclude that the modulation of proline incorporation by both loading and load release is faster than the modulation of sulfate incorporation. Furthermore, the response to unloading is not just the inverse of the response to loading; this nonlinearity suggests that the response to dynamic loading would not be determined simply by the time average component of the dynamic load.