Victims of major electrical trauma frequently suffer extensive skeletal muscle and nerve damage, which is postulated to be principally mediated by electroporation and/or thermally driven cell membrane permeabilization. We have investigated the efficacy oftwo blood-compatible chemical surfactants for sealing electroporated muscle membranes. In studies using cultured skeletal muscle cells, poloxamer 188 (P188; an 8. Membrane damage is often manifested clinically by release of intracellular contents into the intravascular space (5), one of the clinical hallmarks of major electrical trauma. Skeletal muscle and peripheral nerve necrosis appears to be the primary cause of the high amputation rates associated with electrical trauma. We have postulated that, in the majority of victims, cell membrane permeabilization is the most important pathophysiologic event leading to tissue death (4,6,7) and, therefore, effective therapy for victims of electric shock must reestablish cell membrane structural integrity.Because membranes form spontaneously when surfactants (amphiphiles) are mixed in an aqueous solvent at sufficient concentration, we hypothesized that it may be possible to seal damaged cell membranes by exposing them to adequate concentrations of a noncytotoxic nonionic surfactant, possibly by incorporation of the surfactant into the membrane defects. In a preliminary test of this concept, we found that an 8.4-kDa nonionic synthetic surfactant, poloxamer 188 (P188), which has been clinically accepted for human intravenous administration, effectively sealed electroporated membranes of cultured skeletal muscle cells when used in concentrations >0.5 mg/ml (8, 9). We also noted that sealing the membrane enhanced cell survival as measured by vital dye [i.e., carboxyfluorescein (CF) and trypan blue] assays. The ability of P188 to bind to damaged membranes has been suggested in previous studies (10, 11).The purposes of this investigation were to determine whether the observed membrane effects of P188 on isolated cells were relatively specific to its molecular properties by comparing P188 with a neutral polysaccharide known to adsorb on the lipid bilayer (12), to determine whether the P188 and neutral polysaccharide would also reach damaged cell membranes in situ via intravenous administration and seal them after electropermeabilization, and, most important, to determine whether membrane sealing could prevent tissue necrosis following electrical injury. MATERIALS AND METHODS

P188, a triblock copolymer of the form poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) helps seal electroporated cell membranes, arresting the leakage of intracellular materials from the damaged cells. To explore the nature of the interaction between P188 and cell membranes, we have constructed a model system that assesses the ability of P188 to insert into lipid monolayers. Using concurrent Langmuir isotherm and fluorescence microscopy measurements, we find that P188 changes the phase behavior and morphology of the monolayers. P188 inserts into both dipalmitoylphosphatidlycholine and dipalmitoylphosphatidylglycerol monolayers at surface pressures equal to and lower than approximately 22 mN/m at 30 degrees C; this pressure corresponds to the maximal surface pressure attained by P188 on a pure water subphase. Similar results for the two phospholipids indicate that P188 insertion is not influenced by headgroup electrostatics. Because the equivalent surface pressure of a normal bilayer is on the order of 30 mN/m, the lack of P188 insertion above 22 mN/m further suggests the poloxamer selectively adsorbs into damaged portions of electroporated membranes, thereby localizing its effect. P188 is also found to be "squeezed out" of the monolayers at high surface pressures, suggesting a mechanism for the cell to be rid of the poloxamer when the membrane is restored.

The relation between mechanical loading of cartilage and chondrocyte activity in vivo may be mediated by several physical transduction mechanisms including: cell deformation, hydrostatic pressure gradients, fluid flow, streaming currents, and physicochemical changes. We have developed an organ culture system designed to study chondrocyte biosynthetic response to such physical stimuli. This study focuses on the effects of static compression and physicochemical changes. Cartilage disks harvested from the reserve zone of the epiphyseal plate of 1-2-week-old calves were subjected to static compressive stresses of 0-3 MPa in unconfined compression and the incorporation of [35S]sulfate and [3H]proline was measured during the 12-h loading period. Incorporation of both proline and sulfate decreased monotonically with increasing stress. Compressive loading also produces physicochemical changes including a decreased water content and increased matrix fixed-charge density, with a concomitant increase in interstitial counterion concentrations (e.g., K+, H+) and decreased coion concentrations (e.g., SO4(2-). We therefore examined the possibility that specific changes in interstitial mobile ion concentrations may be linked to chondrocyte response to static compression by measuring biosynthesis in the absence of mechanical compression while independently altering the SO4(2-), K+, and H+ composition of the bathing medium. We found that proline and sulfate incorporation were strongly dependent on pH, but independent of [SO4(2-)] and [K+] in the range studied. These results suggest that compression-induced changes in local, interstitial pH may account for the observed biosynthetic response to static compression.

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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.