H. Paul Ehrlich

Cell locomotion forces versus cell contraction forces for collagen lattice contraction: an in vitro model of wound contraction.

Cultured human dermal fibroblasts suspended in a rapidly polymerizing collagen matrix produce a fibroblast-populated collagen lattice. With time, this lattice will undergo a reduction in size referred to as lattice contraction. During this process, two distinct cell populations develop. At the periphery of the lattice, highly oriented sheets of cells, morphologically identifiable as myofibroblasts, show cell-to-cell contacts and thick, actin-rich staining cytoplasmic stress fibers. It is proposed that these cells undergoing cell contraction produce a multicellular contractile unit which reorients the collagen fibrils associated with them. The cells in the central region, referred to as fibroblasts, are randomly oriented, with few cell-to-cell contacts and faintly staining actin cytoplasmic filaments. In contrast it is proposed that cells working as single units use cell locomotion forces to reorient the collagen fibrils associated with them. Using this model, we sought to determine which of these two mechanisms, cell contraction or cell locomotion, is responsible for the force that contracts collagen lattices. Our experiments showed that fibroblasts produce this contractile force, and that the mechanism for lattice contraction appears to be related to cell locomotion. This is in contrast to a myofibroblast; where the mechanism for contraction is based upon cell contractions. Fibroblasts attempting to move within the collagen matrix reorganize the surrounding collagen fibrils; when these collagen fibrils can be organized no further and cell-to-cell contacts develop, which occurs at the periphery of the lattice first, these cells can no longer participate in the dynamic aspects of lattice contraction.

Studies on vascular smooth muscle cells and dermal fibroblasts in collagen matrices: Effects of heparin☆

The incorporation of such tissue-cultured mesenchymal cells as bovine vascular smooth muscle cells (SMS) and human dermal fibroblasts (DF) in a collagen matrix results in the reorganization and distortion of that matrix. A 2 ml collagen matrix populated by 55 000 bovine SMC and having a surface area of 800 mm2 will be reduced to 226 mm2 by 48 h. Under identical conditions, a lattice populated by 55 000 DF will achieve an area of 78 mm2 at 48 h. DF are thus more efficient at reducing the size of a collagen lattice by the process of lattice contraction. Bovine SMC proliferate in a collagen matrix; human DF do not. DF in a collagen matrix have a more elongate morphology than SMC. Actin cytoplasmic filaments were studied using the specific F-actin staining reagent, Rhodamine-phalloidin. DF in collagen matrix exhibit diffuse cytoplasmic staining while, in monolayer, they display prominent staining stress fibers. SMC in monolayer and in matrices show stained clumps at the periphery of the cell. The addition of 200 U/ml heparin to SMC eliminates those actin aggregates and causes the formation of stress fibers. It also causes stress fibers to form in dermal fibroblasts in a collagen lattice. However, the elongation and spreading--and the formation of stress fibers brought about by heparin--lead to an inhibition of lattice contraction. Heparin effectively inhibits cell-mediated lattice contraction in SMC and DF, and it also causes the formation of cytoplasmic stress fibers.

The modulation of contraction of fibroblast populated collagen lattices by types I, II, and III collagen

Human dermal fibroblasts incorporated in a polymerized collagen lattice reduce the size of that matrix. When cell number, collagen concentration, and medium are identical, lattices made with type III collagen contract faster and to a greater degree than those made with type I collagen. The latter contract faster and to a greater degree than those made with type II collagen.

Physiological variables affecting collagen lattice contraction by human dermal fibroblasts

Normal human dermal fibroblasts cultured in collagen lattices can compact that matrix by the process known as lattice contraction. That process is a model of the pathological one of scar contracture or wound contraction and is affected by several factors. Lattice contraction is promoted by the addition of adequate amounts of fetal bovine serum to the medium (maximum contraction with 10% serum). The process requires energy, of which glucose and pyruvate have been shown to be adequate sources. When glucose is used as the substrate, the major pathway of energy generation appears to be anaerobic metabolism. When pyruvate is the only substrate, aerobic metabolism may be crucial. The synthesis of DNA is not required for lattice contraction, while protein synthesis is, although the identities of the specific proteins are unknown. Impairment of calcium ion transport inhibits lattice contraction, and the specific inhibition of calmodulin-calcium interactions by W-7 blocks contraction. W-7 at a concentration of 6 x 10(-6) M blocks lattice contraction completely, while it has no effect at any lower concentration. Impairing dynamic microtubule activity impairs contraction. Disrupting microfilaments by cytochalasin B completely blocks lattice contraction. Microfilament function and calcium-calmodulin may be linked by a mechanism involving myosin-ATPase. The process of cell-mediated lattice contraction requires the production of energy, protein synthesis, and a functional cytoskeleton.

The identification of αA and αB collagen chains in hypertrophic scar

Abstract Hypertrophic scarring develop as a result of full thickness dermal destruction. The connective tissue matrix of this lesion is mostly collagen. Collagen extracted by limited pepsin digestion was partially characterized, using bulk salt and heat-gelling techniques. The different collagen types isolated were Types I, III, and V or AB collagen composed of αA and αB chains. The identity of these collagen types was established by stained protein band patterns on sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The possible location of Type V or AB collagen in hypertrophic scar is discussed.

Human Skin and Post-Burn Scar Hyaluronan: Demonstration of the Association with Collagen and other Proteins

Hyaluronan (HA) extracted from tissues has been demonstrated to have an enhancing effect on the process of wound healing; the question arises whether this effect is due to the HA or to associated collagen and other proteins. In this study, HA has been extracted from human skin and scar tissue under dissociative conditions and isolated by DEAE-cellulose chromatography followed by CsCl gradient and Sepharose CL-6B chromatography. This highly purified HA was found to contain between 4 and 28% protein, with collagen constituting 5% of the total protein. A functional association between HA and a collagen protein complex is proposed.

Heparin modulates human intestinal smooth muscle cell proliferation, protein synthesis, and lattice contraction

The effect of heparin on human intestinal smooth muscle cell proliferation, collagen and noncollagen protein synthesis, and collagen lattice contraction was studied in vitro. Proliferation of serum-stimulated cells was inhibited in a concentration-related fashion by continuous exposure to heparin. The inhibition of proliferation was reversible when heparin was removed from the culture medium. Collagen synthesis was inhibited by 24-h exposure to heparin, but only during that phase of culture (8-12 days) when collagen synthesis was maximal. Noncollagen protein synthesis was down-regulated by 24-h exposure to heparin at all phases of culture tested (5-21 days). Heparin also abolished the contraction of collagen lattices populated by human intestinal smooth muscle cells. These studies demonstrate that heparin plays a significant role in the modulation of human intestinal smooth muscle cell behavior in vitro and suggest that a similar role is played by heparinlike components of the extracellular matrix in vivo.

Promotion of Vascular Patency in Dermal Burns with Ibuprofen

Differences in the vascular response to burn and freeze injuries were investigated as a model for defining the mechanism and cause of vascular occlusion in rats after dermal burns. Concentrations of thromboxane and prostacyclin in wound fluid were elevated in both types of trauma. However, inhibition of prostaglandin synthesis by indomethacin failed to promote vascular patency in burn-injured animals. However, the systemic administration of ibuprofen and imidazole led to increased vascular patency. Ibuprofen promoted vascular patency even when given six hours after burn trauma. These studies indicate that ibuprofen and imidazole promote vascular patency by fostering fibrinolysis rather than by inhibiting prostaglandin synthesis and release.

Dermal vascular patterns in response to burn or freeze injury in rats

Abstract Full thickness injury of rat skin by burning results in wound healing with contraction whereas wounds of similar area and depth caused by freezing heal without contracting. Burns cause cell death and denaturation of the connective tissue matrix; freezing causes cell death, but the matrix is not immediately denatured and appears to maintain its integrity during the initial phases of repair. Differences in vascular perfusion within and surrounding the site of burn and freeze injury are described in the present study. During the first 2 hr after burning, arteriograms and Microfil latex vascular casts showed reduced, but existent perfusion at the injury site; in contrast, the site of freezing showed little perfusion. At 4 to 5 hr following injury, this pattern had reversed. There was loss of perfusion in the burn injury site and reappearance of perfusion in the frozen site. At 24 hr, the burned site was nearly avascular and surrounded by an area of vasodilatation. The frozen site had a patent vasculature within the dermis and little vasodilatation in the surrounding area. The persistent perfusion in the frozen dermis was presumably through vascular channels which were dead, but not occluded. We suspect that factors released from the burned matrix account for some of these differences in pattern of vascular perfusion.

Local increases of subcutaneous β-endorphin immunoactivity at the site of thermal injury

To examine interactions between exogenous opioid analgesia and endogenous opioid generation at a site of burn-induced tissue injury, we measured beta-endorphin (BE) and corticosterone (C) in aliquots of plasma and wound fluid withdrawn from subcutaneous wire mesh chambers beneath the site of a 3-5% surface area burn. After brief inhalational anesthesia at the time of thermal injury, rats received morphine (4 mg/kg, single dose), fentanyl (0.02 mg/kg hourly for 4 h), or no opioid. Systemic hormone responses and behavioral changes were minimal as expected for the minimal percentage burn. In all three groups intrachamber BE and C rose above baseline at 1, 2 and 4 h postburn, then returned to baseline at 24 h. Systemic opioid treatment produced analgesia (by tail flick latency testing) but did not reduce intrachamber hormone responses. Thus local BE and C responses at the site of thermal injury are regulated differently from systemic pituitary-adrenal responses.