Showing papers on "Wound healing published in 1985"

Cutaneous tissue repair: Basic biologic considerations. I

Abstract: Wound repair of the integument is reviewed in the context of new developments in cell biology and biochemistry. Injury of the skin and concomitant blood vessel disruption lead to extravasation of blood constituents, followed by platelet aggregation and blood clotting. These events initiate inflammation and set the stage for repair processes. The macrophage plays a pivotal role in the transition between wound inflammation and repair (granulation tissue formation), since this cell both scavenges tissue debris and releases a plethora of biologically active substances that include growth factors. Although concrete evidence is lacking, growth factors are probably at least partially responsible for the angiogenesis and fibroplasia (granulation tissue) that gradually fill the wound void. If the epidermal barrier is disrupted during injury, reepithelialization begins within 24 hours and proceeds first over the margin of residual dermis and subsequently over granulation tissue. The signals for angiogenesis, fibroplasia, neomatrix formation, and reepithelialization in wound repair are not known, but a number of possibilities are discussed. Matrix remodeling is the last stage of wound repair and gradually increases the scar tensile strength to 70% to 80% of normal skin.

Sustained release of epidermal growth factor accelerates wound repair.

Abstract: Epidermal growth factor (EGF) is a potent mitogen in vitro, but its biological role is less clear. The vulnerary effects of EGF were evaluated in a model of wound repair, the polyvinyl alcohol sponge implanted subcutaneously in rats. EGF was purified to homogeneity by reverse-phase HPLC and quantified by receptor binding assay and amino acid analysis. Preliminary data showed moderate promotion of granulation tissue formation by daily injections of 10 micrograms of EGF. To test the hypothesis that long-term exposure to EGF is required for complete cellular response, the factor was incorporated into pellets releasing 10 or 20 micrograms of biologically active EGF per day, and the pellets were embedded within the sponges. Slow release of EGF caused a dramatic increase in the extent and organization of the granulation tissue at day 7, a doubling in the DNA content, and 33% increases in protein content and wet weight, as compared with placebo controls. Although collagen content was also increased by almost 50%, the relative rate of collagen synthesis remained the same, suggesting that the morphological and biochemical increase in collagen resulted from increased numbers of fibroblasts rather than a specific stimulation of collagen synthesis. These results indicate that the local sustained presence of EGF accelerates the process of wound repair, specifically neovascularization, organization by fibroblasts, and accumulation of collagen.

Stimulation of granulation tissue formation by platelet-derived growth factor in normal and diabetic rats.

Abstract: Subcutaneous implantation of Hunt-Schilling wound chambers in rats induces a wound repair response causing the chamber first to fill with fluid and subsequently with connective tissue. The presence of a type I collagen gel encouraged a more rapid dispersion of cells throughout the chamber but had no effect on the rate of new collagen deposition. Addition of platelet-derived growth factor (PDGF; 50 ng/chamber) to the collagen-filled chambers caused an earlier influx of connective tissue cells, a marked increase in DNA synthesis, and a greater collagen deposition in the chamber during the first 2 wk after implantation. After 3 wk, however, the levels of collagen were similar in PDGF-supplemented and control chambers. Diabetic animals exhibited a decreased rate of repair which was restored to normal by addition of PDGF to the wound chamber. Combinations of PDGF and insulin caused an even more rapid increase in collagen deposition. These results suggest that the levels of various growth factors, particularly PDGF, may be limiting at wound sites and that supplementation of wounds with these factors can accelerate the rate of new tissue formation.

Human endothelial cells are chemotactic to endothelial cell growth factor and heparin.

Abstract: The response of human endothelial cell migration to various extracellular matrix components and growth factors has been assessed. Human endothelial cells demonstrate increased chemotaxis and chemokinesis when placed in a modified Boyden chamber with endothelial cell growth factor (ECGF) used at a concentration of 10(-9) M. Anti-ECGF antibody inhibits the chemotactic response. Heparin (10(-8) to 10(-10) M) was also chemotactic and was shown to potentiate the chemotactic activity of ECGF. Although laminin, fibronectin, the polypeptide (epidermal, fibroblast, and nerve) growth factors, and collagen types I, II, III, IV, and V demonstrate a chemotactic response, these activities were one third to one half less than observed with ECGF. These data suggest that ECGF and heparin may play a significant role as response modifiers of human endothelial cell migration which may be relevant to tumor metastasis, wound healing, and atherogenesis.

Differentiation of Pulpal Cells and Inductive Influences of Various Matrices with Reference to Pulpal Wound Healing

Abstract: Based on recent literature, the dynamics of mesenchymal cells in transplantation of various tissues and matrices, as well as the origin of new odontoblasts which participate in the formation of the dentin bridge, are described. Experiments involving implantation of pulp, periosteum, perichondrium, treated dentin, and bone matrices were performed to emphasize the capability of these cells to produce hard tissue. Light and electron microscopic and autoradiographic studies were carried out to clarify the origin of replacement odontoblasts. It appears that the pulp cells, endothelial cells, and pericytes become undifferentiated mesenchymal cells following pulp exposure. These mesenchymal cells differentiate into odontoblasts, which subsequently produce a dentin matrix. Pulp tissues autografted to non-pulpal sites, elaborated bone (or osteodentin) matrix, but they did not graft to tubular dentin. An experiment on dentin bridge formation, using germ-free rats, demonstrated that the pulp tissue has intrinsic healing potential. Therefore, it was concluded that the ability of pulp tissue to elaborate hard tissues depends on its environment.

Accelerated wound repair, cell proliferation, and collagen accumulation are produced by a cartilage-derived growth factor.

Abstract: Cartilage-derived growth factor (CDGF), a cationic polypeptide of approximately 18,000 mol wt, was prepared from bovine articular cartilage; other sources were bovine and human scapular and costal cartilage. Previous studies have shown that CDGF stimulates the proliferation of cultured mouse fibroblasts as well as chondrocytes and endothelial cells from various sources. In this study, CDGF was shown to stimulate dose-dependently the accumulation of DNA and collagen by rat embryo fibroblasts and a population of fibroblasts derived from granulation tissue. CDGF also stimulated the proliferation of cultured bovine capillary endothelial cells dose-dependently. To evaluate the effects of CDGF in vivo, we implanted polyvinyl alcohol sponges subcutaneously in rats. 6 d postimplantation, sponges were injected with 300 micrograms of partially purified CDGF, a dose which takes into account the cell numbers in the sponges as compared with cell cultures. CDGF rapidly disappeared from the sponges and only approximately 10% of the initial dose was present at 4 h. Despite its transient presence, CDGF caused a relative increase in sponge DNA content of 2.6-fold at 48 h and 2.4-fold at 72 h. We repeated the sponge experiment by using 500-ng injections of CDGF purified to near homogeneity by heparin-Sepharose chromatography. Purified CDGF caused significant increases in sponge collagen, protein, and DNA content at 48 and 72 h after a single injection. The effects of CDGF were abolished by heat and unaffected by reduction of disulfide linkages. Morphologically, CDGF did not evoke an inflammatory response, and its effect on proliferating endothelial cells and fibroblasts was, therefore, probably direct. However, increases in DNA content of sponges could not be fully accounted for by increased DNA synthesis, which suggests that recruitment may be an important component of the in vivo response. Taken together, the effects of CDGF on cultured cells and granulation tissue suggest that the sustained presence of CDGF in vivo may greatly enhance its effects upon wound repair.

Collagen fiber formation in repair tissue: development of strength and toughness.

Abstract: Dermal repair tissue shows a progressive increase in collagen content which may be related to the wound tensile strength. Wound strength and extensibility are lower than those found in normal skin. In animals, wounds closed by metal clips are chosen as a model to study the proliferative and remodeling phases of healing from a mechanical and morphological point of view. During the proliferative phase the low wound strength is associated with formation of collagen fibers of small diameter, later, (days 28-45) an acute change appears corresponding to the remodeling phase, with increased collagen fiber diameters observed by scanning electron microscopy and light microscopy, and increased tensile strength and toughness. By 180 days, wound strength and collagen fiber morphology were close to that observed in the normal skin. These observations show a direct relationship between collagen fiber diameter and tensile strength. In addition, packing density of collagen fibrils (determined by the birefringence retardation per unit thickness under polarized light) was unchanged until day 90, although collagen fiber diameters increased during this time.

Cutaneous tissue repair: practical implications of current knowledge. II.

Abstract: This article reviews the scientific basis for the certain factors that delay wound repair in the clinical setting. A brief history of wound healing is given, followed by a discussion of endogenous local factors (bacterial infection, hypoxia, foreign body, and desiccation) and endogenous systemic factors (nutritional deficiencies, aging, coagulation disorders, and the Ehlers-Danlos syndromes) associated with poor wound repair. Also reviewed are the mechanisms by which exogenously administered agents (glucocorticoids, antineoplastic agents, and anticoagulants) may delay healing. Commonly used topical antimicrobials, their spectrum of activity, and evidence of effects on wound healing are examined. Finally, properties of commercially available wound coverings and wound care in the future are discussed.

Cutaneous wound healing: A model for cell-matrix interactions

Abstract: Molecules of the extracellular matrix may influence the biologic behavior of basal cell keratinocytes. In the unwounded situation, basal cells are in contact with the basement membrane of the dermo-epidermal junction and eventually detach from this structure, move vertically, and differentiate. During wound healing these cells are in contact with an entirely different set of matrix molecules (including elastin, fibronectin, and interstitial collagen). The cells migrate laterally to cover the wound and remain in a proliferative, undifferentiated state. When 25-day-old epidermal cultures were compared, adult human keratinocyte migration across an interstitial collagen matrix was found to be approximately double that across a basement membrane matrix. This finding suggests that adult human keratinocyte migration is influenced by the matrix in contact with the cells. This type of cell-matrix interaction may be important to the understanding of cutaneous wound healing.

Characterization of the adhesion of the human monocytic cell line U937 to cultured endothelial cells.

Abstract: Adhesion of blood-borne monocytes to the vascular endothelium is the first step in the infiltration of this leukocyte into the vessel wall or the interstitial space during inflammation. A significant role for the monocyte in both wound healing and atherogenesis is now well accepted. The molecular interactions involved in monocyte attachment to the endothelium are unknown. To study this phenomenon we have developed an in vitro system that uses the human monocytic tumor cell line U937 as a model for the blood-borne monocyte. 51Cr-labeled U937 cells were found to adhere with high affinity to cultured endothelial cells (ECs) from several sources. Much less binding was observed to either smooth muscle cells or fibroblasts from several species. Conditioned medium and cocultivation experiments ruled out the possibility that target cells could affect U937 cell binding by secretion of factors. Binding of U937 cells to porcine aortic ECs reached equilibrium after 30 min at 37 degrees C and 90 min at 4 degrees C with similar extent of binding at the two temperatures. Binding of U937 to the endothelium reached saturation at 9-12 U937 per porcine aortic EC (semi-confluent) with half-maximal binding at 1.5 X 10(6) U937 cells/ml. Bound cells dissociated with a half-life of 20 h at 37 degrees C. Adhesion of U937 cells was blocked by prior incubation of ECs with normal monocytes but not with platelets, lymphocytes, or neutrophils. Trypsin treatment or detergent solubilization of ECs inhibited U937 cell binding. A striking effect of EC density on monocytic cell adhesion was observed with bovine, rat, and porcine ECs. Confluent cultures of these cells exhibited negligible binding of U937, but when plated sparsely, the same cells were excellent targets for U937 cell adhesion. In addition, when confluent cultures of bovine aortic ECs were "wounded" with a cotton swab and then allowed to recover for 24 h at 37 degrees C, U937 cells were found to adhere most readily to the ECs migrating into the wound and neighboring the wound but not to ECs in the confluent monolayer away from the wound edge. These latter results may have implications for the focal adhesion of monocytes to the vessel wall in vivo.

Mesothelial healing: morphological and kinetic investigations.

Abstract: By using structural, kinetic and irradiation techniques it is possible to show that mesothelial healing is a local event. Initially, macrophages occupy the surface of a wound on the injured visceral layer, while mesothelial proliferation proceeds at the edge of the wound and the opposing parietal surface. Fibrin is formed on the wound surface within 24 h, even in the absence of much haemorrhage. Mesothelial ingrowth begins with isolated cells migrating from the wound edge as well as from the serosal surface apposing the wound where mesothelial cells are actively replicating. The cells presumably slide over a bridge of fibrin and macrophages, a process likely to be enhanced by the serosal fluid. Early colonization by macrophages results in the removal of debris and probably prevents the formation of adhesions during mesothelial restoration.

Cellular migration and morphology in corneal endothelial wound repair.

Abstract: After a mechanical denudation of rabbit corneal endothelial cells, the healing process was followed with wide-field specular microscopy. Individual cell migration and morphologic changes were analyzed by computer-assisted morphometry. The cells surrounding the wound migrated to cover the defect without producing intercellular gaps. The greatest cellular migration and morphologic alterations occurred close to the wound edge. As the cells migrated toward the wound, they elongated and increased their surface area in the direction of the migration. As the healing proceeded, the cells lost their original hexagonal pattern, which returned after coverage was complete. The wound was covered completely by large, irregularly shaped cells showing mitotic figures between 24 and 48 hr. During this period, cellular migration decreased and normal cellular morphology began to recover. When mitosis decreased, the normal cellular pattern rearranged towards a more hexagonal shape. During the healing process, the degree and direction of cellular migration varied from cell to cell. Additionally, changes in cell-to-cell contact (positional changes of neighboring cells) occurred in one-third of migrating cells. Such cellular migration can account for monolayered cells sliding without producing gaps between individual cells.

Modulation of the sis gene transcript during endothelial cell differentiation in vitro.

Abstract: Endothelial cells, which line the interior walls of blood vessels, proliferate at the site of blood vessel injury. Knowledge of the factors that control the proliferation of these cells would help elucidate the role of endothelial cells in wound healing, tumor growth, and arteriosclerosis. In vitro, endothelial cells organize into viable, three-dimensional tubular structures in environments that limit cell proliferation. The process of endothelial cell organization was found to result in decreased levels of the sis messenger RNA transcript and increased levels of the messenger RNA transcript for fibronectin. This situation was reversed on transition from the organized structure to a proliferative monolayer. These results suggest a reciprocity for two biological response modifiers involved in the regulation of endothelial cell proliferation and differentiation in vitro.

Dermal wound repair: role of collagen matrix implants and synthetic polymer dressings.

Abstract: The effects of two different polymeric wound dressings and a new collagen matrix (CM) implant on the healing and scarring of full-thickness excision wounds were studied in swine. The synthetic polymers comprised an occlusive O2-impermeable hydrocolloid dressing (HCD) and an occlusive O2-permeable polyurethane film (PUF). The CM implant consisted of an acellular collagen sponge fabricated from purified bovine tendon type I collagen. Wounds were evaluated for granulation tissue--production capacity by measuring 14C proline incorporation into collagenase-sensitive protein. Epidermal resurfacing and wound contraction were measured by computerized morphometric image analysis of wounds made on a tattooed grid. In comparison with air-exposed wounds, the relative collagen synthetic capacity was greater in the granulation tissue of wounds treated with HCD, PUF, or CM with occlusion. Both HCD and PUF accelerated by 40% the epidermal resurfacing over the granulating wound bed. Wound contraction was significantly reduced by CM but was not altered by the occlusive dressings.

Purification of human platelet-derived growth factor.

Abstract: Publisher Summary This chapter describes a method for the purification of human platelet-derived growth factor (PDGF) from outdated plateletrich plasma (PRP), using commonly available laboratory reagents and yielding a mitogen purified 800,000-fold over the starting material. The chapter discusses the methods of analysis and properties of highly purified PDGF. PDGF has a 30,000 molecular weight glycoprotein, which is released from the platelet during coagulation and has been shown to be one of the principal macromolecules in whole blood serum capable of stimulating deoxyribonucleic acid (DNA) synthesis and cell growth in connective tissue cells in vitro. PDGF plays a physiologic role in wound healing and tissue repair and a pathologic role in the formation of lesions of atherosclerosis. Studies with highly purified PDGF help determine the role of PDGF and other platelet mitogens in these processes and in defining the early cellular changes that occur in response to stimulation by PDGF. However, it is now known that PDGF like molecules are also synthesized by cultured vascular endothelial cells, newborn rat aortic smooth muscle cells, and cells transformed by a wide variety of transforming agents.

Fibrin degradation and angiogenesis: Quantitative analysis of the angiogenic response in the chick chorioallantoic membrane

Abstract: Fibrin deposition and removal is a feature common to major pathological processes such as wound healing, chronic inflammation and tumour invasion: processes involving the ingrowth of new blood vessels. Low molecular weight fibrin degradation products (MW less than 50,000) are now shown to induce angiogenesis in the chick chorioallantoic membrane (CAM). This effect has also been shown by new quantitative assays to be associated with stimulation of both DNA and protein synthesis. Autoradiography indicates that all cell types in the CAM are stimulated to divide, and it is proposed that fibrin degradation products are a pathological growth factor.

Correlation of corneal endothelial pump site density, barrier function, and morphology in wound repair.

Abstract: After transcorneal freezing, physiologic function (pump and barrier) of the regenerating rabbit corneal endothelium was evaluated and compared with the morphologic differentiation that occurs during wound healing. Endothelial pump function was investigated utilizing the specific binding of tritiated ouabain to endothelial Na+/K+ ATPase (pump sites); the permeabilities of isolated de-epithelialized corneas to labeled inulin and dextran were measured to determine endothelial barrier function. Endothelial recovery after transcorneal freezing can be described as a three-stage process. Stage one is characterized by the establishment of an initial coverage of the wound by pleomorphic spindle-shaped cells which form a functional but incomplete barrier and minimal pump site density. In stage two, the cells assume a flattened configuration consisting of irregular polygons and establish nearly normal pump capacity. In stage three, a significant remodeling of the monolayer continues despite the layer's early physiologic recovery.

Biosynthetic human EGF accelerates healing of Neodecadron-treated primate corneas.

Abstract: Topical administration of biosynthetic human epidermal growth factor (h-EGF), given in combination with an antibiotic and synthetic steroid (Neodecadron) accelerated the rate of corneal epithelial regeneration and significantly increased the strength of full-thickness stromal incisions in primates The regenerated epithelial cells of EGF-Neodecadron-treated corneas appeared normal on histologic examination and showed no evidence of hypertrophy or hyperplasia The EGF-Neodecadron-treated stromal incisions were characterized by new collagen formation and a smaller epithelial cell plug than Neodecadron-treated control corneas These results suggest that biosynthetic h-EGF, which lacks the immunologic potential of nonhuman proteins, may be effective in accelerating healing of corneal epithelial defects and stromal incisions in patients whose healing is suppressed by treatment with steroids

Skeletal muscle injury--molecular changes in the collagen during healing.

Abstract: Changes in the collagen types and cross-linking of granulation and scar tissue in the injured site of partially ruptured gastrocnemius muscle were studied after a reproducible contusion injury to the left calf of a rat. In normal i.m. collagen the proportion of Type I collagen was considerably higher than Type III. Following injury there was a rapid increase in the proportion of Type III collagen reaching a maximum at 5 days after injury. After a further 2 days the proportion of Type I had increased significantly resulting in a decrease of the Type III/I ratio to below that of the control. However, as healing progressed there was a gradual shift back to the Type III/I ratio for normal i.m. collagen.

The effect of citric acid and fibronectin application on healing following surgical treatment of naturally occurring periodontal disease in beagle dogs

Abstract: It has recently been suggested that following the exposure of root surface collagen with citric acid, the addition of topically applied fibronectin might promote healing with a fibrous re-attachment. The purpose of this study was to determine the benefit of citric acid demineralization and fibronectin application in the surgical treatment of severe, naturally occurring periodontal disease in Beagle dogs. The 4 treatment modalities employed were: (1) surgery alone (mucoperiosteal flaps); (2) surgery plus fibronectin; (3) surgery plus citric acid; (4) surgery plus citric acid followed by fibronectin application. Coronal and root surface notches were used as biometric and histometric reference points. Final clinical measurements were recorded 6 weeks post surgically, on the day of sacrifice. Significantly increased amounts of connective tissue reattachment were observed in the areas treated with the citric acid/fibronectin combination. Fibrous re-attachment was enhanced at the expense of epithelial downgrowth and occurred directly to both new and old cementum and exposed dentin, often in a functional manner, i.e., perpendicular to the root surface. Areas treated with the surgery and citric acid technique attained moderate amounts of fibrous re-attachment while the other treatment modalities were associated with a long junctional epithelium. The enhanced fibrous re-attachment may be the product of an accelerated coalescing of exposed soft tissue and root surface collagen fibrils, while under the mediating effect of fibronectin.

Compositions and method for improving wound healing

Abstract: The healing of wounds is promoted by contacting the wound surfaces with a suspension of collagen and a glycosaminoglycan that is chemotactic for fibroblasts and/or endothelial cells. Typical glycosaminoglycans that exhibit the desired chemotaxis are heparin, heparan sulfate, and alginate. Two or more glycosaminoglycans can be present in the suspensions. Collagen is present in the suspension in the order of 7-10 mg/ml; while the glycosaminoglycan is present in such lower concentrations, e.g. 250-350 μ/ml. Application of the collagen/glycosaminoglycan suspension to open wounds greatly increases the rate of healing.

Locally acting growth factors for vascular smooth muscle cells: endogenous synthesis and release from platelets.

Abstract: Release of platelet-derived growth factor (PDGF) from platelets has been postulated to stimulate at least some of the cell proliferation seen at sites of tissue damage, both beneficially (wound healing) and perniciously (during formation of atherosclerotic lesions) Two other growth factors have been localized to the platelet: epidermal growth factor and transforming growth factor These factors may function synergistically with PDGF in promoting smooth muscle cell proliferation in the injured vessel wall PDGF-like molecules (PDGF-c) that bind to the PDGF receptor and are at least partially recognized by antiserum against PDGF may also be synthesized by vessel wall cells themselves under certain circumstances Arterial endothelial cells secrete several mitogens, one of which is a PDGF-c Release is greatly stimulated by exposure of the cells to physiologic concentrations of thrombin Also, aortic smooth muscle cells from 2-week-old rats secrete mitogenic levels of PDGF-c In this case, PDGF-c accounts for all the mitogenic activity in conditioned medium (when assayed on 3T3 cells) Smooth muscle cells obtained from adult rat aortae secrete 150-fold less PDGF-c In a third example, when adult rat carotid arteries are damaged with a balloon catheter, smooth muscle cells migrate into the intima of the artery and proliferate By 2 weeks, the number of smooth muscle cells in the artery has doubled When these intimal smooth muscle cells are cultured, they are found to secrete PDGF-c These findings suggest that activation of endogenous synthesis of PDGF-c may contribute to the smooth muscle cell proliferation seen in response to vascular injury

Effect of wound location and the use of topical collagen gel on exuberant granulation tissue formation and wound healing in the horse and pony.

Abstract: Preformed collagen gel was topically applied to cutaneous wounds of the equine dorsal fetlock (thoracic limb) and metatarsal regions to evaluate the effect on exuberant granulation tissue production and wound healing. In 6 horses and 3 ponies (less than 140 cm high at the withers and less than 365 kg), 36 standardized cutaneous limb wounds were surgically induced (4 wounds/animal); 18 wounds were treated topically with collagen gel, and 18 wounds were not treated (controls). Collagen gel was initially applied to the wound at 0, 2, or 7 days after wound formation (groups 1, 2, and 3, respectively). Four measurements were regularly made: amount of wound contraction and the size of the granulation bed, epithelial covering, and total wound. Sequential skin and wound biopsies were evaluated histologically to assess wound healing. Using a computer, data were analyzed for differences in the 4 measurements between treated and control wounds, between fetlock wounds and metatarsal wounds, and among groups 1, 2, and 3. Analyses were performed on days 15 and 45 of wound healing and on the final day of healing. A significant difference (P greater than 0.05) in the production of exuberant granulation tissue, rate of epithelialization, or degree of wound contraction was not detected between the collagen-treated and control wounds. Total healing time and final scar size were similar. Wound healing patterns were significantly different (P less than 0.05) in the fetlock wounds and metatarsal wounds. All wounds enlarged up to day 15 with fetlock wounds enlarging significantly more than did the metatarsal wounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Type IV collagen synthesis by cultured human microvascular endothelial cells and its deposition into the subendothelial basement membrane.

Abstract: Cultured microvascular endothelial cells isolated from human dermis were examined for the synthesis of basement membrane specific (type IV) collagen and its deposition in subendothelial matrix. Biosynthetically radiolabeled proteins secreted into the culture medium were analyzed by sodium dodecyl sulfate gel electrophoresis after reduction, revealing a single collagenous component with an approximate Mr of 180 000 that could be resolved into two closely migrating polypeptide chains. Prior to reduction, the 180 000 bands migrated as a high molecular weight complex, indicating the presence of intermolecular disulfide bonding. The 180 000 material was identified as type IV procollagen on the basis of its selective degradation by purified bacterial collagenase, moderate sensitivity to pepsin digestion, immunoprecipitation with antibodies to human type IV collagen, and comigration with type IV procollagen purified from human and murine sources. In the basement membrane like matrix elaborated by the microvascular endothelial cells at their basal surface, type IV procollagen was the predominant constituent. This matrix-associated type IV procollagen was present as a highly cross-linked and insoluble complex that was solubilized only after denaturation and reduction of disulfide bonds. In addition, there was evidence of nonreducible dimers and higher molecular weight aggregates of type IV procollagen. These findings support the suggestion that the presence of intermolecular disulfide bonds and other covalent interactions stabilizes the incorporation of the type IV procollagen into the basement membrane matrix. Cultured microvascular endothelial cells therefore appear to deposit a basal lamina-like structure that is biochemically similar to that formed in vivo, providing a unique model system that should be useful for understanding microvascular basement membrane metabolism, especially as it relates to wound healing, tissue remodeling, and disease processes.

Endothelial injury and healing in vitro. Studies using an organ culture system.

Abstract: The authors have developed an organ culture system in which segments of rabbit aorta were used for the study of the healing of superficial, intimal injury analogous to that produced in vivo by a nylon monofilament catheter. Aortic segments containing an intact endothelial monolayer were obtained for culture by careful avoidance of the damaging consequences of loss of vascular pressure, vasocontraction, and thrombosis. The segment could be maintained in culture for several weeks. Repair of superficial injury was studied after placing a series of precisely located injuries, 100 mu wide, on each aortic segment over a period of several days. The pattern of healing observed was similar to that observed in vivo. The wounded area was recovered by migrating cells within 24 hours, and during succeeding days these cells underwent a predictable pattern of remodeling which restored the normal morphologic features of the endothelium. The cells within the wound underwent a burst of cell division between 24 and 96 hours after injury and subsequently became quiescent. This organ culture approach potentially allows the investigation of many consequences of endothelial injury in a system which provides far greater control and manipulation of the cellular environment than is possible in vivo.