Accumulation of T lymphocytes and expression of interleukin-2 receptors in nonrheumatic stenotic aortic valves.

OBJECTIVES: Cell-specific antibodies were used to identify immunocompetent cells in a comparison of valves from patients who had symptomatic tricuspid aortic stenosis with subjects who had no evidence of valvular heart disease. BACKGROUND: Nonrheumatic valvular aortic stenosis is the most common valvular heart disease among adults. The biologic processes involved in the development of this disease are poorly understood. METHODS: Tricuspid stenotic aortic valves were obtained from 19 patients undergoing surgery for nonrheumatic valvular aortic stenosis, and 10 control valves were collected at autopsy. The valves were fixed in formaldehyde, cryosectioned and stained with antibodies against fibroblasts, endothelial cells, macrophages, T lymphocytes and interleukin-2 receptors. A subset of valves were also analyzed with antibodies against T-helper cells and cytotoxic T cells. RESULTS: Stenotic valves were characterized by a basal accumulation of calcium deposits and a cell-rich subendothelial thickening. The immunohistologic analysis indicated that the cells in the subendothelial connective tissue were fibroblasts. T lymphocytes appeared to be the most common cell type in the vicinity of the calcium deposits and were also found close to the endothelial lining of the valves. T-helper cells were more frequent than cytotoxic T cells. Expression of interleukin-2 receptors occurred at the same location as T lymphocytes. Control valves lacked subendothelial thickening and contained only few cells reacting with antibodies against lymphocytes and macrophages. CONCLUSIONS: The presence of activated T lymphocytes in tricuspid stenotic valves suggests that immunologic mechanisms may be involved in the etiology of nonrheumatic aortic stenosis.

Combinatorial expression patterns of the connexins 26, 32, and 43 during development, homeostasis, and regeneration of rat teeth.

Gap junctions permit the exchange of regulatory molecules between cells and play important roles during organogenesis. The expression pattern of the gap junction proteins connexin 26, 32, and 43 was studied by immunohistochemistry in the developing, adult, and injured rat teeth. Connexins 32 and 43, but not the connexin 26, were detected during the late stages of embryonic tooth development (bell stage). Expression of connexin 32 was predominant in epithelial cells, whereas connexin 43 was more widely distributed and found in both epithelial and mesenchymal cells. During cytodifferentiation (early postnatal stages), both connexin 32 and 43 were expressed in the epithelial-derived ameloblasts, synthesizing and secreting the enamel matrix proteins. In mesenchyme, connexin 32 was observed only in differentiating odontoblasts, while connexin 43 was expressed in both differentiating and functional odontoblasts, which secrete the dentin matrix. In adult rat teeth, connexin 26 and 43 were expressed in the odontoblastic layer at low and high levels, respectively, while connexin 32 was absent from odontoblasts. Electron microscopy showed that connexin 43 was distributed exclusively at sites of contacts between odontoblasts. However, double immunostaining combined with confocal microscopy suggested an occasional overlap between odontoblasts and calcitonin gene-related peptide-positive nerve fibers. Denervation experiments showed that the expression of connexins in dental pulp was independent of innervation, whereas in injured teeth connexin 43 was upregulated in pulpal fibroblasts. Finally, cultured dental epithelial cells expressed both connexin 32 and 43, and connexin 43 was detected in cultured pulp fibroblasts in vitro, thus mimicking the in vivo distribution pattern of connexins. These results demonstrate that connexins are involved in tooth development and suggest that a given connexin may have distinct roles during odontogenesis and tooth homeostasis.

Conditioned medium from cultured human keratinocytes has growth stimulatory properties on different human cell types.

Evidence for growth-stimulatory properties of keratinocyte-conditioned medium (KCM) on human fibroblasts, endothelial cells, keratinocytes, smooth muscle cells, and a mouse fibroblast cell line (3T3 cells) is presented. On human fibroblasts KCM caused an increase of over 400% in DNA synthesis as revealed by 3H-thymidine incorporation and autoradiography. The proliferative effect was comparable to that of platelet-derived growth factor (PDGF), but was not inhibited by PDGF antibodies and exceeded that of transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and basic fibroblast growth factor (bFGF). Furthermore, KCM was found to stimulate smooth muscle cells, keratinocytes, and endothelial cells more potently than PDGF, EGF/TGF-alpha, and bFGF, respectively. KCM was also potent in stimulating thymidine incorporation in 3T3 cells, whereas EGF showed a twenty-fold weaker stimulatory effect. Because keratinocytes have been shown to secrete TGF-alpha, which binds to the EGF receptor, binding of factors in KCM to the EGF receptor was assayed. The displacement of radiolabeled EGF by KCM corresponded to a low concentration of EGF (0.5 ng/ml), implying that the growth-stimulatory effect of KCM was not mediated via activation of EGF receptors. Taken together, these results suggest the presence of hitherto unidentified growth-stimulatory factor(s), expressed and secreted by cultured human keratinocytes.

Collagenase expression is rapidly induced in wound-edge keratinocytes after acute injury in human skin, persists during healing, and stops at re-epithelialization.

Collagenolytic activity has been reported previously in association with wounds. We used in situ hybridization and immunohistochemistry to localize cellular sites of interstitial collagenase production in acute wounds in human skin at days 1, 2, 4, 6, 9, and 14 after wounding. In vivo, collagenase expression peaked in migrating basal keratinocytes at the wound edge at day 1, then gradually decreased and was undetectable at day 9 when healing was complete. To minimize the effects of crust formation and inflammation, we examined the healing of wounds made with a 3-mm punch in organ-cultured skin. In these in vitro wounds, re-epithelialization occurred by 5-7 d in 10% serum, although remodeling of the connective tissue was minimal. Collagenase expression showed a similar pattern as in the in vivo wounds; it was detected in migrating keratinocytes already 4-6 h after wounding, peaked at 12-24 h, gradually decreased during the next few days, and subsided upon re-epithelialization. In dermal fibroblasts, on the other hand, expression of collagenase started considerably later, after 5-7 d in culture, and persisted after complete re-epithelialization, indicating that collagenase is differentially regulated in different cell types. Our findings also show that collagenase induction in keratinocytes does not require inflammation and occurs as a rapid response to wounding, suggesting that interstitial collagenase is not only necessary for remodeling of the extracellular matrix, but may also have a role in initiating migration of keratinocytes in wound healing.

Long-term alleviation of allodynia-like behaviors by intrathecal implantation of bovine chromaffin cells in rats with spinal cord injury.

Adrenal chromaffin cells produce analgesic substances, such as catecholamines and enkephalins, and intrathecal (i.t.) implantation of either allografted adrenal tissue or xenogenic chromaffin cells produce antinociception in animals. We evaluated the analgesic effect of bovine chromaffin cells in a model of central pain in which rats exhibit chronic allodynia-like behavior after photochemically induced ischemic spinal cord injury. Bovine chromaffin cells or endothelial cells were injected i.t. onto the lumbar spinal cord and their effects on mechanical and cold allodynia-like behaviors were studied for up to 8 weeks. The chronic allodynia-like behavior was stable for months without signs of remission and i.t. implantation of human endothelial cells did not alleviate the chronic allodynia-like behavior for the entire observation period. In contrast, 2 weeks after i.t. implantation of bovine chromaffin cells, the mechanical allodynia was abolished in the spinally injured rats, and the enhanced response to cold stimuli was significantly reduced. The overall effects were significant up to 8 weeks after i.t. implantation, although the anti-allodynic effect decreased towards the end of the observation period. No signs of side-effects were noted after i.t. implantation. The allodynia-like state was temporarily restored by naloxone (0.5 mg/kg) or phentolamine (0.3 mg/kg) injected intraperitoneally. Immunohistochemical examination revealed that tyrosine hydroxylase (TH)-positive chromaffin cells could be identified adjacent to the spinal cord up to 4 weeks after i.t. implantation, whereas at 8 weeks the TH-positive cells were sparse. It is concluded that bovine chromaffin cells stay viable in rat spinal cord for a considerable period of time after i.t. administration and alleviate chronic allodynia-like behavior in spinally injured rats, possibly through activation of opioid and alpha-adrenoceptors. The present results further document a new therapeutic approach for the treatment of chronic neuropathic pain.

Biologically modified LDL increases the adhesive properties of endothelial cells.

Adhesion of monocytes to the arterial endothelium is an important early event in atherosclerosis. Several lines of evidence have suggested that oxidation of low density lipoprotein (LDL) in the arterial wall may initiate the inflammatory-like process that generally is present in atherosclerotic lesions. In vitro, oxidation of LDL can be obtained both by exposure to divalent ions, such as Cu2+, or by incubation with different cell types, including monocytes and endothelial cells. The present study was designed to investigate the possible influence of oxidized LDL on the adhesive properties of endothelial cells. We report here that Cu(2+)-oxidized LDL is as effective as interleukin 1 beta in stimulating the ability of cultured human endothelial cells to bind U937 monocytic cells. The stimulation was inhibited by cycloheximide, indicating that de novo protein synthesis is required. Biologically modified LDL, obtained by incubation with human peripheral blood monocytes, also enhanced the adhesiveness of endothelial cells. This effect was not due to an increased secretion of interleukin 1 beta from the monocytes exposed to LDL. Treatment of endothelial cells for 24 h with native LDL was also found to increase the adhesion of U937 cells. Exposure of endothelial cells to LDL for 24 h resulted in an oxidative modification of LDL. Furthermore, the antioxidant butylated hydroxytoluene inhibited both the endothelial-dependent oxidation of LDL as well as the increased adhesion of U937 cells, suggesting a coupling between these two processes. The results indicate that LDL, modified by exposure to monocytes or endothelial cells in the arterial wall, may increase the adhesive properties of the endothelium.

Mononuclear leukocytes exposed to oxidized low density lipoprotein secrete a factor that stimulates endothelial cells to express adhesion molecules.

In animals fed a hypercholesterolemic diet, development of atherosclerosis is preceded by attachment of mononuclear leukocytes to the arterial endothelium. Early lesions begin to develop as monocytes migrate into the intima and ingest lipids. A major part of these lipids is believed to be derived from oxidatively modified low density lipoprotein (LDL). In the present study we demonstrate that human mononuclear leukocytes exposed to low concentrations of copper-oxidized LDL secrete one or several factors that stimulate the expression of intercellular adhesion molecule-1 (ICAM-1, CD54), vascular cell adhesion molecule (VCAM-1) and endothelial selectin (E-selectin-1, ELAM-1), whereas native LDL was found to be without effect. Exposure of endothelial cells to non-conditioned medium containing oxidized LDL did not influence the expression of adhesion molecules. Incubation of endothelial cells with conditioned medium from mononuclear cells grown in the presence of oxidized LDL also resulted in a three-fold increase in the binding of monocytoid U937 cells. The present findings suggest that mononuclear leukocytes exposed to oxidatively modified LDL in early atherosclerotic lesions may stimulate the recruitment of other leukocytes by secreting cytokines which induce the expression of adhesion molecules on the endothelium.

Spinal axons in central nervous system scar tissue are closely related to laminin-immunoreactive astrocytes.

Although transected central nervous system axons fail to regrow after injuries in adult mammals, they send sprouts into the scar tissue that forms at the lesion. We have investigated the relation between scar cells, laminin-like immunoreactivity and cut spinal axons in two previously characterized spinal cord lesion types. Labeling with antisera to glial fibrillary acidic protein and laminin demonstrated that the scar tissue formed after lesions in the rat and cat dorsal and ventral funiculi showed prominent gliosis and strong laminin-like immunoreactivity four days to one year postlesion. Axonal sprouts in the scar, visualized with antibodies to neurofilament (RT97) or by tracing using fluorescein-conjugated dextran, were ensheathed by a thin layer of strongly laminin-immunoreactive tissue. Immunoelectron microscopy demonstrated that axons in the scar were ensheathed predominantly by astrocytes, and that the surface of the cells outlining the axons in the scar showed strong laminin-like immunoreactivity. Adhesive and neurite orienting properties in the scar tissue were assessed in an in vitro system where PC12 cells were cultured on spinal cord slices from dorsal funiculus-lesioned rats. Very few cells adhered to the spinal cord section except for the part where the scar tissue had formed, where numerous cells were attached. The PC12 cells that had adhered to the scar tissue were mainly seen in parts of the scar that showed laminin-like immunoreactivity and their neurites predominantly followed tissue showing laminin-like immunoreactivity. The close association between axonal sprouts and laminin-like immunoreactivity indicates a role for laminin in axonal growth and/or guidance in the injured spinal cord.

Lining of viable and nonviable allogeneic and xenogeneic cardiovascular tissue with cultured adult human venous endothelium.

With the aim of creating a confluent endothelial lining of cultured adult human saphenous vein endothelial cells on cardiovascular bioprosthetic tissues in vitro, we performed seeding on deendothelialized segments of viable or devitalized (in deionized water) human vein, porcine aorta, and bioprosthetic tissues preserved in glutaraldehyde. After being seeded, specimens were kept for 7 days under culture conditions. On glutaraldehyde-preserved tissue, seeding was performed after 3 weeks of elution of glutaraldehyde. Evaluation was performed with hematoxylin-eosin staining, immunohistochemical staining of von Willebrand's factor and of collagen IV-related antigens, and scanning and transmission electron microscopy. The origin of the cells as derived from culture was verified by vital staining with a carbocyanine dye. Evaluation revealed a confluent lining of cultured human saphenous vein endothelial cells similar to native endothelium on both viable and nonviable human and porcine tissues. Collagen IV-related immunoreactivity was demonstrated close to the endothelial cells, corresponding to a de novo-formed basement membrane. Organelles and a basement membrane were demonstrated by transmission electron microscopy. The human saphenous vein endothelial cells seeded on glutaraldehyde-preserved tissues showed initial adherence but rounded up and detached on the second day of culture, probably because of residual glutaraldehyde. This study demonstrates that the native endothelium of allogenic or xenogeneic viable and nonviable vascular tissue may be replaced by cultured endothelium in vitro. The structural similarities with a native endothelium suggest that in vitro endothelialization with cultured autologous endothelial cells may be used to improve performance of cardiovascular bioprostheses.

Reduction of monocyte adhesion to xenogenic tissue by endothelialization: an adhesion molecule and time-dependent mechanism.

Great interest has been shown for the seeding of autologous endothelial cells on prosthetic materials. We investigated the inflammatory and immunogenic properties of xenogeneic tissue before and after seeding with cultured human great saphenous vein endothelial cells in vitro. Adhesion of monocytes to xenogeneic tissue with or without endothelium and the endothelial cell expression of E-selectin, intercellular adhesion molecule 1, vascular adhesion molecule 1, and major histocompatibility complex class II antigens were investigated 1, 3, and 7 days after seeding. Both monocyte adhesion and endothelial adhesion molecule expression were relatively high 1 day after seeding and were significantly lowered after 3 to 7 days. There was no difference between monocyte adhesion and adhesion molecule expression on viable or nonviable xenogeneic tissue. Monocyte adhesion and adhesion molecule expression increased after interleukin-1 beta or interferon-gamma stimulation of the endothelial cells. The results suggest that human endothelial cells exhibit an early proinflammatory and immunogenic activity immediately after seeding. Three and 7 days after seeding, the endothelialized surface is less adhesive for monocytes as compared with nonendothelialized tissue. These findings have implications when cultured or intraoperatively recruited endothelial cells are used clinically.

In vitro endothelialization of bioprosthetic heart valves provides a cell monolayer with proliferative capacities and resistance to pulsatile flow.

OBJECTIVES: Degeneration of bioprosthetic heart valves has been suggested to be at least partly an immunogenic reaction toward the xenogeneic tissue. An autologous endothelial lining has been proposed to overcome this problem. We examined in vitro endothelialization of such tissue and retention of endothelial cells after exposure to flow resembling the in vivo situation. METHODS: Cultured human saphenous vein endothelial cells were used to in vitro endothelialize photo-oxidized bioprosthetic heart valves. The endothelialized valves were mounted in a specially designed flow device, creating a pulsatile flow through the valve. Maintenance of a confluent cell layer and deposition of basement membrane markers were determined with immunohistochemical labeling. RESULTS: Labeling of the main components of the basement membrane, laminin and collagen type IV, was verified within 6 hours after in vitro endothelialization. Under static conditions, 4-mm wide denudations were completely re-endothelialized in 4 days, which was similar to the growth rate on gelatin-coated cell culture plastic, which served as a control material. After exposure of endothelialized valves to pulsatile flows for 24 hours (80 beats/min, 3.4 L/min), there were minimal cell losses from the bioprostheses. The cell layer adapted to the pulsatile flow, as verified by rearrangement of morphology and intracellular stress fibers. CONCLUSIONS: This study shows the feasibility of in vitro endothelialization of photo-oxidized bioprosthetic heart valves. The cells are able to withstand a pulsatile flow in vitro, to develop basement membrane-like structures, and to re-endothelialize denuded areas. This technology may be used to enhance the performance of bioprosthetic heart valve prostheses.

Rapid visualization of viable and nonviable endothelium on cardiovascular prosthetic surfaces by means of fluorescent dyes.

Increasing interest in endothelialization of synthetic and tissue cardiovascular prostheses in vitro emphasizes the need for simple and rapid methods to evaluate presence of endothelium on surfaces. Scanning electron microscopy is a commonly used method for this purpose. In this study we investigated alternative and more rapid staining methods. Human saphenous vein endothelial cells in culture and on cardiovascular prosthetic materials (pyrolytic carbon, cusps of bioprosthetic heart valves, pig aorta, and expanded polytetrafluoroethylene) were labeled by exposing them to medium containing 5-chloromethylfluorescein diacetate or 1,1-dioctadecyl-3,3,3',3'-tetramethylindo carbocyanine perchlorate. For comparison, specimens were also fixed and processed for scanning electron microscopy. A bright fluorescence of endothelial cells labeled with 5-chlormethylfluorescein diacetate or 1,1-deoctadecyl-3,3,3',3'-tetramethylindo carbocyanine perchlorate wre clearly visualized in culture, on pyrolytic carbon, and on expanded polytetrafluoroethylene. Unfixed, prelabeled cells could be visualized immediately and unlabeled cells could be investigated for viability within 1 hour. Cells seeded on biologic tissue specimens could be visualized within 15 minutes with a modified hematoxylin-eosin staining. We suggest the use of these methods for rapid visualization of endothelium present on surfaces of cardiovascular prosthetic materials where they can partly replace the use of scanning electron microscopy.

Effects of neuropeptides on growth of cultivated rat molar pulp fibroblasts.

The effect of the neuropeptides substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) on DNA synthesis of dental pulp cells was investigated in cells grown from molar tooth bud explants from 4-6 days old rat pups. A concentration response-assay of the proliferative response of pulpal cells was performed with SP, NPY, NKA, CGRP and VIP (0.01 to 1 nM) in the presence of EGF (10 ng/ml), hydrocortisone (0.4 microgram/ml) and 3% FCS, using [3H]thymidine incorporation. The results showed that SP, NKA and CGRP, but not NPY and VIP, increased the cell number in a concentration-dependent manner, with maxima at 10(-10)-10(-9) M (SP, NKA) and 10(-7) M (CGRP). No potentiating effect was noted when cells were simultaneously stimulated with SP and CGRP. The finding that SP, NKA and CGRP have growth regulatory properties on pulpal cells in vitro suggests that sensory neuropeptides may be involved during pulpal development or in wound healing after pulpal injury.

In vitro endothelialization of photooxidatively stabilized xenogeneic pericardium.

The possibility of improving the performance of heart valve bioprostheses and vascular biografts by means of preendothelialization with cultured autologous cells has been suggested. Such culture techniques are available, but the glutaraldehyde-preserved heart valve prostheses used clinically appear cytotoxic. Recently, dye-mediated photooxidation has been reported to stabilize pericardial tissue, possibly through the cross-linking of collagen fibrils. We have seeded cultured adult human saphenous vein endothelial cells (HSVECs) onto photooxidatively stabilized tissue and investigated the morphologic characteristics 7 days later. A confluent lining of cultured HSVECs similar to native endothelium was demonstrated by scanning electron microscopy. The presence of von Willebrand's factor, an integrin located at the interendothelial cell contacts (PECAM/CD 31), and the basement membrane component collagen type IV was demonstrated using monoclonal antibodies. The results were similar for the HSVECs seeded onto both bovine and porcine pericardial tissues. The results clearly indicate that the dye-mediated photooxidation technique produces a tissue that is cell compatible. Provided the HSVECs remain attached and retain antithrombotic and antiinflammatory properties, this appears to be a feasible way of endothelializing bioprosthetic heart valves before implantation.

Endothelialization of mechanical heart valves in vitro with cultured adult human cells.

Previous studies have shown that in vitro endothelialization of synthetic vascular prostheses with cultured autologous cells leads to a reduction in their thrombogenicity. It is possible that pre-endothelialization of mechanical heart valve prostheses would have a similarly advantageous effect. In this study we have therefore investigated the possibility of creating an endothelium layer on mechanical heart valve prostheses by seeding in vitro with cultured adult human endothelial cells. After harvest, endothelial cells were cultured for two to three weeks before seeding of six valves (three Monostrut, three CarboMedics bileaflet) with 1.5-2 x 10(5) cells/cm2. The valves were then kept under culture conditions for seven days. A confluent lining of cultured endothelial cells was observed in the scanning electron microscope on the parts covered with pyrolitic carbon, e.g. disc, leaflet, flange, and on carbon coated sewing rings. Untreated sewing rings showed a discontinuous endothelial lining. On the metallic surface only a few scattered cells were observed. Two endothelialized bileaflet valves were implanted in the mitral position in pig and permitted to perform physiologic work for one hour. A mainly continuous endothelium remained on the carbon covered sewing rings but the endothelium detached from the leaflets and flanges. This study demonstrates that mechanical heart valves do not contain cytotoxic compounds. It also demonstrates that cultured adult human endothelial cells are able to form a monolayer when seeded on monostrut and bileaflet valve prostheses. A significant number of cells remain on the sewing ring after pulsatile flow in the mitral position but not on the polished pyrolitic carbon surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro endothelialization of commercially available heart valve bioprostheses with cultured adult human cells.

The possibility of improving the performance of heart valve bioprostheses by preendothelialization with autologous cells has been suggested. In this study we used cultured adult human vein endothelial cells to endothelialize cusps isolated from commercially available porcine valve bioprostheses. We also describe a method for primary endothelialization of the intact heart valve bioprosthesis. After detoxification of the glutaraldehyde, the cusps or valves were seeded at high density with cultured cells. To obtain an even distribution on the intact valve bioprosthesis, a device was designed which permits application of cells from different directions during rotation. Evaluation was performed by hematoxylin-eosin staining, scanning electron microscopy and immunohistochemistry of the von Willebrand factor and the human basement membrane constituent collagen IV. The endothelial cells were vital-stained during culture by the addition to the culture medium of carbocyanine dye. This made it possible to verify that the endothelium was derived from culture. A confluent lining of cultured endothelial cells in close proximity to a de novo formed basement membrane was observed on the isolated cusps 7 days after seeding. The intact heart valve bioprosthesis showed an even distribution of seeded cells with areas of cells spreading to confluency as evaluated after 24 h. However, 7 days after seeding only unspread and probably dead cells were observed.

Characterization of a new in vitro model for studies of reepithelialization in human partial thickness wounds.

Reepithelialization of artificial partial thickness wounds made in biopsies of human skin was determined after 3, 5, or 7 d of incubation, submerged or elevated to the air-liquid interface. The biopsies were reepithelialized within 5-7 d, with a more complete epidermal healing in wounds exposed to air. Both types of wounds showed similar time-course in deposition of basement membrane components, as detected by immunofluorescence labeling. Laminin and collagen type VII were deposited underneath the migrating tips, whereas collagen type IV was detected after reepithelialization. Markers of terminal differentiation showed a pattern close to normal in the air-liquid incubated wounds after reepithelialization. Involucrin was detected in the suprabasal regions of the migrating epidermis and thereafter in the upper half of neo-epidermis in the air-liquid incubated wound. Filaggrin could not be detected in the submerged wounds at any time during healing, whereas wounds exposed to air showed a well-differentiated epidermis by Day 7. Tritiated thymidine-incorporation indicated proliferation of epidermal and dermal cells during reepithelialization and a maintained viability, as shown by cultivation of endothelial- and fibroblast-like cells obtained from the dermis 7 d after wounding. Reepithelialization in this human in vitro model is supported by a matrix close to normal with the possibility of extracellular influences and cell-cell interactions and, in addition, the technique is simple and reproducible. Therefore, we suggest this model for studies of regeneration in culture and as a complement to in vivo studies on epidermal healing.

Cell adhesion and short-term patency in human endothelium preseeded 1.5-mm polytetrafluoroethylene vascular grafts: an experimental study.

It has been shown that endothelialization improves short-term patency of 1.5-mm expanded polytetrafluoroethylene vascular grafts. A model for endothelialization of 1.5-mm expanded polytetrafluoroethylene vascular grafts with human endothelial cells is described. In this model, the adherence of endothelial cells was increased significantly in grafts coated with serum proteins and collagen. By means of this protocol, graft patency was tested after implantation in two animal models: the rat aorta and the rabbit common carotid artery. Anastomosis was performed with a 3M Precise Microvascular Anastomotic System. In both animal models, no significant loss of endothelial cells in the precoated grafts (rat, n = 8) were noted 1 hour after blood flow restoration. All uncoated grafts showed significant endothelial cell loss. In the rabbit model, all nonendothelialized grafts (n = 8) clotted 5 to 25 minutes after flow restoration. Seven (n = 8) endothelialized grafts showed no clotting during 1 hour's observation: one clotted immediately for a patency rate of 87.5 percent. These results indicate that endothelialization of 1.5-mm grafts is practical. Furthermore, adhesion of endothelial cells to the graft walls is not affected by short-term, pulsatile, high-pressure blood flow.

A biodegradable bovine collagen membrane as a dermal template for human in vivo wound healing.

A bovine collagen membrane was used as a template for dermal regeneration in human full thickness wounds. Healing was allowed for 7, 21, or 42 days. The formation of neodermis, basement membrane, and terminal differentiation were assessed histologically and immunohistochemically. The collagen template was neovascularised within 7 days, and from day 21 small vessels were detected throughout the transplanted area. The procollagen content decreased whereas the number of fibroblasts increased with time. Collagen type IV was not detected after 7 days but was deposited with time from the wound edges and inwards over the transplanted area. Re-epithelialisation was complete at day 7 and terminal differentiation was similar to normal human skin from day 21. We have shown the time course of dermal and epidermal healing with the aid of a ready-to-use biodegradable collagen membrane. This material may be used as a true dermal template-because of the evidence of dermal regeneration and, in addition, its availability and ease of handling.

Direct mounting of cultured epidermal grafts. A way to enhance the transplantation area.

Cultured epidermal grafts were prepared in a new way, not allowing the epidermal sheets to shrink before mounting. Using this procedure, the graft area was increased by about 100%. Furthermore, this procedure is also quicker than the conventional technique. The directly mounted grafts take just as well as conventionally prepared grafts and their macroscopical and histological appearances are similar.