On the role of type IX collagen in the extracellular matrix of cartilage: type IX collagen is localized to intersections of collagen fibrils.

The tissue distribution of type II and type IX collagen in 17-d-old chicken embryo was studied by immunofluorescence using polyclonal antibodies against type II collagen and a peptic fragment of type IX collagen (HMW), respectively. Both proteins were found only in cartilage where they were co-distributed. They occurred uniformly throughout the extracellular matrix, i.e., without distinction between pericellular, territorial, and interterritorial matrices. Tissues that undergo endochondral bone formation contained type IX collagen, whereas periosteal and membranous bones were negative. The thin collagenous fibrils in cartilage consisted of type II collagen as determined by immunoelectron microscopy. Type IX collagen was associated with the fibrils but essentially was restricted to intersections of the fibrils. These observations suggested that type IX collagen contributes to the stabilization of the network of thin fibers of the extracellular matrix of cartilage by interactions of its triple helical domains with several fibrils at or close to their intersections.

Neoplastic modulation of extracellular matrix: proteoglycan changes in the rabbit mesentery induced by V2 carcinoma cells.

Previous studies have shown that the invasion of V2 carcinoma cells in the rabbit mesentery is associated with marked extracellular matrix synthesis leading eventually to an overall increase in mesenteric mass. The purpose of the present study was to investigate the structural and biochemical composition of the extracellular matrix in tumor-free parts of rabbit mesenteries at various stages after intraperitoneal implantation of V2 carcinoma cells. The overall thickness of the tumor-implanted mesenteries increased progressively and peaked at about Day 14, when it was about 8 times greater than the untreated or liver-implanted controls. This was mainly the result of an accumulation of extracellular matrix components. In particular, there was a marked increase in both collagen fibers and proteoglycan granules, as well as filaments, probably hyaluronic acid, as visualized by ruthenium hexammine trichloride. Stereological analysis showed a 6-fold increase in collagen fibers and a significant increase in the density and average diameter of proteoglycan granules. Biochemical analysis revealed a marked elevation in uronic acid content in the tumor-implanted mesenteries. Specifically, they contained 2.6 and 8.6 times the amount of hyaluronic acid and chondroitin sulfate, respectively, than did controls. Furthermore, the relative percentage of chondroitin sulfate was elevated markedly (26 versus 6% in controls). However, the content of heparan or dermatan sulfate did not vary significantly. Stereological analysis of the fibroblasts showed that their absolute number had doubled and that the cell volume of the individual fibroblast had increased markedly. This suggests that the fibroblasts were responsible for the excessive production of the extracellular matrix. These results support the concept that carcinoma cells can modulate their surrounding extracellular environment by stimulating the synthesis of connective tissue in the host mesenchymal cells.

Superior late patency of small-diameter Dacron grafts seeded with omental microvascular cells: an experimental study.

The purpose of the study was to investigate the effect of omental microvascular cell seeding on the patency of small-diameter Dacron prostheses usable for coronary artery bypass grafting. In a canine carotid artery model, each dog (n = 64) received one seeded and one similar nonseeded Dacron prosthesis (internal diameter = 4 or 6 mm). Enzymatically harvested omental microvascular cells (omentum = 27.6 +/- 5.9 g [+/- the standard deviation]; range, 17 to 50 g) were seeded prior to implantation. The seeding density was 1.91 +/- 0.26 [+/- the standard error] x 10(6) cells/cm2 of graft surface. Dipyridamole (75 mg/d) and acetylsalicylic acid (325 mg/d) were administered orally for 4 weeks postoperatively. The prostheses were explanted between 2 and 52 weeks after placement. The results were assessed by angiography; light, scanning electron, and transmission electron microscopy; and morphometry. The seeded grafts developed a uniform luminal monolayer of endothelial cells with minimal platelet or cellular deposition. These grafts also had a significantly higher overall patency rate and significantly larger thrombus-free surface areas than the nonseeded grafts. The overall actuarial patency rates at 1 week, 5, 12, 26, and 52 weeks were 100%, 98%, 93%, 93%, and 93%, respectively, for seeded Dacron grafts and 100%, 91%, 61%, 54%, and 18%, respectively, for nonseeded grafts. The patency rates of Dacron grafts usable for coronary artery bypass grafting are significantly improved by seeding with omental microvascular cells in a canine model.

Endothelial cell seeding improves patency of synthetic vascular grafts: manual versus automatized method.

Lack of an endothelial surface is the most important variable causing the relatively poor patency of synthetic bypass grafts. This study was designed to investigate the effect of endothelial cell seeding on small-diameter Dacron grafts seeded with microvascular endothelial cells from omentum, and to evaluate two methods (manual vs automatized) for one-stage seeding in a canine carotid artery model. In 30 mongrel dogs microvascular endothelial cells were harvested from omentum, either by a manual or an automatized method, and seeded onto 6-mm internal diameter Dacron prostheses prior to the graft interposition into the common carotid arteries. Non-seeded Dacron grafts were used as control grafts. All dogs received dipyridamole (75 mg/day) and acetylsalicylic acid (325 mg/day) for 4 weeks. The prostheses were explanted between 2 and 26 weeks after insertion. The results were assessed by patency, angiography, light and scanning electron microscopy, transmission electron microscopy, and morphometry. Endothelial cell seeding improved the patency rate significantly, regardless of the seeding methods used. The overall actuarial patency rates at 5, 12, and 26 weeks were 98%, 94% and 94%, respectively, for the seeded Dacron grafts, and 92%, 62% and 54%, respectively, for the non-seeded grafts. The automatized method yielded more endothelial cells per gram of omental tissue than the manual method (P = 0.0002), but there was no difference (P = 0.34) between the seeding densities per square centimeter of the graft surface. The harvesting and seeding by the automatized method took 55 min for the whole procedure, 20 min less than the manual method. We concluded that one-stage endothelial cell seeding with omental microvascular endothelial cells improved the patency of small-diameter Dacron grafts in a canine model. The automatized method obtained excellent results comparable to the manual procedure, and also reduced the time necessary for the cell seeding.

The pocket epithelium: a light- and electronmicroscopic study.

The POCKET epithelium is important for the pathogenesis of gingivitis and periodontitis. However, this epithelial variant has never been adequately described. The bioptic material with supraalveolar pockets originated from previous studies in which cotton floss ligatures were placed around the crowns of premolars in eight dogs. After periods of 4 to 21 days or up to 5 months, block biopsies comprising dental and gingival tissues were taken on the buccal side. The tissues were processed for light- and electron microscopic examination. The observations revealed that the pocket epithelium (1) does not attach to the tooth, (2) forms irregular ridges and, over connective tissue papillae, thin coverings which occasionally ulcerate, (3) consists of cells only some of which show a tendency to differentiate, (4) presents a basal lamina complex with discontinuities and multiplications, and (5) is infiltrated mainly by lymphocytes, T- and B-blasts and plasma cells, and is transmigrated by neutrophilic granulocytes. It is concluded that the mosaic-like structure of the pocket epithelium reflects the heterogeneity of the adjacent plaque, that this structure together with the absence of membrane coating granules is the basis for an extremely high permeability, and that epithelial ridges may conduct and collect foreign substances which thereby become more easily recognizable for leukocytes.

The effect of cholera toxin and epidermal growth factor on the in-vitro growth of human oral epithelial cells.

Human epithelial cells isolated from adult gingival and infant palatal biopsies were cultured using 3T3 feeder cells. The colony-forming efficiency was about 0.8 per cent with cholera toxin and epidermal growth factor (EGF). The cell yield of cultures from infant palates depended on the concentration of cholera toxin and the presence of EGF in the culture medium; the culture lifetime and the number of cell generations were higher for oral epithelial cells originating from infants than from adults; the mean thickness of well-developed areas was 15 micron in control cultures and slightly smaller with cholera toxin and EGF. It is concluded that cultivation of epithelial cells from the human oral mucosa is easier with culture media containing cholera toxin and EGF. The same is true for cells originating from infants rather than from adults.

Absolute volume of differentiating cells in the epithelium of the human hard palate.

In previous studies the differentiation of the epithelium in the human hard palate has been described stereologically using parameters expressed per unit tissue volume. Since single epithelial cells represent the true biological units of this tissue, it became necessary to estimate the absolute size of such cells in order to transform density data into absolute data. Therefore, in the present study, a stereological method (originally developed for myocyte volume determination) was tested in terms of its applicability to stratified epithelia; the absolute size of differentiating epithelial cells was determined in the epithelium of the human hard palate. The results suggest that (1) rather precise determination of epithelial cell size is possible by using the modified myocyte volume determination, and (2) the average cell volumes are 926 +/- 148, 4,111 +/- 1,619, 4,394 +/- 551 microns3 for the stratum basale, the upper stratum spinosum and the stratum granulosum, respectively. The results are discussed with respect to methodology and to differentiation phenomena in the epithelium of the human hard palate.

Exfoliative cytology and ultrastructure of superficial epithelial cells from the normal human oral mucosa.

Superficial, mature epithelial cells were collected from clinically normal hard palate, cheek and lip of 5 volunteers. These cells were stained using 3 different exfoliative cytology methods and prepared for electron microscopy. In total, 209 individual cells were examined both by the light and electron microscopes and classified as orange, red or blue cells exhibiting an oral keratin or a filament pattern. The combination of data sets revealed that type and degree of terminal differentiation are not related to the staining characteristics. Independent of the staining methods, cells displaying an oral keratin pattern stained either orange, red or blue, and cells displaying a filament pattern stained either red or blue. It is concluded that the staining characteristics of superficial oral epithelial cells do not reflect their status of terminal differentiation as seen ultrastructurally.

Pathomorphologic features of the ulcerative stage of oral aphthous ulcerations.

Macroscopic, histopathologic, and immunohistochemical features of eight 1- to 7-day-old minor (Mikulicz) aphthae, one herpetiform ulcer, and one ulcer from a patient with Behcet's syndrome were studied. In addition to light and electron microscopy, methods included the peroxidase-antiperoxidase (PAP) technique to disclose binding of IgA, IgG, IgM, Clq, and C3. Observations revealed the presence and distribution of extravasates of erythrocytes at and around the ulcers, extravascular neutrophilic granulocytes undermining the oral epithelium of the ulcer margin, the presence of numerous macrophages loaded with phagolysosomes containing debris of neutrophilic granulocytes, particular pathomorphologic features of a Behcet lesion and a herpetiform lesion, and the occurrence in diseased and normal oral mucosa of particular stratum spinosum cells binding nonselectively all immune components tested in this study, probably by leakage and passive diffusion of serum proteins. The observations fit the concept of immune complex vasculitis being essential in the pathogenesis of oral aphthous ulcerations.

Ultrastructural evidence for contacts between migrating L5222 rat leukemia cells and extracellular matrix components of the rat mesentery.

The nature of interactions between cells migrating through tissues and their structural surroundings are largely unknown. We have therefore examined the ultrastructural relationship between L5222 rat leukemia cells, moving through the loose connective tissue of the mesentery, and components of the extracellular matrix (ECM). Ultrathin tissue sections, fixed in the presence of ruthenium hexammine trichloride (RHT), revealed the following: Constitutents of fibrillar and nonfibrillar elements of the ECM are in contact with the plasma membrane of L5222 cells. Linear nonfibrillar ECM elements contact the plasma membrane at point-like sites, often associated with root-like structures present within the submembraneous microfilament mesh. Aggregates of ECM material are connected to patch-like cell membrane sites, associated with a condensed, plate-like part of the microfilament mesh. Point-like and patch-like contacts are more numerous at the anterior part of polarized migrating L5222 cells than on the posterior end. In round resting leukemia cells they are evenly distributed around the cell periphery. We suggest that the ECM-cell membrane contacts represent tissue adhesion sites. We therefore hypothesize that in migrating cells a coordinate interaction occurs between the contact sites and the continuous microfilament meshwork which results in a simultaneous backward movement of ECM-membrane contacts on the cell body and in a net forward movement of the whole cell. Since Dembo et al. (1981) present a similar mechanism for in vitro locomotion of granulocytes, we assume that blood cell locomotion in vivo and in vitro depends on similar molecular mechanisms: force generation by the cell, transmembraneous linkage between cytoskeletal and ECM elements, and membrane fluidity. The major difference in blood cell locomotion through a three-dimensional tissue or on a plane substratum would then be given by the distribution of contact sites, occurring around the cell periphery or limited to the ventral cell surface, respectively.

Stereologic analysis of leukocyte infiltration in oral ulcers of developing Mikulicz aphthae.

Biopsy specimens of 1- to 7-day-old oral ulcers from patients with minor (Mikulicz), herpetiform, and Behcet's aphthae and of nonulcerated oral mucosa of aphthous patients were subjected to a quantitative, stereologic, electron microscopic analysis of the connective tissue infiltrate residing both at the center of and lateral to the ulcers. The data representing volume fractions and the numerical density of cellular and other infiltrate components demonstrated that (1) the infiltrate under the epithelium lateral to the ulcer is different from that at the ulcer's center, (2) at both sites, composition of the gross infiltrate does not change with age of the ulcer, (3) a large population of leukocytes (about 18% in the lateral and 23% in the central region) belong to the monocyte/macrophage series, (4) blast-forming T-lymphocytes are consistently present, blast-forming B-lymphocytes and plasma cells are very rare, and (5) mast cells are several times more numerous than in normal mucosa. In a comparison of the infiltrates of Mikulicz aphthae with those of herpetiform and Behcet's ulcers, it appears that the pathogenesis of the various oral ulcerations may well be diverse.

[Development of small-diameter vascular prostheses covered with human endothelial cells: Possibilities and limits of in vitro-tests]

There is an increasing demand for small-diameter vascular prostheses for the replacement of arteriosclerotic coronary arteries. They may be replaced by autologous blood vessels, usually parts of the saphenous vein. Prostheses of synthetic materials and an inner diameter of less than 4 to 6 mm are unsatisfactory and, therefore, not implanted for coronary arteries. A substantial improvement is, however, expected for prostheses covered with human autologous endothelial cells. It has to be proved that this new type of vascular prostheses is an adequate replacement for small arteries. Tests of the new prosthesis should comprise cell and tissue compatibility of the synthetic materials as well as normal function of the endothelial cells. The aim of the present paper was to reduce the number of animal experiments in this development by establishing new in vitro tests for endothelial cell compatibility of synthetic materials and for the adherence of endothelial cell on the prosthesis. Physiologically haemodynamic streaming conditions are in vitro produced by self-constructed circulatory systems. First results demonstrate that physiologic shear stress is achieved. Limits and relevance of the in vitro tests are discussed in relation to animal experiments and clinical studies.

An improved procedure for enzymatic harvesting of highly purified canine venous endothelial cells for experimental small diameter vascular prostheses.

We developed a new device, the vein holder, to improve yield and purity of enzymatic harvests of venous endothelial cells. External jugular veins of mongrel dogs were dissected by a no-touch technique. In vitro length and circumference of the vein segments were decreased to about half of the in situ dimensions. The vein holder enabled mounting of the veins at 80% of their in situ length during endothelial cell harvesting. Trypan blue staining and scanning electron microscopic observations revealed that vein eversion as well as the new vein holder technique successfully removed the endothelium. Endothelial cell harvests by the eversion technique were, however, low and varied in size, viability, and purity. In contrast, the defined handling by the new vein holder technique regularly provided markedly increased amounts of endothelial cells. Most of the cells attached and developed cultures consisting of endothelial cells only, as shown by the uptake of DilAcLDL. Prostacyclin production of confluent cultures was similar to that of native veins. It is concluded that minimal handling, defined mounting, and prevention of overfilling the vein markedly improves endothelial cell harvests, providing greater amounts of viable and purified endothelial cells.

Morphology of peritumoral proteoglycan alterations in the rabbit mesentery invaded by V2 carcinoma cells.

After intraperitoneal implantation into Swiss Silver rabbits, V2 rabbit carcinoma cells invade the mesentery where they form nodules of different size and texture: compact (less than 120 microns in diameter), loose (120-250 microns) and mixed (above 200 microns). Together with tumor development, certain changes take place in the loose connective tissue of the mesentery. Application of TEM, together with use of safranin O, has shown that, in areas free of tumor growth, collagen bundles become thick and heavy and proteoglycan density is increased. Concurrently, the number of fibrocytes, now transformed to fibroblasts, increases. Small, compact nodules are surrounded by a concentrically arranged extracellular matrix. Its overall density is similar to that of nodule-free areas. In the immediate vicinity of large, loose nodules, all constituents of the extracellular matrix disappear. Adjacent connective tissue is partly destroyed but still contains collagen fibers and proteoglycans. These findings suggest the following: The presence of V2 carcinoma cells induces marked alterations in the structured and non-structured components of the extracellular matrix. These changes are, at the same time, progressive and regressive and the occurrence of one or the other depends on local tumor progression. Progressive alterations may result from an increased activity of fibroblasts. Since degradative effects, on the other hand, are only seen in the immediate vicinity of larger tumor aggregates, it is assumed that a minimal number of tumor cells is essential for destruction of extracellular matrix.

Tumor-associated desmoplasia in the rabbit mesentery characterized by morphological, biochemical and cytophotometric methods.

Intraperitoneal implantation of V2 carcinoma cells in the rabbit leads to invasion of the mesentery and to structural tissue alterations which are concomitantly of a destructive and a desmoplastic type. In this report, we describe the desmoplastic changes which are characterized by the increased formation of collagen and of proteoglycans resulting in an increased thickness of the membrane. Biochemical data indicate that the total amount of collagen increases with time after implantation, whereas the relative amount per unit of dry weight, as well as the contributions of type I (15-25%) and type III (6-8%), stay within the same range. The increased synthesis of extracellular matrix is accompanied by a change in the appearance of the fibroblasts which now show the morphologic features of synthesizing cells. Also, an appreciable number have entered the S-phase. We propose that the desmoplastic changes are tumor-associated, since implantation of epithelial cells from normal rabbit liver does not result in similar alteration. Our findings are discussed in view of the role played by tumor and/or host cells in the increased production of extracellular matrix, of possible factor(s) elaborated by the tumor cells, and of the general significance of desmoplastia for tumor spread.

Seeding with omental cells prevents late neointimal hyperplasia in small-diameter Dacron grafts.

BACKGROUND: The influence of complete endothelialization of a prosthetic graft on development of late neointimal hyperplasia is unknown. This study was designed to investigate the effect of complete coverage with endothelial-like cells on late neointimal hyperplasia in small-diameter Dacron grafts seeded with omental cells in a canine model. METHODS AND RESULTS: Four-mm-ID Dacron grafts were seeded with cells from omentum and implanted in the carotid arteries in 24 mongrel dogs. Each dog received one seeded and one nonseeded graft. The graft patencies were assessed by angiography at 1, 5, 12, 26, and 52 weeks after surgery. The prostheses were explanted at 5, 12, 26, and 52 weeks after surgery and underwent microscopic studies. The actuarial patency rates at 1, 5, 12, 26, and 52 weeks were 100%, 95%, 95%, 95% and 95% for seeded grafts and 100%, 86%, 49%, 40%, and 13% for nonseeded grafts, respectively. The seeded grafts exhibited a uniform endothelial-like luminal monolayer without the development of late neointimal proliferation or anastomotic neointimal hyperplasia. Neointimal tissue thickness increased up to 6 months; no additional progression of the subendothelial tissue thickness was observed, in fact there was an insignificant decrease. CONCLUSIONS: Seeding with omental cells prevents development of late neointimal hyperplasia of small diameter prosthetic vascular grafts in a canine model.

A compliant small-diameter vascular prosthesis lined with functional venous endothelial cells.

The long-term patency of small-diameter vascular grafts is still unsatisfactory. In contrast to native arteries, they are inelastic and lack active antithrombogenicity. To improve long-term patency, a new 4 mm internal diameter prosthesis was developed which is compliant and lined with functional endothelial cells (ENC). The wall of this prosthesis consists of a microporous polyurethane-siloxane copolymer reinforced with a polyester network. It displays compliance (13.2 x 10(-4) mmHg-1) comparable to native arteries, is nonkinkable (minimum radius of curvature = 5 mm), burst resistant, and easily suturable. Using a lining procedure, coverage of prostheses by ENC was in excess of 95%. The ENC populations were found to be highly pure (by factor VIII-related antigen, DilAcLDL uptake) and to produce about 0.3 ng prostacyclin per cm2. In vitro tests of shear stress resistance demonstrated that ENC monolayers on the new elastic prosthesis remain intact for 3 hr in physiologically pulsating culture medium (Vmax = 50 cm/sec). Lined prostheses implanted for 24 hours in mongrel dogs as an arteriovenous shunt demonstrated the antithrombogenicity of the cultured ENC. The results suggest that small-diameter vascular prostheses which are compliant, porous, and actively antithrombogenic are feasible.

Internal-mammary coronary artery grafts: is their superiority also due to a basically intact endothelium?

The internal mammary artery (IMA) is a superior conduit for coronary artery revascularization and many factors have been suggested for explanation of this superiority. IMA and saphenous vein grafts have been systematically analysed with scanning electron microscopy (SEM) in a series of 11 patients undergoing coronary artery revascularization. At the time of implantation endothelial damage is almost absent in internal-mammary-artery (IMA) grafts; small areas of exposed subendothelial matrix may be present but are essentially non-thrombogenic as reflected by the lack of clots in these areas. In contrast the endothelium of harvested human saphenous veins (SV) shows large thrombogenic defects with exposed collagenous fibrils. The extent and deepness of the defects deteriorated in the period between removal of the vein and its attachment to the aorta. We conclude that long-term superiority of IMA grafts may also be due to the lack of primary intimal defects.

Late neutrophil infiltration of canine endothelial cell seeded Dacron grafts.

Neutrophil infiltration is known to affect the endothelial monolayer of seeded vascular grafts. The aim of this study was to develop an in vitro system allowing the monitoring of neutrophil (PMN) adherence after graft implantation. Dacron prostheses were seeded with autologous canine microvascular cells from omental adipose tissue and implanted for 35 days. In vitro, mature monolayers of canine homologous venous endothelial cells (CHVENC) were exposed to heparinized whole blood samples taken at days one and four postoperatively, followed by weekly tests. PMNs adherent to the CHVENC were counted per culture area. Results showed the feasibility of PMN monitoring, and demonstrated a late PMN adhesion, reaching its maximum about 20 days after implantation and decreasing to normal values after five weeks. It is concluded that in vitro tests can be used for noninvasive studies of host plasma factors and leukocyte activation.

[Effect of hemodynamic conditions and prosthesis structure on endothelialization of clinical and experimental small lumen vascular prostheses]. / Einfluss der hämodynamischen Bedingungen und der Prothesenstruktur auf die Endothelialisierung von klinischen und experimentellen kleinlumigen Gefässprothesen.

Seeding of small-diameter vascular prostheses (ID less than or equal to 6 mm) with autologous microvascular cells (AMVC) results in a complete endothelial cell layer on the luminal surface. The purpose of this study was to examine the influence of the blood flow velocity (due to 4 or 6 mm ID) and the structure of inner graft surface (crimped, uncrimped) on the endothelialization. AMVC were harvested from omental adipose tissue (mean: 0.56 X 10(6) cells/g tissue) from 10 mongrel dogs (mean: 27.9 kg). During preclotting, the 4 mm uncrimped and the 6 mm crimped double velour Dacron prostheses (Meadox Medicals, Inc.) were seeded with 1.0 X 10(6) cells/cm2 graft surface. Grafts were implanted into the carotid arteries (N = 5 in each group). The animals received antiplatelet therapy. After five weeks, all seeded prostheses were patent. The thrombus free surface (TFS) of seeded prostheses was 99.9% (4 mm) and 90.5% (6 mm). Scanning electron microscopy revealed an athrombogenic layer of endothelial cells on a smooth surface. -It is concluded that in canine experiments endothelialization of 4 and 6 mm grafts after seeding with AMVC is not affected by blood flow velocity or graft structure.