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PubMed2000

Higher numbers of autologous fibroblasts in an artificial dermal substitute improve tissue regeneration and modulate scar tissue formation.

Cultured skin substitutes are increasingly important for the treatment of burns and chronic wounds. The role of fibroblast numbers present in a living-skin equivalent is at present unknown. The quality of dermal tissue regeneration was therefore investigated in relation to the number of autologous fibroblasts seeded in dermal substitutes, transplanted instantaneously or precultured for 10 days in the substitute. A full-thickness porcine wound model was used to compare acellular dermal substitutes (ADS) with dermal substitutes seeded with fibroblasts at two densities, 1x10(5) (0-DS10) and 5x10(5) cells/cm(2) (0-DS50), and with dermal substitutes seeded 10 days before operation at the same densities (10-DS10 and 10-DS50) (n=7 for each group, five pigs). After transplantation of the dermal substitutes, split-skin mesh grafts were applied on top. Wound healing was evaluated blind for 6 weeks. Cosmetic appearance was evaluated and wound contraction was measured by planimetry. The wound biopsies taken after 3 weeks were stained for myofibroblasts (alpha-smooth muscle actin), and after 6 weeks for scar tissue formation (collagen bundles organized in parallel and the absence of elastin staining). Collagen maturation was investigated with polarized light. For wound cosmetic parameters, the 10-DS50 and 0-DS50 treatments scored significantly better than the ADS treatment, as did the 10-DS50 treatment for wound contraction (p<0.05, paired t-test). Three weeks after wounding, the area with myofibroblasts in the granulation tissue, determined by image analysis, was significantly smaller for 0-DS50, 10-DS10, and 10-DS50 than for the ADS treatment (p<0.04, paired t-test). After 6 weeks, the wounds treated with 0-DS50, 0-DS10, and 10-DS50 had significantly less scar tissue and significantly more mature collagen bundles in the regenerated dermis. This improvement of wound healing was correlated with the higher numbers of fibroblasts present in the dermal substitute at the moment of transplantation. In conclusion, dermal regeneration of experimental full-skin defects was significantly improved by treatment with dermal substitutes containing high numbers of (precultured) autologous fibroblasts.

Animals↗
PubMed1998

New face of an old enzyme: alkaline phosphatase may contribute to human tissue aging by inducing tissue hardening and calcification.

Tissue nonspecific alkaline phosphatase (AP) plays a well-known role in bone mineralization. This role was first suggested by a human AP deficiency disease, hypophosphatasia. Further studies with AP gene knockout mice have also suggested a role for AP in mineralization. However, AP is also expressed in other human tissues besides bone and cartilage, and this raises a question as to whether AP may also play a role in pathological mineralization such as dystrophic and vascular calcification. In vitro studies carried out in our laboratory indicate that a variety of cell types stably expressing membrane-bound AP can affect extracellular mineralization regardless of the tissue from which the cell lines originated (e.g. fibroblasts, vascular endothelial cells, or renal epithelial cells). This AP-mediated extracellular mineralization is both substrate/dependent and culture environment/dependent and may be consistent with a putative role for AP in pathological mineralization in tissues other than bone and cartilage. In this regard, it is interesting to note that high levels of AP are observed in vascular endothelia of small arterioles in brain and heart. It is probable that expression of AP in small arterioles of brain and heart may also contribute to the vascular hardening and calcification observed in humans. This in turn could be related to vascular aging, vascular disease, and the resultant weakening of and/or rupture of vessel walls.

Aging↗
PubMed1997

Analysis of tissue factor and tissue factor pathway inhibitor expression in human colorectal carcinoma cell lines and metastatic sublines to the liver.

To investigate the expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in human colorectal carcinomas, Northern blot analysis was performed in a series of human colorectal carcinoma cell lines and in normal or tumoral colorectal tissues. Of 16 human colorectal carcinoma cell lines examined, most expressed TF mRNA, though the levels of expression varied significantly. Considerably higher expression was observed in the cell line CaR-1, while lines established from metastatic lesions tended to express abundant TF mRNA. By contrast, TFPI mRNA levels were low in these high TF-expressing cell lines. TFPI was expressed abundantly in WiDr and in a few other cell lines which expressed a very low level of TF mRNA. Immunocytochemically, both proteins were stained predominantly on the cell surface; however, diffuse cytoplasmic staining for TF also was observed in CaR-1 cells. In addition, the cell surface TF activity was significantly higher in CaR-1 cells than in WiDr cells, confirming the results of mRNA analysis. The level of TF mRNA in colorectal carcinoma tissue in vivo and its ratio to the normal counterpart also varied significantly among the cases. To search for a possible role of TF/TFPI in metastasis of colorectal carcinoma cells, the expression of these genes was compared between a rectal adenocarcinoma cell line, RCM-1, and its highly metastatic subline, RCM-1 L-10. Compared with the parent line, RCM-1 L-10 expressed 7.5-fold higher levels of TF mRNA, whereas TFPI expression was not altered significantly or even decreased slightly. The higher cellular TF activity was confirmed in the metastatic subline in comparison with the parent line.

Adenocarcinoma↗
PubMed1998

Engineering the tissue which encapsulates subcutaneous implants. II. Plasma-tissue exchange properties.

This study assesses the plasma-tissue exchange characteristics of the capsular tissue that forms around implants and how they are affected by implant porosity. The number of vessels and their permeability to rhodamine were measured by intravascular injection of the fluorophore tracer into Sprague-Dawley rats that hosted for 3-4 months polyvinyl alcohol (PVA) and polytetrafluoroethylene (PTFE) subcutaneous implants. Rats were implanted with four pore sizes of PVA--a nonporous PVA (PVA-skin), and 5, 60, and 700 micron mean pore sizes (PVA-5, PVA-60, and PVA-700, respectively)--and two pore sizes of PTFE: 0.50 (PTFE-0.5) and 5.0 (PTFE-5) mean micron pore sizes. Photodensitometric image analysis was used to quantify the local tracer extravasation and, hence the permeability coefficients of isolated vessels around the implants. The number of functional vessels within 100 microm of the implants highlighted by the lissamine-rhodamine tracer were counted with fluorescence microscopy and with H&E stained sections using brightfield microscopy. The permeability of vessels did not vary substantially with implant pore size but generally were lower than those measured for surrounding subcutis. Pore size, however, had a dramatic effect on the vascular density of tissue-encapsulating implants: the number of microvessels (under 10 microm in radius) within the tissue surrounding the porous implants was higher than the number around nonporous implants. Pore sizes on the order of cellular dimensions incited optimal neovascularization; the vascular density around PVA-60 implants was six times higher (p < .001) and three times higher (p < .001) than those around PVA-0 implants in the fluorescent images and in brightfield, respectively. Moreover, brightfield microscopy showed the number of vessels around PVA-60 implants was almost double those in normal subcutis. The results suggest that optimal vascular density around long-term implants, such as sensors, biofluid cell constructs, and immunoisolated cell systems, may be engineered with pore size.

Animals↗
PubMed2001

Detection of novel gene expression in paraffin-embedded tissues by isotopic in situ hybridization in tissue microarrays.

Correlating altered gene expression patterns with particular disease states is a critical step in understanding disease processes and developing treatment strategies. Many thousands of novel gene sequences have recently been annotated in public and private databases and are now available for analysis. Tissue-specific expression patterns of these sequences can be evaluated physically on DNA arrays and other high throughput assays, or virtually by bioinformatics mining of expressed sequence tag (EST) databases. As a secondary screening tool, in situ hybridisation (ISH) not only confirms tissue specificity, but also reveals what is often valuable information about cell-type expression patterns of nov16l sequences. Due to their availability and long-term stability at room temperature, formalin-fixed paraffin-embedded clinical specimens provide an invaluable resource for evaluating expression patterns of novel human genes. We describe a high-throughput approach for identifying and quantifying the expression of novel genes in paraffin-embedded human tissues using isotopic in situ hybridisation and tissue microarrays (TMA).

Blotting, Northern↗
PubMed1975

Further observations of Fc receptors in human malignant tissue and normal lymphoid tissue.

Twenty human malignant solid tumors of various histologic types were tested for the presence of Fc receptor using cryostat sections or single cell suspensions of fresh tissue. Sheep erythrocytes sensitized by various amounts of rabbit IgG antibodies served as indicator cells (EA). All tumors possessed Fc receptor, but to varying degrees; eight reacted more strongly than normal spleen without any relation to histologic type. The tumors which gave the strongest reactions in sections also formed the highest percent of EA rosettes in suspensions, thus indicating surface localization of receptors. The reactions with spleen sections localized to the B cell and monocytic areas; the latter also showed high avidity in reactions with uncomplexed IgG. Rabbit antisera to tumors, spleen, and peripheral lymphocytes (polyvalent ALS) in inhibited the reactions, while a T-cell-specific ALS did not. Absorptions of the antisera with lymphocytes or tissue sediments of spleen and tumors removed the inhibiting activity, tissue sediments of muscle and kidney only reduced the titers. Again, results with spleen sections paralleled those obtained with tumor sections. Apparently, the tumor Fc receptor is very similar to the Fc receptors present in normal lymphoreticular tissues.

Absorption↗
PubMed2000

Surface microarchitectural design in biomedical applications: in vivo analysis of tissue ingrowth in excimer laser-directed micropored scaffold for cardiovascular tissue engineering.

A micropatterned microporous segmented polyurethane film (20 x 12 mm in size, 30 micrometer thick) with four regions was prepared by excimer laser microprocessing to provide an in vivo model of transmural tissue ingrowth in an open cell-structured scaffold specially designed for cardiovascular tissue engineering. Three microporous regions had the same circular micropores (30 micrometer diameter) but different pore density arrangements (percentage of total pore area against unit area was 0.3%, 1.1%, and 4.5%), and the other region remained nonporous. The covered stent, prepared by wrapping the regionally different density-microporous film on an expandable metallic stent (approximately 3.1 mm in diameter), was delivered to the luminal surface of canine common carotid arteries and placed after expansion of the stent to a diameter of approximately 8 mm using a balloon catheter. At 4 weeks of implantation, all the covered stents (n = 10) were patent. The luminal surfaces of the covered stents were almost confluently endothelialized both in nonporous and microporous regions. Histological examination showed that the neointimal wall was formed by tissue ingrowth from host through micropores (transmural) and anastomotic sites. Thrombus formation occurred frequently in the lowest density porous region and nonporous region. With an increase in pore density, the thickness of the neointimal wall decreased. This study demonstrated how the micropore density of implanted devices influences tissue ingrowth in arterial implantation.

Angioplasty, Balloon↗
PubMed2005

New infusion device for trans-tissue, sustained local delivery of anticancer agent to surgically resected tissue: potential use for suppression of local recurrence of pancreatic cancer.

Local recurrence is a major cause of death of patients who have undergone resection for pancreatic cancer. To reduce the incidence of local recurrence, a new drug-infusion device, which is fixed on resected tissues immediately after surgery, was devised for trans-tissue, sustained local delivery. The drug-infusion device proposed here has the following functions: tight adhesion of the device on resected tissues, sustained drug infusion to the tissue, drug reloading, and easy removal from the body after use. The fabricated prototype experimental device, described here, was a thin infusion pouch made of a thin elastomer film (segmented polyurethane), which is connected to an elastomeric tube for drug reloading. The suppressive effect of the device delivering the anticancer agent, gemcitabine (GEM), was examined using subcutaneous tumor-bearing athymic mouse. A low-dose, local sustained GEM delivery using the device significantly suppressed the tumor growth and regrowth after surgery. The preliminary model experimental result appears to provide a promising therapeutic procedure for increased survival rate of the patients undergoing surgery for pancreatic cancer.

Animals↗
PubMed1988

Pulsed CO2 laser tissue ablation: effect of tissue type and pulse duration on thermal damage.

Tissue removal by infrared lasers is accompanied by thermal damage to nonablated tissue. The extent of thermal damage can be controlled by a choice of laser wavelength, irradiance, and exposure duration. The effect of exposure duration has been studied in vivo by using CO2 lasers with pulse widths that vary from 2 microseconds to 50 msec. Pulse widths of 50 msec, typical of a shuttered, continuous-wave CO2 laser, produce damage regions 750 micron wide in normal guinea pig skin; the use of a 2-microseconds-long pulse reduced this damage zone to as little as 50 micron. Using 2-microseconds-long pulses, in vitro studies showed that the minimum zone of thermal damage varied significantly with tissue type. The thermal denaturation of these tissues has been studied and correlated with damage. The effect of denaturation temperature and pulse duration on the width of the damage zone is explained by a simple model.

Animals↗
PubMedDate not supplied

Quantitation of metabolites in NMR spectra from isolated tissues, using 14N spectroscopy and nitrate to determine tissue volume.

Quantification of metabolites is a goal of many biomedical NMR studies. To obtain absolute measurements of metabolite concentrations is often both difficult and time-consuming. In this paper a method for determining metabolite concentrations directly is described and validated. It is applicable to studies of amphibian muscles, and with suitable precautions, to other isolated organs and tissues. The method is based upon using 14N NMR and nitrate-containing solutions to determine what fraction (F) of the sensitive volume of the RF coil is occupied by tissue. As the concentration of nitrate is known it can be used to calibrate other 14N metabolites in the tissue. Moreover, once F is determined, it can be used to calibrate metabolites in spectra from other nuclei e.g., 31P or 31C. All that is required is that a spectrum from a standard for that nucleus is obtained. Thus this method does not require any 'internal' (intrinsic to the tissue) standard, and is extremely quick and simple to use.

Adenosine Triphosphate↗
PubMed1999

Concurrent increase of oxidative DNA damage and lipid peroxidation together with mitochondrial DNA mutation in human lung tissues during aging--smoking enhances oxidative stress on the aged tissues.

Although mutation of mitochondrial DNA (mtDNA) in human tissues has been established to associate with intrinsic aging, the impact of environmental factors on the formation and accumulation of mtDNA mutations and oxidative DNA damage in human tissues is poorly understood. We have investigated the levels of mtDNA with the 4977-bp deletion and A3243G point mutation, oxidative DNA damage (indicated by the formation of 8-hydroxy-2'-deoxyguanosine, 8-OH-dG), and lipid peroxides in lung tissues from smokers and nonsmokers of subjects of different ages. The results showed concurrent age-dependent increase of the 4977-bp deleted mtDNA (P < 0.001), 8-OH-dG (P < 0.05), and lipid peroxides (P < 0.05) in the human lung. In the group of subjects above 60 years old, smokers had more extensive DNA damage and lipid peroxidation than did the nonsmokers. However, the levels of mtDNA with the 4977-bp deletion and A3243G point mutation in the lung of smokers were not significantly different from those of the age-matched nonsmokers. Taken together, these results suggest that accumulation of mtDNA with the 4977-bp deletion together with oxidative DNA damage and lipid peroxides is associated with aging and that smoking enhances oxidative damage in human lung tissues.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1997

Developmental fates of larval tissues after metamorphosis in ascidian Halocynthia roretzi. I. Origin of mesodermal tissues of the juvenile.

Cell lineages during ascidian embryogenesis are invariant. In a previous study, the developmental fate of each blastomere during embryogenesis of the ascidian Halocynthia roretzi was analyzed in detail by intracellular injection of a tracer molecule, horseradish peroxidase (Nishida, H., Dev. Biol. 121, 526-541, 1987). In the present study, the developmental fates of various larval mesodermal tissues after metamorphosis in H. roretzi were traced by labeling each precursor blastomere of larval tissues. Mesenchyme cells of larvae gave rise to tonic cells of juveniles. Trunk ventral cells gave rise to body-wall (atrial siphon and latitudinal mantle) muscle, heart, and pericardium of juvenile. Trunk lateral cells gave rise to blood (coelomic) cells as well as body-wall (oral siphon and longitudinal mantle) muscle. Larval muscle and notochord cells did not contribute to juvenile tissues. It was revealed that it is predictable to which juvenile tissue a blastomere of the 110-cell embryo will give rise. Therefore, in ascidian development, developmental fates after metamorphosis were almost invariant with regard to cell type of descendants.

Animals↗
PubMed1985

Nucleoside phosphotransferase in animal tissues. Tissue distribution and kinetic properties.

Amphibian, avian and mammal tissues contain a nucleoside phosphotransferase clearly different from those previously described in vegetables and bacteria. Whatever the animal source, the enzyme showed many similar characteristics as far as substrate specificity, dependence upon Mg2+, instability at 37 degrees C, and the protecting effect of nucleotides were concerned. Moreover, when submitted to gel filtration, the enzyme behaved in all cases as a dissociable high molecular weight protein, whose degree of association was controlled by nucleotides. In amphibian and avian tissues multiple forms of the enzyme seem to be present which differ for the substrate concentration at half-maximal velocity (S0.5); the concentration of nucleotide effector which affords half-maximal protection at 37 degrees C (P0.5); and the Hill coefficient for monophosphate donor. Within each single species, the higher the interaction coefficient was, the lower S0.5 and P0.5 values were. In mammalian tissues one form of nucleoside phosphotransferase seems to prevail where cooperative interactions are almost absent and whose S0.5 as well as P0.5 values do not vary significantly from one tissue to another.

Animals↗
PubMed1981

The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissue.

The influence of fixation and tissue preparation on the immunohistochemical localization of human fibronectin in gastrointestinal tract tissue has been examined using indirect immunoperoxidase technique. The most optimal staining result with strong intensity and well defined localization was obtained on frozen sections of unfixed material. Nearly identical results with improved morphology were obtained when staining paraffin sections of tissue fixed in 96% ethanol, 96% + 1% acetic acid and absolute acetone. All other fixatives tested, 10% neutral buffered formalin. Lillie's AAF, Bouin's fixative, Clarke's fixative, 4% formaldehyde, 4% formaldehyde + 0.5% cetylpyridiniumchloride (F-CPC), 4% formaldehyde +0.1% glutaraldehyde gave unsatisfactory results. However, proteolytic digestion with pepsin of paraffin sections prior to staining of buffered formalin and F-CPCfixed material gave results comparable with those obtained on unfixed frozen sections are regards definition of the staining whereas staining intensity was decreased in some degree. No improvement was observed when using proteolytic digestion of tissue fixed in other fixatives.

Colon↗
PubMed1992

Amino acid metabolism in several tissues of the obese Zucker rat as indicated by the tissue accumulation of alpha-amino[1-14C]isobutyrate.

Measurements of the tissue accumulation of alpha-amino[1-14C]isobutyrate [1-14C]AIB in lean (+/?) and obese (fa/fa) Zucker rats showed an augmented tissue/plasma ratio in the liver of the obese animals. In contrast, brown adipose tissue AIB accumulation was lower in the fa/fa animals. In response to a 24 h starvation period AIB accumulation was significantly elevated in the liver and plasma of the lean animals and was unchanged in the liver of the fa/fa animals. The circulating concentration of alanine and branched-chain amino acids was elevated in the fa/fa animals as compared to their lean counterparts. These observations suggest that amino acid uptake is not involved in the impaired muscle development observed in the obese Zucker rat and that the ability of brown adipose tissue for amino acid utilization is decreased in the obese animals suggesting that this may partially explain the impaired thermoregulatory capacity observed in brown adipose tissue of obese Zucker rats.

Amino Acids↗
PubMed1987

Analysis of tissue lymphocytes by double fluorescent staining--gastric cancer tissue and regional lymph nodes.

An immunohistochemical study was performed on human lymphocytes in the tissue of gastric cancer, and also in the regional lymph nodes, by double fluorescent staining, using monoclonal antibodies. Leu3a+8+ cells (inducer T cells) which consist about 30 per cent of Leu 3a+ cells were seen in the tissue surrounding the gastric cancer. The other 70 per cent of Leu 3a+ cells were Leu3a+8- cells (helper T cells). In the lymph nodes they were noted in T cell areas in almost the same proportions, while in germinal centers, only Leu3a+8- cells were found. On the other hand, OKT8+Leu15- cells (cytotoxic T cells) were noted in a large number, while OKT8+Leu15+ cells (suppressor T cells) were few. Further, an increase of OKT8+Leu15- cells was seen around gastric cancer or metastatic cancer in lymph nodes. These immunohistochemical findings suggest that cytotoxic T cells are the main component in the tissue of gastric cancers and the regional lymph nodes. Increases in inducer T cells and helper T cells are probably required to induce cytotoxic T cells around the cancer tissue.

Antibodies, Monoclonal↗
PubMed1978

Cartilage tissue differentiation from mesenchymal cells derived from mature muscle in tissue culture.

Under the influence of biochemical components of bone matrix gelatin (BMG), cartilage differentiates in tissue culture from the connective tissue cell outgrowths of mature muscle. Proliferation and differentiation begin within 24 hr with synthesis of hyaluronate, continue with high levels of synthesis of DNA and hyaluronidase, and culminate in production of large quantities of chondroitin sulfate. The addition of hyaluronic acid to the culture medium during the first 48 hr of culture depresses, whereas chondroitin sulfate enhances, subsequent production of cartilage. These observations on the cell biosynthetic products prior to the appearance of mature cartilage suggest that the BMG--modified connective tissue outgrowths of mature muscle exhibit the developmental potential of embryonic axial mesenchyme. Whether muscle harbors embryonic cells in a programmed but not yet activated readiness (protodifferentiated state) to differentiate into cartilage, or simply contributes a population of temporarily dedifferentiated fibroblasts, is not known, but in any event, BMG switches the pathway of further development from fibrous connective tissue to cartilage.

Bone Matrix↗
PubMed1999

Elemental composition changes between breast tissue with and without silicone gel sheeting and hypertrophic scar tissue.

Hypertrophic scars occur after dermal trauma and are characterized by being elevated above normal skin level as a result of an abundance of collagen. The application of silicone gel sheeting (SGS) has been found to be an effective method of treatment, causing them to regress much quicker than they would do naturally. Normal skin and hypertrophic scar tissue were characterized using proton-induced X-ray emission (PIXE). Skin tissue that had been covered in SGS was also analyzed. For each element and sample type, the concentrations in the epidermis were plotted against the dermis. By considering the concentrations of breast tissue with and without SGS, it could be seen if the SGS changed the compositional structure of the skin. It was found that for the elements P, S, Cl, and K the SGS has no effect on the structure of the skin, as both breast types (with and without SGS) have regression lines that overlap. However, this work shows that there are significant differences for P in the dermis and Cl in the epidermis between the breast tissue with SGS and its control. Therefore, this work shows that the effect the SGS has on concentration occurs similarly for both the epidermis and dermis.

Breast↗