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Molecular cloning of an ovine ovarian tissue inhibitor of metalloproteinases: ontogeny of messenger ribonucleic acid expression and in situ localization within preovulatory follicles and luteal tissue.

Secretion of a tissue inhibitor of metalloproteinases (TIMP-1) is initiated by ovine preovulatory follicles after the gonadotropin surge. In addition, TIMP-1 is a major secretory product of ovine corpora lutea. We have isolated an approximately full-length cDNA clone for TIMP-1 from an ovine luteal cDNA library. The 887-basepair cDNA obtained was 95%, 86%, and 77% identical to the reported nucleotide sequences of bovine, human, and mouse TIMP-1 cDNAs, respectively. Total cellular RNA was isolated from preovulatory follicles collected before (presurge; n = 5) or 12-14 h after (postsurge; n = 4) a LHRH-induced gonadotropin surge (36 h after prostaglandin F2 alpha-induced luteolysis); from luteal tissue collected on days 3, 7, 10, 13, and 16 postestrus (n = 5, 5, 4, 5, and 5, respectively); and from purified populations of small (n = 4) and large (n = 3) luteal cells. Concentrations of TIMP-1 mRNA (picograms per micrograms tissue DNA) were increased in preovulatory follicles after exposure to a gonadotropin surge (P < or = 0.01). TIMP-1 mRNA was localized primarily to the granulosa layer of postsurge follicles by in situ hybridization. Concentrations of TIMP-1 mRNA in luteal tissue did not differ throughout the luteal phase (P = 0.07). However, TIMP-1 mRNA was localized predominantly to specific cells located in the connective tissue surrounding and within day 3 corpora lutea. In situ hybridization of day 10 corpora lutea localized TIMP-1 mRNA predominantly to specific cells that were randomly dispersed throughout the luteal tissue. TIMP-1 mRNA was expressed by purified populations of both small and large luteal cells collected from day 10 corpora lutea. Concentrations of TIMP-1 mRNA (picograms per micrograms DNA) were greater in the large luteal cell populations (P < or = 0.0001). We conclude that 1) expression of TIMP-1 mRNA by the granulosa layer of ovine preovulatory follicles increased after the gonadotropin surge, whereas TIMP-1 mRNA concentrations during the luteal phase remained constant; 2) during the luteal phase, TIMP-1 mRNA was localized to specific cells surrounding (day 3) or located within (day 10) the corpus luteum; and 3) expression of TIMP-1 mRNA was greatest in large luteal cells.

Amino Acid Sequence↗

Modulation of adipose tissue expression of murine matrix metalloproteinases and their tissue inhibitors with obesity.

The potential role of the matrix metalloproteinase (MMP) system in the pathophysiology of the adipose tissue was investigated in a mouse model of nutritionally induced obesity. mRNA levels of 16 MMPs and 4 tissue inhibitors of MMPs (TIMPs) were measured by semiquantitative RT-PCR in adipose tissue isolated from mice maintained for 15 weeks on a standard or high-fat diet. In mice on standard diet, with the exception of MMP-8, all MMP and TIMP transcripts were detected in both gonadal and subcutaneous depots. In obese mice, the expression of MMP-3, -11, -12, -13, and -14 and TIMP-1 mRNAs was upregulated, whereas that of MMP-7, -9, -16, and -24 and TIMP-4 was downregulated. Most MMP and TIMP mRNAs were expressed at higher levels in stromal-vascular cells than in mature adipocytes. Analysis of adipose tissue by in situ fluorescent zymography revealed MMP-dependent proteolytic activities, demonstrating the presence of active MMPs in the intact tissue. In vitro conversion of adipogenic 3T3-F442A cells into mature adipocytes was associated with substantial modulations of MMP and TIMP expression. Moreover, this in vitro adipogenesis was reduced in the presence of a synthetic MMP inhibitor. Thus, the adipose tissue expresses a large array of MMPs and TIMPs, which modulate adipocyte differentiation.

3T3 Cells↗

Tissue distribution and residues of clenbuterol, salbutamol, and terbutaline in tissues of treated broiler chickens.

To examine the tissue distribution and residues after withdrawal of various beta-agonists (i.e., clenbuterol, salbutamol, and terbutaline) 160 1-d-old broiler chickens were assigned to four groups. During treatment (16 to 35 d), the birds were fed a control diet or a diet containing 1 ppm of clenbuterol, 10 ppm of salbutamol, or 10 ppm of terbutaline. After d 35 all groups received the control diet. Five birds of each group were then slaughtered and tissues were collected on d 0, 1, 2, 3, 7, 14, and 43 following withdrawal of beta-agonists from the feed. Extraction of beta-agonists from the tissues was carried out by a new method using hetero-bifunctional solid phase extraction. The amount of beta-agonists in the extracts was measured by an enzyme immunoassay (EIA). The highest concentrations of beta-agonists were found in feathers: 224 ng of clenbuterol/g, 1,140 ng of salbutamol/g, and 1,159 ng of terbutaline/g. Clenbuterol accumulated above plasma levels in all tissues that were investigated (liver, kidney, stomach, muscle, fat, feather, eye). Salbutamol was most concentrated in feather, eye, liver, and kidney; terbutaline accumulated only in feather, liver, and kidney. Overall, clenbuterol showed the highest accumulation in the tissues analyzed. A withdrawal period of greater than 2 wk was required for residues in edible tissues to decline below detectable levels.

Adipose Tissue↗

[Regeneration of tooth and periodontal tissue using tissue engineering concepts].

In the context of minimally invasive surgery, the next logical step is to provide a biological replacement for missing tissue without the need for a harvesting operation. Tissue engineering is defined as the fabrication of living parts for the body from cells in the laboratory. Donor cells such as stem cell or cultivated, differentiated cells are seeded on a appropriately configurated scaffold replicating extracellular matrix. Growth factors are added to the in-vitro system to encourage stem cell proliferation. The engineered structure is then transplanted to the recipient. In this article, the regeneration strategies for tooth and periodontal tissue using tissue engineering concepts. Culture expanded mesenchymal stem cell is useful for periodontal tissue and tooth regeneration with bioabsorbable matrix such as b-TCP and collagen fiber. In the near future, these regenerated tissue will be accepted in clinical situations.

Animals↗

[Repeated tissue expansion for repairing extensive scalp and facial soft tissue defects: analysis of 18 cases].

OBJECTIVE: To summarize our experiences with 18 cases of repeated tissue expansion for treating extensive scalp and facial soft tissue defects. METHOD: Tissue expansion was performed for 1 or 2 times after the primary expansion for repairing large scalp and facial soft tissue defects ranging from 9 cm x 5 cm to 16 cm x 12 cm using re-expanded flaps. The indications, complications and experiences in the treatment were discussed. RESULT: Satisfactory results were obtained in all the 18 cases. On the follow-up conducted 6 months postoperatively, the repaired defects presented excellent appearance and functional recovery without additional scars. CONCLUSION: Repeated tissue expansion is an effective technique for repairing large tissue defects in the head and face.

Adolescent↗

A novel approach to periodontal tissue regeneration with mesenchymal stem cells and platelet-rich plasma using tissue engineering technology: A clinical case report.

Tissue engineering represents one of the most exciting advances in regenerative medicine. However, little has been reported on the application of tissue engineering for regeneration of periodontal tissues. Therefore, the aim of this study was to show how a technique based on tissue engineering principles can be applied to periodontology. Mesenchymal stem cells (MSCs) were isolated from a patient's iliac crest marrow aspirates. Platelet-rich plasma (PRP) was isolated from peripheral blood. Full-thickness periodontal flaps were elevated and the root surfaces were scaled and planed. A MSCs-PRP gel was prepared and applied to the root surface and adjacent defect space. The primary outcome measures were changes in pocket depth, clinical attachment level, bleeding on probing, and defect bone fill. Re-examination demonstrated that the treatment, including the application of MSCs-PRP gel at periodontal sites with angular defects, resulted in a 4-mm reduction in probing depths and a 4-mm clinical attachment gain, while bleeding and tooth mobility disappeared. Radiographic assessments showed that the bone defect had been reduced in depth. Interdental papillae supported by this tissue engineering technology regenerated. The use of MSCs in PRP gel might be helpful for periodontal tissue regeneration, treatment of esthetically sensitive sites, and reduction of patient morbidity.

Alveolar Bone Loss↗

Tissue compliance meter for objective, quantitative documentation of soft tissue consistency and pathology.

A new instrument is described, the tissue compliance meter (TCM), for quantitative and objective recording of soft tissue consistency. This quality is appreciated at present only by the subjective method of palpation. Use of the TCM therefore offers a method to quantify palpation of tissue consistency and to document findings objectively. The handheld instrument allows immediate and simple reading of the depth of penetration of a rubber disc at a known pressure. The relation between the achieved penetration and employed pressure expresses the compliance. The TCM consists of a rubber disc with the surface of 1 cm2 attached to a force gauge. The depth of penetration of the rubber tip is indicated by a disc which slides on the shaft of the force gauge. Normal values were established for men and women over muscles which are frequently affected by spasm. Tissue compliance measurement can document changes in soft tissue consistency which occur in muscle spasm, spasticity, swelling, tumors, lumps, hematomas, etc. Use of the TCM provides the most sensitive and earliest objective indication of either healing and resolution in soft tissue pathology or occurrence of complications. Changes in muscle tone such as reduction of spasm, tension, or spasticity can be recorded. The effects of different types of physical therapy can thus be documented objectively.

Connective Tissue↗

The structure and function of dermal connective tissue in normal individuals and patients with inherited connective tissue disorders.

Normal, human dermis is a dense, interwoven collagen and elastic matrix organized into papillary and reticular regions. the papillary dermis is a narrow zone beneath the epidermis which includes an even narrower subepidermal connective tissue band beneath the basal lamina. The reticular dermis has superficial intermediate and deep reticular zones. Each region is distinguished by the organization of the fibrous connective tissue. In this review, the structure, composition and function of each region of the normal dermis is surveyed and alterations in the tissue that have been recognized ultrastructurally in skin from patients with inherited connective tissue diseases are discussed. A molecular defect in a connective tissue molecule can be expressed phenotypically at one or more levels of dermal organization and can modify the structure of other matrix components. In some cases, the entire interwoven pattern of the dermis is altered; in others, the dermis retains the interwoven pattern and alterations are expressed as defects in the association of fibrils into fibers and fiber bundles. Abnormalities in the size, packing and relationships of fiber bundles also can occur. These changes involve the collagen matrix primarily. Depletion or exaggeration of elastin, or of the amorphous ground substance can also influence the organization of collagen and of the overall dermal architecture. The alterations described are illustrated with scanning electron micrographs of skin from patients with inherited connective disorders and are discussed in terms of the mechanical abnormalities of the tissue.

Collagen↗

A clinical/statistical comparison between the subpedicle connective tissue graft method and the guided tissue regeneration technique in root coverage.

Subepithelial connective tissue grafts and guided tissue regeneration have been shown to be effective means to obtain root coverage. The purpose of this study is to compare statistically the results obtained with these techniques 1 year after the surgical procedures were performed. Thirty-six gingival recessions belonging to Class I and Class II of the Miller classification were treated: 18 cases with subepithelial connective tissue grafts (SCTG) and 18 cases with guided tissue regeneration (GTR). Each patient was randomly assigned to a group. At baseline, the group treated with subepithelial connective tissue grafts presented a mean recession of 4.88 mm, whereas the group treated with guided tissue regeneration presented a mean recession of 5.88 mm (P = .082). After 1 year, the mean root coverage was 77.08% in the SCTG group and 80.88% in the GTR group. The difference was not statistically significant (P > .05). The mean root coverage was 3.83 mm for the SCTG group and 4.61 mm for the GTR group. The mean gain in probing attachment level was 3.05 mm for the SCTG group and 5.55 mm for the GTR group. The difference was statistically significant (P = .01). In conclusion, the mean root coverage obtained was similar for the two groups, whereas the clinical attachment gain was greater in the GTR group. Therefore, it appears that the GTR technique is preferable when severe mucogingival defects are present and gain of clinical attachment level is mandatory.

Connective Tissue↗

Matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase expression profiles in tracheal aspirates do not adequately reflect tracheal or lung tissue profiles in neonatal respiratory distress: observations from an animal model.

OBJECTIVE: Matrix metalloproteinase (MMP)/tissue inhibitor of matrix metalloproteinase (TIMP) expression in tracheal aspirates (TA) is commonly assayed to represent the protein profile in the lung. This study tests the hypothesis that the profile of MMPs 2, 7, and 9 and the profile of TIMPs 1 and 2 will be different in TA, tracheal tissue, and lung tissue in neonatal respiratory distress. DESIGN: Interventional laboratory study. SETTING: An academic medical research facility in northeastern United States. SUBJECTS: Oleic acid-injured, spontaneously breathing newborn piglets. INTERVENTIONS: Ten piglets (3-4 days old, 2.4 +/- 0.4 kg) were instrumented, injured by intravenous administration of oleic acid, and supported on continuous positive airway pressure of 2-5 cm H2O, with or without exogenous surfactant, depending on physiologic requirements. MEASUREMENT AND MAIN RESULTS: After 6 hrs, TA, trachea, and lung were obtained for MMP/TIMP analysis by substrate zymography/reverse zymography. TA contained less active (p < .01) and more latent (p < .05) MMP-2 than trachea and lung, and the active/latent ratio was less in TA than in both tissues (p < .01). TA and trachea contained more total (p < .05) and active (p < .01) MMP-9 than did the lung; TA contained more active MMP-9 than trachea (p < .01). MMP-7 was greater in all forms relative to total protein (p < .01) from both tissues compared with TA. Trachea contained more latent MMP-7 than lung (p < .01). TIMP-1 was different across protein sources (p < .01) where TA < trachea < lung. The active MMP-2/TIMP-2 ratio was lower in TA than in lung (p < .01); the MMP-9/TIMP-1 ratio had a significant trend (p < .01) where TA > trachea > lung. CONCLUSIONS: The MMP/TIMP profiles in TA do not adequately represent the profiles in either trachea or lung. Thus, MMP/TIMP profiles from TA are limited and should be interpreted for trends rather than actual tissue levels.

Analysis of Variance↗

The soft-tissue shaving procedure for removal of adipose tissue. A new, less traumatic approach than liposuction.

OBJECTIVE: To introduce the soft-tissue shaving cannula, a new, precise alternative to conventional liposuction that embodies an entirely different method of soft-tissue extraction and that appears to be less traumatic and more precise than methods currently used in liposuction. DESIGN: Nonrandomized, nonblinded comparison of the soft-tissue shaver and conventional liposuction devices in fresh cadavers, followed by clinical use of the liposhaver in selected patients undergoing cosmetic facial liposuction. INTERVENTIONS: Conventional liposuction devices and the soft-tissue shaving cannulas in fresh cadavers (< 8 hours old). A submental lipectomy and a melolabial fold liposhaving were performed in a clinical setting. OUTCOME MEASURES: Subjective evaluation by the operating surgeons. RESULTS: The fat was cleanly shaved and the contour result was even, without dimpling or asymmetry. CONCLUSIONS: Our early experiences suggest that this new liposhaving technique may offer a precise, less traumatic alternative to conventional liposuction. Fat can be shaved in an open fashion under direct vision. It does not rely on a vacuum seal. The soft-tissue shaving cannula shaves fat one layer at a time. The depth of each layer removed, the depth of soft-tissue injury, and the optimal settings and sizes for various procedures remain to be reported.

Adipose Tissue↗

Tissue engineering strategies for adipose tissue repair.

Tissue engineering is a relatively young field that combines engineering, clinical science, and life sciences to, in part, repair or regrow tissues. Adipose tissue has recently become a focus area for tissue engineering, encouraged by the large number of reconstructive, cosmetic, and correctional indications that could be addressed with clinically translatable adipose tissue engineering strategies. This review discusses the three aspects of an adipose construct, namely cell types, scaffold, and microenvironment, and presents current tissue engineering strategies under pursuit.

Adipose Tissue↗

Stability, tissue metabolism, tissue distribution and blood partition of azosemide.

Stability of azosemide after incubation in various pH solutions, human plasma, human gastric juice, and rat liver homogenates, metabolism of azosemide after incubation in 9000 g supernatant fraction of various rat tissue homogenates in the presence of NADPH, tissue distribution of azosemide and M1 after intravenous (i.v.) administration of azosemide, 20 mg kg-1, to rats, and blood partition of azosemide between plasma and blood cells from rabbit blood were studied. Azosemide seemed to be stable for up to 48 h incubation in various pH solutions ranging from two to 13 at an azosemide concentration of 10 micrograms mL-1; more than 93.4% of azosemide was recovered, and a metabolite of azosemide, M1, was not detected. However, the drug was unstable in pH1 solution: 75.8% of azosemide was recovered and 2.16 micrograms mL-1 of M1 (expressed in terms of azosemide) was formed after 48 h incubation in pH 1 solution at an azosemide concentration of 10 micrograms mL-1. Azosemide was stable in both human plasma and rat liver homogenates for up to 24 h incubation at an azosemide concentration of 1 microgram mL-1, and in human gastric juice for up to 4 h incubation at an azosemide concentration of 10 micrograms mL-1. However, all rat tissues studied had metabolic activity for azosemide in the presence of NADPH, with heart having a considerable metabolic activity: approximately 22% of azosemide disappeared and 9.32 micrograms of M1 was formed per gram of heart (expressed in terms of azosemide) after 30 min incubation of 50 micrograms of azosemide in 9000 g supernatant fraction of heart homogenates. The tissue to plasma ratios of azosemide (T/P) were greater than unity only in the liver (1.26) and kidney (1.74); however, M1 showed high affinity for all tissues studied except the brain and spleen when each tissue was collected at 30 min after i.v. administration of azosemide to rats. The equilibrium plasma to blood cell concentration ratios of azosemide were independent of azosemide blood concentrations: the values were 2.78-4.25 at azosemide blood concentrations of 1, 10, and 20 micrograms mL-1 in three rabbits. There was negligible 'blood storage effect' of azosemide, especially at low blood concentrations of azosemide, such as 1 and 10 micrograms mL-1.

Animals↗

An improved method for discrimination of cell populations in tissue sections using microscopy-based multicolor tissue cytometry.

BACKGROUND: In tissue context, researchers and pathologists lack a generally applicable standard for quantitative determination of cytological parameters. Increasing knowledge of disease-specific markers calls for an appropriate in situ tissue cytometry. METHODS: Microscopy-based multicolor tissue cytometry (MMTC) permits multicolor analysis of single cells within tissue context. RESULTS: Tissue specimens stained for CD45/CD3/CD4/CD8 were analyzed. Specificity as well as reproducibility of MMTC is demonstrated and a novel MMTC-based function to improve visual discrimination of subpopulations is introduced. CONCLUSIONS: Our data demonstrate that MMTC constitutes an important step toward automated and quantitative fluorometry of solid tissues and cell monolayers.

Algorithms↗

Expression profiling of 68 glycosyltransferase genes in 27 different human tissues by the systematic multiplex reverse transcription-polymerase chain reaction method revealed clustering of sexually related tissues in hierarchical clustering algorithm analysis.

We have developed an experimental system to study the expression of 68 human glycosyltransferase genes. Using this system, we examined the expression of those genes in 27 different tissues by the technique which we named systematic multiplex reverse transcription-polymerase chain reaction (SM RT-PCR). The panoramic view of a total of 1836 (68 x 27) expression data demonstrates that some glycosyltransferase genes are differentially expressed whereas some others are ubiquitously expressed. The data gathered provide more information on glycosyltransferase gene expression in tissues than any other paper published, and surpass in quantity all the information combined from previous publications. Although the expression profiling of glycosyltransferase genes alone may not directly explain the repertoires of oligosaccharides synthesized, it is an important step toward a better understanding of the gene expression network involved in oligosaccharide synthesis/degradation. Our modestly high-throughput gene expression study and the data analysis using a hierarchical clustering algorithm have allowed us to investigate the correlation between tissues and glycosyltransferase gene expression. Similar patterns of glycosyltransferase gene expression were observed in functionally and anatomically related tissues. All, but one, sexually related tissues formed a cluster in a tissue dendrogram, suggesting the involvement of sex hormones in the transcriptional control of many glycosyltransferase genes. Once established, the SM RT-PCR is cost- and time-efficient and requires small amounts of RNA as template. It is especially useful for the simultaneous analyses of multiple samples. Because of its simple design, the SM RT-PCR may offer an easy alternative in studying the expression of many other families of genes, as well as groups of related/unrelated genes, in various biological phenomena.

Algorithms↗

Liver tissue characterization by in vitro NMR: tissue handling and biological variation.

The extraction of reliable and useful relaxation time data for tissue characterization by NMR requires strict protocols, optimized for each type of biological tissue, which include parameters like storage duration and temperature as well as measurement parameters. Spin-lattice relaxation times in liver tissue vary not only with NMR frequency but also with their "time-after-excision characteristics," while spin-spin relaxation times are almost independent of most parameters which influence T1 at 20 MHz in normal liver tissue (e.g., species, sex, circadian rythm, starvation). T2, however, being more sensitive to water content and pH changes, is well suited for detecting nonspecific tissue alterations (e.g., due to ischemia, chemical toxins). Following the suggestions outlined herein, investigation of at least 120 min of time-after-excision (storage) effects allows the significant distinguishing of various physiological differences in normal liver tissue as well as improvement of early detection of liver pathologies.

Animals↗

Prevention of tissue injury and postsurgical adhesions by precoating tissues with hyaluronic acid solutions.

The effectiveness of inhibiting serosal tissue damage and preventing surgical adhesions by precoating tissues with dilute solutions of hyaluronic acid (HA) was evaluated in a rat cecal abrasion model. This study was performed at three independent laboratories using the same protocol. Three hundred and seventy-five adult rats were divided into five treatment groups (125 animals at each study site): 0.1% HA, 0.25% HA, 0.4% HA, phosphate-buffered saline solution (PBS), and no solution. The abdominal cavity of each animal was precoated with 4 ml of test solution or no solution, prior to a controlled abrasion of the cecum. One week later, the animals were sacrificed and adhesions were scored on a 0-4 scale. The data were pooled because no statistical difference was found in the trends at the three study sites. The PBS precoating and no tissue precoating treatment groups had the same high incidence of cecal adhesions, which was significantly higher than the incidence of adhesions in the HA treatment groups. As the HA concentration in the precoating solution increased from 0% (PBS group) to 0.4% HA, the mean incidence of cecal adhesions decreased in a concentration-dependent manner from 1.6 +/- 0.11 to 0.7 +/- 0.09 (P < 0.001). The percentage of animals with no cecal adhesions increased from 11% in the PBS group to 50% in the 0.4% HA treatment group (P < 0.001). In a separate histological study employing 150 rats, HA solutions significantly inhibited serosal tissue damage and ameliorated the inflammatory response due to abrasion and desiccation compared to that with no coating or precoating with buffered saline. Together, these studies demonstrate that tissue precoating with dilute HA solutions reduces damage to serosal tissues during surgery and thereby limits formation of postsurgical adhesions.

Administration, Topical↗

Dual role of diffusion in tissue gas exchange: blood-tissue equilibration and diffusion shunt.

The role of diffusion in inert gas washout from tissue is investigated using simple mathematical models incorporating diffusive blood-tissue equilibration and diffusion shunt due to diffusive gas transfer between precapillary and postcapillary vessels with counter-current blood flow. With increasing diffusivity blood-tissue equilibration is improved, but simultaneously the diffusion shunt by veno-arterial back diffusion is increased. Similarly, with decreasing blood flow, the extent of diffusion limitation in blood-tissue transfer is diminished, but at the same time veno-arterial diffusive shunting is enhanced. Diffusion shunt slows inert gas washout (local tissue clearance) and thus, if not taken into account, leads to an underestimation of capillary blood flow calculated from the washout rate constant. Diffusion shunting of O2 diminishes the efficacy of blood-tissue transfer, but its extent is predicted to be smaller compared to that for inert gases, because the chemical combination of O2 in blood decreases diffusive shunting.

Animals↗