Explant culture of human fetal small intestine

Human fetal intestine (10-14 wk gestation) has been cultured as explants in a serum-free Leibovitz L-15 medium for periods up to 9 days. As determined by light microscopy, the overall architecture of the intestinal explant was maintained throughout the culture period. At the ultrastructural level the villus absorptive cells remained tall with well-defined brush border, apical tubular system, and supranuclear and infranuclear accumulations of glycogen. All other epithelial cell types were also preserved. The incorporation of [3H]thymidine and [3H]leucine continued during the culture period, reflecting a sustained synthesis of deoxyribonucleic acid and proteins. The hydrolytic activities of the brush border membrane were established based on data obtained throughout the course of the culture of a large number of intestinal specimens. Sucrase, maltase, glucoamylase, trehalase, lactase, alkaline phosphatase, and gamma-glutamyl transpeptidase activities increased during the 9 days of culture even though different patterns were recorded. These observations clearly established that human fetal small intestine can be maintained in organ culture for at least 9 days in a serum-free medium.     

Factor XIII modulates intestinal epithelial wound healing in vitro.

BACKGROUND: An acquired deficiency of blood coagulation factor XIII has been proposed to cause an impairment of intestinal wound healing and hemostasis in patients with inflammatory bowel diseases. Substitution of factor XIII seems to result in a rapid improvement of intestinal wound healing. Our aim was therefore to characterize the role of factor XIII in the modulation of intestinal wound healing in vitro. METHODS: Factor XIII was added to subconfluent cultures of two non-transformed small-intestinal epithelial cell lines (IEC-6, IEC-18) and three human colon cancer-derived epithelial cell lines (T84, CaCo-2, HT-29) with subsequent assessment of cell proliferation with a colorimetric 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenylformazan (MTT) assay. The effects on epithelial cell migration in vitro were assessed with an in vitro wounding model of confluent IEC-6 cell monolayers. RESULTS: Factor XIII caused a modest inhibition of proliferation of IEC-6 and IEC-18 cells. However, factor XIII significantly stimulated proliferation of T84, CaCo-2. and HT-29 cell lines. In addition, thrombin-activated factor Xill promoted intestinal epithelial cell restitution in vitro on average 2.5-fold. The modulatory effects of factor XIII could not be significantly blocked by anti-transforming growth factor beta (TGFbeta). CONCLUSIONS: Factor XIII may promote intestinal epithelial wound healing by enhancement of epithelial cell restitution through a TGFbeta-independent pathway. This may explain previously described beneficial effects of factor XIII in the treatment of active ulcerative colitis.     

Leprdb diabetic mouse bone marrow cells inhibit skin wound vascularization but promote wound healing

Bone marrow stem cells participate in tissue repair processes and may have roles in skin wound repair. Diabetes is characterized by delayed and poor wound healing, and type 1 diabetes seems to lead to stem cell dysfunction. Hence, stem cell dysfunction could contribute to poor healing, and stem cell-based therapies may be efficacious in diabetic wounds. We investigated the potential of exogenous stem cells to promote skin healing and possible effects of type 2 diabetes on stem cell function. Mouse bone marrow cells from nondiabetic and diabetic mice were enriched for putative stem cells and injected under skin wounds of nondiabetic or type 2 diabetic Leprdb mice. Using histology and morphometry, vascularization and healing in treated and untreated mice were analyzed. We anticipated a correlation between improved wound healing and vascularization, because therapies that increase tissue vascularization tend to enhance wound healing. Our data indicate that exogenous nondiabetic bone marrow-derived cells increase vascularization and improve wound healing in Leprdb mice but have little effect on nondiabetic controls. In contrast, Leprdb-derived marrow cells inhibit vascularization but promote wound healing in Leprdb mice. Thus, adult stem cell function may be impaired by type 2 diabetes; the ability to promote vascularization and wound healing are distinct functions of bone marrow cells; and neovascularization and wound healing may not be tightly coupled. Additionally, we observed little incorporation of injected cells into wound structures, suggesting that improved healing is mediated through mechanisms other than direct differentiation and incorporation of the cells.     

Effects of granulocyte-macrophage colony-stimulating factor on incisional wound healing in an experimental diabetic rat model

The exact nature of poor wound healing in diabetes is uncertain. Neutrophils play a critical role in the host defense mechanism, and it is suggested that impaired neutrophil functions cause healing difficulties with or without infections in diabetic patients. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is used clinically when given systematically to increase the circulating neutrophils, but its wound-healing effects have not been systematically studied. This study was undertaken to examine the effects of GM-CSF on incisional wound healing in an experimental diabetic rat model. Forty rats were randomly divided into three groups, group I receiving saline as control, diabetes-induced group II receiving saline and diabetes-induced group III receiving GM-CSF. The anesthetized rats in all groups were wounded 21 days after diabetes induction by streptozotocin. Blood neutrophil counts and neutrophil fractions were also determined three days after wounding. Tensile strengths of wounded skin and the hydroxyproline (hyp) level of the wound were determined and wound healing processes were evaluated by light and electron microscopy, fourteen days after wounding. Neutrophil counts and phagocytosis were significantly increased in group III and neutrophil counts decreased in group II (p < 0.05). Although the hydroxyproline level of wound tissue significantly decreased in group II as compared with group III (p < 0.05), there was no differences of tensile strength between group II and III (p < 0.05). Wound score in group II was less than that in groups I and III (p < 0.05). It is concluded that PMN may have a role in modulating wound healing. GM-CSF may be useful for creating better wound healing healing. GM-CSF may be useful for creating better wound healing in risky patients such as diabetics.     

Impact of remote ischemic preconditioning on wound healing in small bowel anastomoses

AIM: To investigate the influence of remote ischemic preconditioning (RIPC) on anastomotic integrity. METHODS: Sixty male Wistar rats were randomized to six groups. The control group (n = 10) had an end-to-end ileal anastomosis without RIPC. The preconditioned groups (n = 34) varied in time of ischemia and time of reperfusion. One group received the amino acid L-arginine before constructing the anastomosis (n = 9). On postoperative day 4, the rats were re-laparotomized, and bursting pressure, hydroxyproline...     

Polyamines as an inhibitor on erythropoiesis of hemodialysis patients by in vitro bioassay using the fetal mouse liver assay

The pathogenesis of anemia in patients with chronic renal failure has been greatly attributed to erythropoietin (EPO) deficiency. Recently, however, there has been some thought that uremic inhibitors might suppress the activity of EPO and reduce the maturation of erythropoiesis. Polyamines are well known to be involved in the regulation of cellular proliferation and differentiation. Furthermore, the polyamine levels in the serum or erythrocytes are elevated in chronic hemodialysis patients, and can be lowered immediately by hemodialysis. In the present study, we first measured the polyamines levels (putrescine, spermidine, spermine) by high performance liquid chromatography (HPLC) in 20 chronic hemodialysis patients, and investigated the effects of polyamines on erythropoiesis by in vitro bioassay using fetal mouse liver cells. The direct effects of polyamines in erythroid colony formation in the medium with and without EPO were evaluated. Each polyamine level in chronic hemodialysis patients was higher than in the healthy subjects, and a significant negative correlation was found between polyamines and erythropoiesis. Polyamines inhibited the activity of EPO, but they did not have any direct effect on colony formation of the fetal mouse liver cells. These results suggest that polyamines have inhibitory effects on the proliferation or maturation of erythroid precursor cells and are intimately involved in the pathogenesis of renal anemia in chronic hemodialysis patients.     

Sugar alcohols enhance calcium transport from rat small and large intestine epithelium in vitro

We compared the effect of a variety of sugar alcohols on calcium absorption from the rat small and large intestine in vitro. An Ussing chamber technique was used to determine the net transport of Ca across the epithelium isolated from the jejunum, ileum, cecum, and colon of rats. The concentration of Ca in the serosal and mucosal Tris buffer solution was 1.25 mM and 10 mM, respectively. The Ca concentration in the serosal medium was determined after incubation for 30 min and the net Ca absorption was evaluated. The addition of 0.1–200 mM erythritol, xylitol, sorbitol, maltitol, palatinit, or lactitol to the mucosal medium affected net Ca absorption in the intestinal preparations. Differences in Ca transport were observed between portions of the intestine, but not between sugar alcohols tested. We concluded that sugar alcohols directly affect the epithelial tissue and promote Ca absorption from the small and large intestine in vitro.     

Developmental characteristics of adapting mouse small intestine crypt cells

BACKGROUND & AIMS: Following massive small bowel resection (SBR), the remnant intestine undergoes an adaptive process characterized by increases in a number of physiologic and morphologic parameters. These changes are the result of a stimulus that increases crypt cell mitosis and augments cellular progression along the villus axis. To better define this process, we identified patterns of gene expression specifically within adapting intestinal crypt cells following SBR. METHODS: Laser capture microdissection was used to isolate mouse intestinal crypt cells following SBR or sham operation. Multiple biological and technical complementary DNA microarray replicates allowed rigorous statistical analyses for identification of important expression profiles. Major groups of genes were classified as to site of action, functional pathway, and possible regulatory groups. RESULTS: A total of 300 genes differentially expressed at significant levels within adapting crypt enterocytes were analyzed. Comparison of this list of differentially expressed adapting crypt cell genes with a generalized mouse gene expression database (from 82 developing and adult mouse tissues) showed the greatest overlap with developing and immature intestinal tissues. We identified prominent groups of genes involved with cell growth, signal transduction, and nucleic acid binding. Genes not previously shown to be involved with adaptation or development and maturation were identified. CONCLUSIONS: Identification of similar genes coordinately regulated during both adaptation and development, processes that share key morphologic features, provides a basis for new mechanistic insights into these shared characteristics.     

Mesalamine promotes intestinal epithelial wound healing in vitro through a TGF-beta-independent mechanism

Objective. Treatment with 5-aminosalicylic acid (5-ASA) derivatives is one of the main principles in the therapy of uncomplicated mild to moderate inflammatory bowel diseases (IBD). The beneficial effect of 5-ASA in the treatment of IBD is attributed to its anti-inflammatory and anti-oxidant properties within the inflamed gut. The aim of this study was to investigate whether 5-ASA also modulates intestinal epithelial wound repair in vitro. Material and methods. The effects of 5-ASA on cell migration and proliferation, two key processes in mucosal healing, were studied in the non-transformed small-intestinal epithelial cell line IEC-6 using an in vitro wounding model and colorimetric MTT assays. Furthermore, the effects of 5-ASA on epithelial cell viability were determined by Trypan blue exclusion and flow cytometry-based cell cycle analysis. Results. Clinically relevant concentrations of 5-ASA caused a significant dose-dependent enhancement of epithelial cell migration and proliferation in vitro. An about 2-fold enhancement of intestinal epithelial cell proliferation and migration was observed for pharmacological doses of 100 µg/ml 5-ASA. Neutralizing antibodies against TGFβ did not modulate 5-ASA effects on IEC-6 cell proliferation and migration, indicating that the effects of 5-ASA were TGFβ independent. Trypan blue viability tests and cell cycle analysis did not reveal any toxic or apoptotic effects of pharmacological 5-ASA concentrations on IEC-6 cells. Conclusions. 5-ASA promotes the rapid re-establishment of mucosal integrity in vitro by enhancing epithelial restitution and proliferation, suggesting that 5-ASA in addition to the well-characterized effects on the intestinal inflammatory cascade may also directly stimulate epithelial wound healing.     

Glycoxidation and wound healing in diabetes: an interesting relationship

Impaired diabetic wound healing (WH) constitutes a serious diabetic complication with increased morbidity, mortality and health expenditure. The exact pathogenetic mechanisms have not been fully clarified. A variety of hyperglycemia and oxidative stress related factors, have been proposed, including advanced glycaction end products (AGE). The existing literature data, support the role of AGE in the pathogenesis of diabetic complications, namely micro- and macro- angiopathy which underlie delayed diabetic WH. In addition, a large body of evidence support a direct negative effect of AGE in the WH process by their interference with various components involved in the cascade following skin injury. Endogenously formed or exogenously derived AGE, in a similar manner, affect negatively the WH process in diabetes. It is obvious that further studies are needed to clarify the exact role of AGE in the impaired diabetic WH and offer possible new therapeutic strategies.     

Mesalamine promotes intestinal epithelial wound healing in vitro through a TGF-beta-independent mechanism

OBJECTIVE: Treatment with 5-aminosalicylic acid (5-ASA) derivatives is one of the main principles in the therapy of uncomplicated mild to moderate inflammatory bowel diseases (IBD). The beneficial effect of 5-ASA in the treatment of IBD is attributed to its anti-inflammatory and anti-oxidant properties within the inflamed gut. The aim of this study was to investigate whether 5-ASA also modulates intestinal epithelial wound repair in vitro. MATERIAL AND METHODS: The effects of 5-ASA on cell migration and proliferation, two key processes in mucosal healing, were studied in the non-transformed small-intestinal epithelial cell line IEC-6 using an in vitro wounding model and colorimetric MTT assays. Furthermore, the effects of 5-ASA on epithelial cell viability were determined by Trypan blue exclusion and flow cytometry-based cell cycle analysis. RESULTS: Clinically relevant concentrations of 5-ASA caused a significant dose-dependent enhancement of epithelial cell migration and proliferation in vitro. An about 2-fold enhancement of intestinal epithelial cell proliferation and migration was observed for pharmacological doses of 100 microg/ml 5-ASA. Neutralizing antibodies against TGFbeta did not modulate 5-ASA effects on IEC-6 cell proliferation and migration, indicating that the effects of 5-ASA were TGFbeta independent. Trypan blue viability tests and cell cycle analysis did not reveal any toxic or apoptotic effects of pharmacological 5-ASA concentrations on IEC-6 cells. CONCLUSIONS: 5-ASA promotes the rapid re-establishment of mucosal integrity in vitro by enhancing epithelial restitution and proliferation, suggesting that 5-ASA in addition to the well-characterized effects on the intestinal inflammatory cascade may also directly stimulate epithelial wound healing.     

COMP-angiopoietin-1 promotes wound healing through enhanced angiogenesis, lymphangiogenesis, and blood flow in a diabetic mouse model

Microvascular dysfunction is a major cause of impaired wound healing seen in diabetic patients. Therefore, reestablishment of structural and functional microvasculature could be beneficial to promote wound healing in these patients. Angiopoietin-1 (Ang1) is a specific growth factor functioning to generate a stable and functional vasculature through the Tie2 and Tie1 receptors. Here we determined the effectiveness of cartilage oligomeric matrix protein (COMP)-Ang1, a soluble, stable, and potent form of Ang1, on promotion of healing in cutaneous wounds of diabetic mice. An excisional full-thickness wound was made in the dorsal side of the tail of diabetic (db/db) mice, and mice were then treated systemically with adenovirus (Ade) encoding COMP-Ang1 or with control virus encoding beta-gal (Ade-beta-gal) or treated topically with recombinant COMP-Ang1 protein or BSA. Time course observations revealed that mice treated with Ade-COMP-Ang1 or COMP-Ang1 protein showed accelerated wound closure and epidermal and dermal regeneration, enhanced angiogenesis and lymphangiogenesis, and higher blood flow in the wound region compared with mice treated with control virus or BSA. COMP-Ang1 promotion of wound closure and angiogenesis was not dependent on endothelial nitric oxide synthase or inducible nitric oxide synthase alone. Taken together, these findings indicate that COMP-Ang1 can promote wound healing in diabetes through enhanced angiogenesis, lymphangiogenesis, and blood flow.     

In vitro osmoregulation of taurine in fetal mouse hearts

Regulation of taurine transport and accumulation in explanted fetal mouse hearts is shown to be under osmotic control. All osmotic agents studied, both ionic (NaCl, LiCl, choline Cl) and nonionic (sucrose, glucose) stimulated [3H]-taurine transport during an incubation of 19 h. Hyperosmotic stimulation of transport achieved statistical significance by 3 h in the presence of sucrose (P less than 0.05). After 1 h, 40 mM NaCl engendered a 56% increase in [3H]-taurine transport (P less than 0.01). The NaCl stimulation at 1 h may relate more to the transport system's absolute sodium ion requirement than hyperosmotic stimulation. Incremental addition of NaCl or sucrose linearly stimulates [3H]-taurine transport in an incubation of 19 h. Total taurine, measured by HPLC, increased 25% with addition of either 40 mM NaCl or 80 mM sucrose. Hyperosmotic stimulation of transport was not blocked with propranolol but was additive to beta-adrenergic stimulation of transport. Osmotic stimulation occurred with a large increase in Vmax (0.41----0.81 nmol/mg tissue/h) but only a small change in Km (0.51----0.43 mM). After 1 h preincubation with a hyperosmotic addition phenylalanine transport was measured, but was not different from control. Phenylalanine accumulation measured during 19 h incubation similarly was not altered. Streptozotocin induced diabetic rats had elevated plasma osmolarities (295 +/- 2.1----322 +/- 1.3 mosmol) and cardiac taurine (24.3 +/- 1.2----36 +/- 1.0 mumol/g wet wt.). The data presented demonstrates that mammalian cardiac taurine is regulated by the osmotic environment of the heart, suggesting an osmoregulatory function for intracellular taurine and physiological relevance in disease states such as diabetes.     

Mind-body techniques in wound healing

Even the most skilled and resourceful wound care provider encounters stagnation of wound healing from time to time. Patients with chronic, nonhealing wounds often display negative thought patterns and behavioral tendencies that, in turn, hinder biological and emotional healing. An increasing body of research supports the negative effect of stress on wound healing. The impact of deeper, emotion-based "wounds" as complicating factors in conventional wound healing are being further explored. It is theorized that emotions such as lack of self worth, guilt, and anger are strongly correlated to the chronic, nonhealing wound. Mind-body techniques such as affirmations, creative visualization, relaxation, and conscious breathing are suggested for incorporation into the treatment program. These techniques seek to empower and engage the patient by promoting greater personal awareness and assertion in the healing process. Wound care providers are seen as facilitators of the innate healing potential inside each individual. Mind-body techniques are offered as a complement for a more comprehensive wound healing strategy.     

Human intestine matures as nude mouse xenograft

This report describes a novel system for the study of the development and function of human intestine. Human fetal bowel transplanted into a subcutaneous tunnel on the back of athymic nude (nu/nu) mice develops a new microcirculation within 4 weeks. Tissues undergo morphological development, become similar to adult human bowel tissue, and may survive for 6 months after transplantation. Monoclonal antibody immunoperoxidase staining shows that the epithelial and some endothelial elements are of human phenotype, but the circulating blood cells and migrating mucosal lymphocytes are of mouse origin.     

Amines protect in vitro the celiac small intestine from the damaging activity of gliadin peptides

Proteins and peptides responsible for the celiac small intestinal lesion inhibit both the enterocyte recovery of in vitro cultured flat celiac mucosa and the in vitro development of fetal rat intestine. They also agglutinate K 562 (S) cells. Using these three in vitro systems (cultured human celiac and rat fetal intestine and cell agglutination), it is shown that several small-molecular-weight amines, mostly the polyamines spermidine and spermine, prevent and reverse K 562 (S) cell agglutination induced by gliadin peptides, whereas they do not prevent cell agglutination induced by concanavalin A and wheat germ agglutinin. Some of these amines also protected in vitro developing fetal rat intestine and flat celiac mucosa from the damaging effect of gliadin peptides. This protective effect may be related to the trophic activity exerted by amines on the intestine and/or the effect of amines on the functions of intestinal brush border or intracellular membranes involved in the intestinal handling of gliadins.     

Submucosa of canine small intestine as an alternative medium-diameter autogenous arterial graft.

BACKGROUND: Small intestinal canine submucosa has been used in previous studies as a large diameter arterial graft and has shown acceptable patency rates. The aim of our experimental study was to assess its effectiveness when it is used as an autogenous medium-sized diameter arterial graft (5-7 mm). METHODS: Fifteen mongrel dogs were included and underwent laparotomy under general anaesthesia. The mucosa, tunica muscularis and serosa were removed from a resected intestinal segment. The remaining tube, which consisted of the submucosa and the basilar tunica mucosa, represented the experimental graft which was used to replace a proportional gap of the canine infrarenal aorta. Ascertainment of peripheral pulses, measurement of the intra-aortic pressures, aortography and in vivo/in situ observation before the sacrifice of the animals, were the procedures used for verification of the graft's patency. RESULTS: The resistance to thrombogenicity of the graft was considered satisfactory: nine out of 10 grafts remained patent for postoperative intervals ranging from one day to one year; one graft showed partial obstruction due to a technical perioperative error. The grafts showed also excellent physical characteristics (ease of handling and suturing, blood impermeability and durability), resistance to infection and showed no tendency to develop myointimal hyperplasia. CONCLUSIONS: Small intestinal canine submucosa showed satisfactory haemodynamic properties, long-term patency and resistance to infection, when used as a medium-diameter arterial substitute.