Biodegradable and injectable cure-on-demand polyurethane scaffolds for regeneration of articular cartilage.

This paper describes the synthesis and characterization of an injectable methacrylate functionalized urethane-based photopolymerizable prepolymer to form biodegradable hydrogels. The tetramethacrylate prepolymer was based on the reaction between two synthesized compounds, diisocyanato poly(ethylene glycol) and monohydroxy dimethacrylate poly(epsilon-caprolactone) triol. The final prepolymer was hydrated with phosphate-buffered saline (pH 7.4) to yield a biocompatible hydrogel containing up to 86% water. The methacrylate functionalized prepolymer was polymerized using blue light (450 nm) with an initiator, camphorquinone and a photosensitizer, N,N-dimethylaminoethyl methacrylate. The polymer was stable in vitro in culture media over the 28 days tested (1.9% mass loss); in the presence of lipase, around 56% mass loss occurred over the 28 days in vitro. Very little degradation occurred in vivo in rats over the same time period. The polymer was well tolerated with very little capsule formation and a moderate host tissue response. Human chondrocytes, seeded onto Cultispher-S beads, were viable in the tetramethacrylate prepolymer and remained viable during and after polymerization. Chondrocyte-bead-polymer constructs were maintained in static and spinner culture for 8 weeks. During this time, cells remained viable, proliferated and migrated from the beads through the polymer towards the edge of the polymer. New extracellular matrix (ECM) was visualized with Masson's trichrome (collagen) and Alcian blue (glycosaminoglycan) staining. Further, the composition of the ECM was typical for articular cartilage with prominent collagen type II and type VI and moderate keratin sulphate, particularly for tissue constructs cultured under dynamic conditions.

Evaluation of photo-crosslinked fibrinogen as a rapid and strong tissue adhesive.

Tissue adhesives and sealants are commonly used in surgery either as an adjunct to, or replacement for, sutures. Previously, we have shown that fibrinogen can be crosslinked rapidly to give a high-strength bond in the presence of a ruthenium(II) complex, a persulfate and irradiation with visible light, and that the crosslinked fibrinogen is nontoxic to cells in vitro. This approach addresses limitations to current fibrin sealants that typically have relatively slow curing times and low bond strengths. In the present study, we have evaluated the efficacy and safety of this new biological scaffold sealant in various animal models. When placed as solid implants into rats, the crosslinked fibrinogen persisted for at least 8 weeks but was fully resorbed by 18 weeks with minimal inflammatory responses. When used as a tissue adhesive for repair of skin incisions in rats or as an arterial haemostat in pig, the photo-crosslinked fibrinogen sealed tissue or arrested bleeding within 20 s of application. For the skin incisions, the fibrinogen sealant promoted rapid tissue vascularization and cellular infiltration with no adverse foreign body cell generation. New collagen deposition occurred and with time the matrix had remodelled to acquire large mature collagen fiber bundles which were accompanied by maximum regenerated tensile strength. This biomaterial system may find useful applications in surgical procedures where rapid curing and/or high strength tissue sealing is required.

Rapid onset of ulcerative typhlocolitis in B6.129P2-IL10tm1Cgn (IL-10-/-) mice infected with Helicobacter trogontum is associated with decreased colonization by altered Schaedler's flora.

Infection with Helicobacter trogontum, a urease-positive helicobacter isolated from subclinically infected rats, was evaluated in B6.129P2-IL10(tm1Cgn) (interleukin-10(-/-) [IL-10(-/-)]) and C57BL/6 (B6) mice. In a first experiment, IL-10(-/-) mice naturally infected with Helicobacter rodentium had subclinical typhlocolitis but developed severe diarrhea and loss of body condition with erosive to ulcerative typhlocolitis within 1 to 3 weeks of experimental infection with H. trogontum. A second experiment demonstrated that helicobacter-free IL-10(-/-) mice dosed with H. trogontum also developed severe clinical signs and typhlocolitis within 2 to 4 weeks, whereas B6 mice colonized with H. trogontum were resistant to disease. In a third experiment, using helicobacter-free IL-10(-/-) mice, dosing with H. trogontum resulted in acute morbidity and typhlocolitis within 8 days. Acute typhlocolitis was accompanied by signs of sepsis supported by degenerative hemograms and recovery of Escherichia coli and Proteus spp. from the livers of infected mice. Quantitative PCR data revealed that H. rodentium and H. trogontum may compete for colonization of the lower bowel, as H. trogontum established higher colonization levels in the absence of H. rodentium (P < 0.003). H. trogontum-induced typhlocolitis was also associated with a significant decrease in the levels of colonization by five of eight anaerobes that comprise altered Schaedler's flora (P < 0.002). These results demonstrate for the first time that H. rodentium infection in IL-10(-/-) mice causes subclinical typhlocolitis and that infection with H. trogontum (with or without H. rodentium) induces a rapid-onset, erosive to ulcerative typhlocolitis which impacts the normal anaerobic flora of the colon and increases the risk of sepsis.

Post-immunisation gastritis and Helicobacter infection in the mouse: a long term study.

BACKGROUND AND AIMS: Helicobacter pylori is a major cause of peptic ulcers and gastric cancer. Vaccine development is progressing but there is concern that immunisation may exacerbate Helicobacter induced gastritis: prophylactic immunisation followed by challenge with H felis or H pylori can induce a more severe gastritis in mice than seen with infection alone. The aim of this study was to investigate the relationship between immunity to Helicobacter infection and post-immunisation gastritis. METHODS: (1) C57BL/6 mice were prophylactically immunised before challenge with either H felis or H pylori. Histopathology and colonisation were assessed one month post-challenge. (2) C57BL/6 mice were prophylactically immunised against H felis infection and gastritis assessed up to 18 months post-challenge. RESULTS: Prophylactic immunisation induced a reduction in bacterial colonisation following H felis challenge which was associated with increased severity of active gastritis with neutrophil infiltration and atrophy. However, immunised mice challenged with H pylori SS1 had little evidence of pathology. Long term follow up showed that post-immunisation gastritis was evident at three months. However, from six months onwards, although immunised/challenged mice still developed gastritis, there was no significant difference between inflammation in these mice and infected controls. Post-immunisation gastritis was not associated with the serum antibody response. Immunisation prevented the formation of secondary lymphoid aggregates in the gastric tissue. CONCLUSION: The H felis mouse model of post-immunisation gastritis is the most extreme example of this type of pathology. We have shown in this model that post-immunisation gastritis is a transient event which does not produce long term exacerbation of pathology.

Role of Helicobacter pylori cag region genes in colonization and gastritis in two animal models.

The Helicobacter pylori chromosomal region known as the cytotoxin-gene associated pathogenicity island (cag PAI) is associated with severe disease and encodes proteins that are believed to induce interleukin (IL-8) secretion by cultured epithelial cells. The objective of this study was to evaluate the relationship between the cag PAI, induction of IL-8, and induction of neutrophilic gastric inflammation. Germ-free neonatal piglets and conventional C57BL/6 mice were given wild-type or cag deficient mutant derivatives of H. pylori strain 26695 or SS1. Bacterial colonization was determined by plate count, gastritis and neutrophilic inflammation were quantified, and IL-8 induction in AGS cells was determined by enzyme-linked immunosorbent assay. Deletion of the entire cag region or interruption of the virB10 or virB11 homolog had no effect on bacterial colonization, gastritis, or neutrophilic inflammation. In contrast, these mutations had variable effects on IL-8 induction, depending on the H. pylori strain. In the piglet-adapated strain 26695, which induced IL-8 secretion by AGS cells, deletion of the cag PAI decreased induction. In the mouse-adapted strain SS1, which did not induce IL-8 secretion, deletion of the cagII region or interruption of any of three cag region genes increased IL-8 induction. These results indicate that in mice and piglets (i) neither the cag PAI nor the ability to induce IL-8 in vitro is essential for colonization or neutrophilic inflammation and (ii) there is no direct relationship between the presence of the cag PAI, IL-8 induction, and neutrophilic gastritis.

Comparison of gastritis and gastric epithelial proliferation in Helicobacter heilmannii-infected nude and BALB/c mice.

Gastric mucosal hypertrophy/nodular hyperplasia occurs as a consequence of Helicobacter infection in mice and humans. The pathogenesis of this hyperplastic response is not understood. To determine the role of host cellular immunity in gastric mucosal hypertrophy/hyperplasia, 6-8-week-old female euthymic BALB/c (n = 30) or NIH athymic nude (n = 40) mice were inoculated with H. heilmannii. Equal numbers of uninoculated mice of each strain served as controls. Mice from each group were euthanatized at 0.5, 6, 12, and 18 months postinoculation (PI). Lymphoplasmacytic gastritis and lymphoid follicle development were less severe in nude mice than in euthymic mice at <6 months PI. The prevalence of gastritis at 0.5, 6, 12, and 18 months PI was 0%, 17%, 67%, and 88%, respectively, in infected nude mice and 33%, 83%, 71%, and 100%, respectively, in infected BALB/c mice. CD4+ T cells in infected nude mice were evident at > or =6 months PI but were less numerous than in infected BALB/c mice at comparable time intervals. However, numbers of CD4+ T cells increased substantially throughout the experiment in infected BALB/c mice. The prevalence of nodular mucosal hyperplasia at 0.5, 6, 12, and 18 months PI was 0%, 0%, 33%, and 20%, respectively, in infected nude mice and 0%, 33%, 80%, and 80%, respectively, in infected BALB/c mice. Nodular hyperplasia occurred in association with the appearance of chronic lymphoplasmacytic inflammation and CD4+ T cells at 12 and 18 months PI in nude mice. H. heilmannii-associated gastritis and mucosal remodeling is reduced in immunodeficient mouse strains lacking normal CD4+ T cell numbers as compared with the response in immunocompetent mice. Additionally, CD4 immunocompetence is an integral aspect of the mucosal hypertrophy/nodular hyperplasia in experimental H. heilmannii-associated disease of mice.

Murine splenocytes induce severe gastritis and delayed-type hypersensitivity and suppress bacterial colonization in Helicobacter pylori-infected SCID mice.

The goal of this study was to evaluate the role of host immunity in gastritis and epithelial damage due to Helicobacter pylori. Splenocytes from H. pylori-infected and uninfected C57BL/6 mice were adoptively transferred to H. pylori-infected and uninfected severe combined immunodeficient (SCID) mice. Transfer was verified by flow cytometry, and all mice were evaluated for the presence of delayed-type hypersensitivity (DTH) by footpad inoculation with sterile H. pylori sonicate and for humoral immunity by enzyme-linked immunosorbent assay. The severity of gastritis and gastric epithelial damage was quantified histologically, epithelial proliferation was determined by proliferating cell nuclear antigen staining, and colonization was quantified by culture. C57BL/6 mice, but not nonrecipient SCID mice, developed moderate gastritis in response to H. pylori. In contrast, recipient SCID mice developed severe gastritis involving 50 to 100% of the gastric mucosa and strong DTH responses not present in C57BL/6 mice. DTH, but not serum anti-H. pylori immunoglobulin G, correlated with adoptive transfer, gastritis, and bacterial clearance. Severe gastritis, but not bacterial colonization, was associated with epithelial metaplasia, erosions, and an elevated labeling index. This study demonstrates that (i) adaptive immunity is essential for development of gastritis due to H. pylori in mice, (ii) T-cell-enriched lymphocytes in SCID mice induce DTH and gastritis, which is more severe than donor gastritis, and (iii) the host inflammatory response, not direct bacterial contact, causes epithelial damage. The greater severity of gastritis in recipient SCID mice than in donor C57BL/6 mice suggests that gastritis is due to specific T-cell subsets and/or the absence of regulatory cell subsets in the transferred splenocytes.

RFLP and RAPD analysis of in vivo genetic interactions between strains of Helicobacter pylori.

BACKGROUND: The nature of the in vivo interactions between co-colonizing strains of Helicobacter pylori has not been well studied. This study was designed to investigate the in vivo interaction between strains of H. pylori using restriction fragment length polymorphism analysis (RFLP) and random amplified polymorphic DNA fingerprinting (RAPD) analysis. MATERIALS AND METHODS: Mice were orally inoculated with either a single strain or a mixture of strains, and killed at various time intervals up to 12 months after inoculation. RESULTS: Inoculum and recovered bacteria were identified by RAPD and RFLP. After 12 months of colonization, bacteria recovered from mice given a single strain were identical to the inoculum. In contrast, bacteria recovered from mice given a mixture of strains developed minor but consistent differences in their RAPD patterns. This variation occurred in spite of the fact that only single strains were recovered from mice given a mixture of strains. CONCLUSION: These findings support the development of genetic heterogeneity of H. pylori during long-term colonization either by genetic drift or by in vivo recombination. Such genomic modification in vivo could explain the naturally occurring heterogeneity of H. pylori.

The role of gastric Helicobacter and N-methyl-N'-nitro- N-nitrosoguanidine in carcinogenesis of mice.

BACKGROUND: The aim of this study was to determine whether gastric epithelial proliferation due to gastric Helicobacter infection in mice represents a preneoplastic lesion. MATERIALS AND METHODS: Helicobacter heilmannii infected and uninfected mice were treated with 150 microg/ml N-methyl-N'-nitro-N-nitrosoguanidine in drinking water for either 20 or 38 weeks. Mice were killed 12 or 18 months after bacterial inoculation. RESULTS: All infected mice developed lymphoplasmocytic gastritis and epithelial hyperplasia. Proliferative gastric lesions were characterised by nodular hypertrophy of the glandular mucosa, multifocal epithelial hyperplasia, and elevated BrdU labeling index. Intestinal metaplasia and true atrophy were not present but proliferative glands were poorly differentiated, lined by mucus-type epithelial cells with no parietal, chief or other specialized cell types. Neoplasms developed only in MNNG-treated mice. of the 180 treated mice the following neoplasms developed: 14 squamous cell carcinomas of mouth and forestomach; 37 hemangiosarcomas of the intestinal serosa; and 15 splenic lymphomas. No tumors were present in the glandular gastric mucosa, and infection did not affect tumor incidence. p53 overexpression occurred in 79% of hemangiosarcomas and 71% of squamous cell carcinomas but not in normal or proliferative gastric glandular mucosa. CONCLUSIONS: Gastric proliferative lesions in Helicobacter-infected mice are not preneoplastic, and the combination of an alkylating agent and non-neoplastic proliferation does not result in gastric carcinogenesis in mice.

Gastrin release and gastric acid secretion in the rat infected with either Helicobacter felis or Helicobacter heilmannii.

Helicobacter pylori infection in humans has been shown to be associated with changes in gastric physiology, including exaggerated basal and meal-stimulated gastrin levels. This has been suggested to be due to the direct effects of the bacterium through inflammation and its urease enzyme. The gastric bacteria Helicobacter felis and Helicobacter heilmannii colonize the antrum of rats in large numbers and induce no significant inflammatory response. Thus, the direct effect of Helicobacter infection on gastric physiology, independent of gastritis, could be studied. Basal, freely fed and stimulated acid and gastrin levels were recorded from animals infected with H. felis, H. heilmannii or uninfected controls over a 30 week period. No significant difference was found between freely fed gastrin over 7 weeks or fasting gastrin over 24 weeks or basal and stimulated acid over 30 weeks between all three groups. Triple therapy did not alter gastrin or acid output. The antrum of all Helicobacter-infected rats was well colonized; triple therapy cleared H. felis but not H. heilmannii. Very little inflammation was seen in control or Helicobacter-infected animals. In conclusion, Helicobacter-induced effects on gastric physiology are unlikely to be due to direct bacterial effects, but are best explained by other factors (i.e. inflammatory damage).

The importance of local acid production in the distribution of Helicobacter felis in the mouse stomach.

BACKGROUND/AIMS: Helicobacter felis colonizes the gastric mucosa of rodents. Preliminary studies showed differences in the distribution of the organism in different parts of the stomach that seemed related to the secretory capacity of the mucosa. The aim of this study was to determine the localization of H. felis in the mouse stomach and to investigate the influence of acid-suppressive agents. METHODS: Specific-pathogen-free BALB/c mice were infected with H. felis. Colonization was assessed in longitudinal sections of gastric tissue from animals untreated or treated with omeprazole or ranitidine. RESULTS: In untreated H. felis-infected animals, the preferred ecological niche was the antrum and cardia equivalent. The density of colonization correlated with the number of parietal cells per gland. Partial acid suppression with ranitidine produced a slight increase in the colonization of the body but was restricted to the upper portions of the gastric gland. Omeprazole treatment produced a greater colonization of the body with bacteria traversing the entire gland. Some reduction in antral colonization occurred. CONCLUSIONS: These results are consistent with the hypothesis that local acid output is a crucial determinant in the distribution of Helicobacter species in the stomach. Differences in local acid output may explain the different patterns of Helicobacter pylori-induced gastric pathology.

Local acid production and Helicobacter pylori: a unifying hypothesis of gastroduodenal disease.

We aimed to produce a unifying hypothesis to explain the different locations of peptic ulcer and gastritis observed in different populations. The pre-Helicobacter pylori literature on patterns of gastroduodenal disease was reviewed and compared with recent human and animal findings on H. pylori infection. Early observations revealed that duodenal and non-pre-pyloric ulcers tend to be mutually exclusive. In duodenal ulcer patients, gastritis is usually restricted to the antrum, while gastric ulcer patients experience more severe pangastritis. The manipulation of acid output by surgery or acid suppressive therapy alters the distribution of gastritis. Recent experimental evidence in humans and animals has shown that these changes parallel changes in the distribution and cellular responses to H. pylori infection. We propose that the most important factor in the ecology of the H. pylori-infected stomach is local acid production. Local acid production determines the location and severity of inflammation and the clinical outcome of this bacterial infection. Priority research areas should be the investigation of the in vivo behaviour of H. pylori in the acid and the non-acid producing areas of the stomach and the measurement of acid output in populations known to have different patterns of gastroduodenal disease.