Developing models of the small intestine

Inflammatory bowel disease (IBD) is a chronic autoimmune disease characterised by inflammation of the gastrointestinal tract. The pathogenesis of IBD is not fully understood and curative therapies are lacking. Consequently, development of robust intestine models, representative of the pathogenesis of IBD remains an unmet need. Thus, the overall aims of the studies presented in this thesis were to develop a number of models of small intestine including: genetically engineered murine model, epithelial cell culture models, and an intestinal stem cell organoid model which could reflect or be used to study the pathogenesis of IBD. Interleukin 1 (IL-1) is an important mediator of inflammation and tissue damage in IBD. The balance between IL-1 and IL-1Ra as a natural inhibitor plays a vital role in a variety of diseases. Here, this thesis investigated whether changes seen during IBD could be induced spontaneously by the removal of IL-1Ra in mice that lack a functional IL-1rn gene. Data presented from this thesis highlighted the importance of IL-1 in the pathogenesis of inflammatory bowel disease. In addition, the potential of L-pNIPAM hydrogel scaffolds, which were developed by the research team at Sheffield Hallam University, was utilised to develop long-term 3D co-cultures of layered Caco-2 and HT29-MTX cells under conditions representative of inflammation by treatment with IL-1β, TNFα, and hypoxia (1% O2) for 1 week was investigated. In vitro cell culture studies in this thesis have demonstrated that L-pNIPAM hydrogel supported long-term 3D co-culture model and stimulation with factors seen during inflammation recapitulated features of IBD. Finally, the potential of L-pNIPAM hydrogel scaffolds to develop 3D intestinal stem cell organoid model was investigated. The in vitro study demonstrated the ability of L-pNIPAM hydrogel as scaffold to support organoid formation and cell differentiation in vitro from small intestinal crypts and Lgr5+ stem cells isolated from mice

Long-term in vitro 3D hydrogel co-culture model of inflammatory bowel disease

The in vitro study of the pathogenesis of inflammatory bowel disease (IBD) requires a cell model which closely reflects the characteristics of the in vivo intestinal epithelium. This study aimed to investigate the application of L-pNIPAM hydrogel as a scaffold to develop a long-term 3D co-culture model of Caco-2 and HT29-MTX cells under conditions analogous to inflammation, to determine its potential use in studying IBD. Monocultures and co-cultures were layered on L-pNIPAM hydrogel scaffolds and maintained under dynamic culture conditions for up to 12 weeks. Treatments with IL-1β, TNFα, and hypoxia for 1 week were used to create an inflammatory environment. Following prolonged culture, the metabolic activity of Caco-2 monoculture and 90% Caco-2/10% HT29-MTX co-cultures on L-pNIPAM hydrogels were increased, and finger-like structures, similar in appearance to villi were observed. Following treatment with IL-1β, TNFα and hypoxia, ALP and ZO-1 were decreased, MUC2 increased, and MUC5AC remained unchanged. ADAMTS1 was increased in response to hypoxia. Caspase 3 expression was increased in response to TNFα and hypoxic conditions. In conclusion, L-pNIPAM hydrogel supported long-term co-culture within a 3D model. Furthermore, stimulation with factors seen during inflammation recapitulated features seen during IBD

Tissue engineering laboratory models of the small intestine.

In recent years, three-dimensional (3D) cell culture models of the small intestine have gained much attention. These models support cell proliferation, migration, and differentiation, and encourage tissue organization which is not possible in two-dimensional (2D) culture systems. Furthermore, the use of a wide variety of cell culture scaffolds and support substrates have revealed considerable differences in cell behavior and tissue organization. These systems have been used in combination with intestinal stem cells, organoid units or human colonic adenocarcinoma cell lines such as Caco-2 and HT29-MTX to generate a number of in vitro and in vivo models of the intestine. Here, we review the current 2D and 3D tissue engineering models of the intestine to determine the most effective sources of intestinal cells and current research on support scaffolds capable of inducing the morphological architecture and function of the intestinal mucosa

Use of hydrogel scaffolds to develop an in vitro 3D culture model of human intestinal epithelium

The human intestinal cell lines: Caco-2 and HT29-MTX cells have been used extensively in 2D and 3D cell cultures as simple models of the small intestinal epithelium in vitro. This study aimed to investigate the potential of three hydrogel scaffolds to support the 3D culture of Caco-2 and HT29-MTX cells and critically assess their use as scaffolds to stimulate villi formation to model a small intestinal epithelium in vitro. Here, alginate, l-pNIPAM, and l-pNIPAM-co-DMAc hydrogels were investigated. The cells were suspended within or layered on these hydrogels and maintained under static or dynamic culture conditions for up to 21days. Caco-2 cell viability was increased when layered on the synthetic hydrogel scaffolds, but reduced when suspended within the synthetic hydrogels. In contrast, HT29-MTX cells remained viable when suspended within or layered on all 3D cultures. Interestingly, cells cultured in and on the alginate hydrogel scaffolds formed multilayer spheroid structures, whilst the cells layered on synthetic hydrogels formed villus-like structures. Immunohistochemistry staining demonstrated positive expression of enterocyte differentiation markers and goblet cell marker. In conclusion, l-pNIPAM hydrogel scaffolds supported both cell lines and induced formation of villus-like structures when cells were layered on and cultured under dynamic conditions. The ability of the l-pNIPAM to recapitulate the 3D structure and differentiate main cell types of human intestinal villi may deliver a potential alternative in vitro model for studying intestinal disease and for drug testing. Forty percent of hospital referrals are linked to disorders of the digestive tract. Current studies have utilised animal models or simple cultures of isolated cells which do not behave in the same manner as human intestine. Thus new models are required which more closely mimic the behaviour of intestinal cells. Here, we tested a number of scaffolds and conditions to develop a cell culture model which closely represents the 3D environment seen within the human small intestine. We successfully created structures seen within the intestine which have not previously been possible with other culture models. These models could be used to investigate tissue engineering, drug discovery, and used asan alternative to in vivo animal models in drug toxicity studies. [Abstract copyright: Copyright © 2017. Published by Elsevier Ltd.

Histological evaluation of the effects of bone morphogenetic protein 9 and angiopoietin 1 on bone healing

Objectives: Bone healing remains a critical clinical orthopedic problem. Bone, which is a greatly vascularized tissue, depends on the tight temporal and spatial link between blood vessels and bone cells. Thus, angiogenesis is crucial for skeletal growth and bone fracture healing. The purpose of this study was to evaluate the efficacy of the local application of osteogenic and angiogenic factors such as bone morphogenetic protein 9 (BMP9) and angiopoietin 1 (Ang1), respectively, and their combination as an osteoinducer in the process of bone healing. Methods: Forty-eight male albino rats, weighing 300 e400 g and aged 6e8 months, were utilized in this study. The animals underwent surgery on the medial side of the tibia bone. In the control group, an absorbable hemostatic sponge was locally applied to the bone defect, while experimental groups were separated into three groups. In Group I, 1 mg BMP9 was locally applied, Group II was treated with 1 mg Ang1, and Group III was treated with local application of a combination (0.5 mg BMP9 and 0.5 mg Ang1). All experimental groups were fixed with an absorbable hemostatic sponge. The rats were sacrificed on days 14 and 28 after surgery. Results: Local application of BMP9 alone, Ang1 alone, and their combination to a tibia defect caused osteoid tissue formation and significantly increased the number of bone cells. A gradual decrease in the number of Q3 trabecular bone, an increase in trabecular area, and no significant difference in the bone marrow area were noted. Conclusion: The combination of BMP9 and Ang1 has therapeutic potential in promoting the healing process of bone defects. Osteogenesis and angiogenesis are regulated by BMP9 and Ang1. These factors act together to accelerate bone regeneration more efficiently than either factor alone

Histological evaluation of the effects of bone morphogenetic protein 9 and angiopoietin 1 on bone healing

Objectives: Bone healing remains a critical clinical orthopedic problem. Bone, which is a greatly vascularized tissue, depends on the tight temporal and spatial link between blood vessels and bone cells. Thus, angiogenesis is crucial for skeletal growth and bone fracture healing. The purpose of this study was to evaluate the efficacy of the local application of osteogenic and angiogenic factors such as bone morphogenetic protein 9 (BMP9) and angiopoietin 1 (Ang1), respectively, and their combination as an osteoinducer in the process of bone healing. Methods: Forty-eight male albino rats, weighing 300 e400 g and aged 6e8 months, were utilized in this study. The animals underwent surgery on the medial side of the tibia bone. In the control group, an absorbable hemostatic sponge was locally applied to the bone defect, while experimental groups were separated into three groups. In Group I, 1 mg BMP9 was locally applied, Group II was treated with 1 mg Ang1, and Group III was treated with local application of a combination (0.5 mg BMP9 and 0.5 mg Ang1). All experimental groups were fixed with an absorbable hemostatic sponge. The rats were sacrificed on days 14 and 28 after surgery. Results: Local application of BMP9 alone, Ang1 alone, and their combination to a tibia defect caused osteoid tissue formation and significantly increased the number of bone cells. A gradual decrease in the number of Q3 trabecular bone, an increase in trabecular area, and no significant difference in the bone marrow area were noted. Conclusion: The combination of BMP9 and Ang1 has therapeutic potential in promoting the healing process of bone defects. Osteogenesis and angiogenesis are regulated by BMP9 and Ang1. These factors act together to accelerate bone regeneration more efficiently than either factor alone

Use of l-pNIPAM hydrogel as a 3D-scaffold for intestinal crypts and stem cell tissue engineering

Intestinal stem cells hold great potential in tissue regeneration of the intestine, however, there are key limitations in their culture in vitro. We previously reported a novel synthetic non-biodegradable hydrogel as a 3D culture model for intestinal epithelium using Caco2 and HT29-MTX cells. Here, we investigated the potential of this system as a 3D scaffold for crypts and single intestinal stem cells to support long-term culture and differentiation. Intestinal crypts were extracted from murine small intestines and Lgr5+ stem cells isolated by magnetic activated cell sorting. Crypts and stem cells were suspended within Matrigel or L-pNIPAM for 14 days or suspended within Matrigel for 7 days then released, dissociated, and suspended within, or on L-pNIPAM hydrogel for 28 days. Cellular behaviour and phenotype were determined by histology and immunohistochemistry for stem cell and differentiation markers: Lgr5, E-cadherin MUC2 chromograninA and lysozymes. Isolated crypts and Lgr5+ intestinal stem cells formed enteroids with a central lumen surrounded by multiple crypt-like buds when cultured in Matrigel. In contrast, when crypts and stem cells were directly suspended within, or layered on L-pNIPAM hydrogel under dynamic culture conditions they formed spherical balls of cells, with no central lumen. When enteroids were initially formed in Matrigel from crypts or single Lgr5+ intestinal stem cells and dissociated into small fragments or single cells and transferred to L-pNIPAM hydrogel they formed new larger enteroids with numerous crypt-like buds. These crypt-like buds showed the presence of mucin-producing cells, which resembled goblet cells, scattered throughout their structures. Immunohistochemistry staining also showed the expression of Lgr5 and differentiation markers of all the main intestinal cell types including: enterocytes, goblet cells, enteroendocrine and Paneth cells. This demonstrated that L-pNIPAM hydrogel supported long-term culture of crypts and Lgr5+ stem cells and promoted intestinal cell differentiation

Studying Effects of Calcium Oxide Nanoparticles on Dentinogenesis in Male Wistar Rats

This study aimed to evaluate potential impacts of calcium oxide nanoparticles (CaO-NPs) at different dosages on predentin thickness, number of blood vessels, periodontal ligament thickness, and blood glucose level of Wistar rats. Twelve rats were randomly gathered into four groups, untreated (control) and CaO-NP-treated groups at three concentrations (25, 50, and 100 mg/kg of the body weight) over a period of 60 days. Histological investigation was performed on twenty-four lower incisor teeth extracted from all the tested groups under a light microscope, and an automatic Fujifilm was used to measure the blood glucose level. The results showed that regular nanoparticle treatment significantly increased predentin and periodontal ligament thicknesses, a gradual decrease in vascularization in the pulp tissue, and an increase in the blood glucose level as the dosages of nanoparticles administered to the rats increased. Administration of the CaO-NPs at low dosage (25 mg/kg) could be beneficial for the growth and integrity of teeth and dentinal tissues in rats

Studying Effects of Calcium Oxide Nanoparticles on Dentinogenesis in Male Wistar Rats

From Hindawi via Jisc Publications RouterHistory: publication-year 2021, received 2021-03-27, accepted 2021-07-18, pub-print 2021-07-26, archival-date 2021-07-26Publication status: PublishedThis study aimed to evaluate potential impacts of calcium oxide nanoparticles (CaO-NPs) at different dosages on predentin thickness, number of blood vessels, periodontal ligament thickness, and blood glucose level of Wistar rats. Twelve rats were randomly gathered into four groups, untreated (control) and CaO-NP-treated groups at three concentrations (25, 50, and 100 mg/kg of the body weight) over a period of 60 days. Histological investigation was performed on twenty-four lower incisor teeth extracted from all the tested groups under a light microscope, and an automatic Fujifilm was used to measure the blood glucose level. The results showed that regular nanoparticle treatment significantly increased predentin and periodontal ligament thicknesses, a gradual decrease in vascularization in the pulp tissue, and an increase in the blood glucose level as the dosages of nanoparticles administered to the rats increased. Administration of the CaO-NPs at low dosage (25 mg/kg) could be beneficial for the growth and integrity of teeth and dentinal tissues in rats

Cross-Linguistic Interference in the Syntactic and Semantic Acquisition of Arabic Language

The present study aims to investigate cross-linguistic interference in the syntactic and semantic acquisition of Arabic. An essay writing test is used to collect data. A total of 30 English and Spanish speakers of Arabic participates in this quantitative study. The participants are selected from three language centers in Jordan via convenient sampling procedures. The analysis of data reveals the following main findings: L1 transfer errors made by English and Spanish speakers of Arabic at the syntactic level account for 45% and 22.1%, respectively, while the percentage of intralingual errors made by English and Spanish speakers of Arabic at syntactic level account for 55%, and 77.9% respectively. On the other hand, the findings demonstrate that the percentage of L1 transfer errors made by English and Spanish speakers of Arabic at the semantic level are 34.85% and 14.3%, respectively, while the total percentage of intralingual errors made by English and Spanish speakers of Arabic at semantic level account for 65.15%, and 85.7 % respectively. The primary source of errors made by English and Spanish speakers of Arabic at both syntactic and semantic levels is intralingual interference