Browsing by Subject "collagen"
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Item Advanced Problem Solving in the Biotherapeutics Industry: Parameters influencing the delivery of a novel cell therapy product and exploration of a new method for determining activity of Clostridium histolyticum collagenase, a wound debridement enzyme(2015-05-01) Harris, Melanie A.; Jerry W. Simecka; Patricia A. GwirtzBiotechnology is a multi-faceted industry with many unique challenges that require knowledge in a broad range of topics. When working in the wound care field it is necessary to not only create a product in the laboratory, but also effectively bring it to the patient. This task requires many skilled people who can test it for efficacy, design and conduct clinical trials, confirm quality and consistency, design packaging, consider transportation issues and so on. The following investigation focuses on the testing of a cellular product and its accompanying device under various conditions as well as the exploration of a new assay capable of the activity of a wound debridement enzyme. The results of the product/device testing have generally confirmed the comparability of the cellular product devices as well as their resistance to various temperatures encountered in the clinical environment. A new modified assay for the testing of collagenase has been established as precise and comparable to current methods, though it requires more testing to confirm robustness.Item Arginine Supplementation Promotes Extracellular Matrix and Metabolic Changes in Keratoconus(MDPI, 2021-08-13) McKay, Tina B.; Priyadarsini, Shrestha; Rowsey, Tyler; Karamichos, DimitriosKeratoconus (KC) is a common corneal ectatic disease that affects 1:500-1:2000 people worldwide and is associated with a progressive thinning of the corneal stroma that may lead to severe astigmatism and visual deficits. Riboflavin-mediated collagen crosslinking currently remains the only approved treatment to halt progressive corneal thinning associated with KC by improving the biomechanical properties of the stroma. Treatments designed to increase collagen deposition by resident corneal stromal keratocytes remain elusive. In this study, we evaluated the effects of arginine supplementation on steady-state levels of arginine and arginine-related metabolites (e.g., ornithine, proline, hydroxyproline, spermidine, and putrescine) and collagen protein expression by primary human corneal fibroblasts isolated from KC and non-KC (healthy) corneas and cultured in an established 3D in vitro model. We identified lower cytoplasmic arginine and spermidine levels in KC-derived constructs compared to healthy controls, which corresponded with overall higher gene expression of arginase. Arginine supplementation led to a robust increase in cytoplasmic arginine, ornithine, and spermidine levels in controls only and a significant increase in collagen type I secretion in KC-derived constructs. Further studies evaluating safety and efficacy of arginine supplementation are required to elucidate the potential therapeutic applications of modulating collagen deposition in the context of KC.Item Dental Tissue Changes in Juvenile and Young Adult Mice with Osteogenesis Imperfecta(2022-05) Moore, Jacob C.; Handler, Emma; Menegaz, Rachel A.; Gonzales, Lauren A.Osteogenesis imperfecta (OI) encompasses a heterogeneous family of heritable connective tissue disorders characterized by insufficient or malformed type I collagen protein causing bone fragility, skeletal deformity, and significant dental issues. The most prominent oral characteristic of OI patients, dentinogenesis imperfecta (DI), is characterized by dentition with significant discoloration and structural defects. During normal dental development, specialized cells secrete layers of collagen-rich matrix, which are then mineralized to form the two hard tissues of the tooth – the enamel, the protective tissue that forms the crown of the tooth, and the dentin, which sits internal to the enamel and forms the bulk of the tooth. Importantly, the matrix on which dentin forms is primarily composed of type I collagen. In DI, the secretion of malformed type I collagen in the developing dentin matrix disrupts the normal regulation and organization of this process, causing issues such as hypomineralization, disorganized dentin tubule structure, and dentin hypertrophy. These abnormal structural properties result in the disease phenotype of DI, including discoloration, enamel attrition, and spontaneous dental fractures. This practicum aims to investigate the dental effects of OI by comparing mineralized dental tissue volumes of mice with a type I collagen mutation with wild-type mice with littermates at the juvenile and adult life stages. The animal model under study, the oim mouse (B6C3FE a/a-Col1a2OIM/J), produces abnormal type I collagen due to a mutation in the COL1A2 gene. Mice that are homozygous for this mutation demonstrate a severe OI phenotype, while heterozygotes demonstrate a mild OI phenotype. Prior studies demonstrate that adult oim mice have dental issues similar to those of humans with OI, including reduced dentin tubule density and dentin cross-sectional area. However, the effects of these mutations on dental tissues across the juvenile and young adult periods have not yet been characterized.Item Development of an Osteoinductive Bone Graft(2010-12-01) Sule, Anupam A.; Slobodan Dan DimitrijevichBone is a unique tissue that serves multiple functions. One of its unique features is the ability to heal by formation of new bone, whereas most other tissues undergo the process of scar formation. When a large amount of bone is lost the only treatment available is the use of bone grafts. Multiple bone graft substitutes are being developed to address the shortage of autologous bone graft. 3-D models are being developed to further our understanding of the cellular processes taking place in vivo. In this study I examined the strategy of designing a 3-Dmodel of hard tissue and a potential bone graft substitute using collagen type I and several different porous scaffolds. Factors influencing collagen gel contraction by human mesenchymal stem cells (hMSC) during the process of osteogenic differentiation were studied and it was shown that collagen type I gels prepared in accordance with our patented technology contract far less than any other collagen gels reported in literature. The validity of MTT assay to track proliferation of hMSC in various 3-D matrices was established and allowed me to show that human mesenchymal stem cells (hMSC) proliferated, differentiated along an osteogenic lineage and mineralized the extracellular matrix (ECM). Higher cell seeding density and greater serum concentration in the culture medium, caused increased collagen type I gel contraction. Late passage cells and osteoblasts caused a greater collagen type I gel contraction than undifferentiated early passage hMSC. hMSC that had been transduced to constitutively express human telomerase reverse transcriptase (hTERT), and which had thereby acquired an extended in vitro life span (telomerized hMSC or TMSC), contracted the collagen gel lesser than hMSC. A Collagen type I Gel - Collagen type I foam Scaffold combination (CGCS) was investigated as a 3-D in vitro model to allow extrapolation of soft tissue results to those characteristic of hard tissue. Deep penetration of MSC into the CGCS with uniform distribution was achieved by the use of collagen type I gel, as the cell carrier. Collagen type I gel improved seeding efficiency and facilitated retention of cells that penetrated deep into the scaffold. Longterm survival, proliferation, viability and in situ osteogenic differentiation within the CGCS were demonstrated. A model that demonstrated migration of cells in and out of CGCS was assembled and tested. A need for the presence of fibrillar collagen gel for mineralization process to take place highlighted the benefit of adding collagen gel to the 3-D models. Porous Beta-tricalcium phosphate (-TCP) was used as the scaffold and impregnated with collagen gel to generate Collagen Gel Impregnated Porous Scaffolds (CGIPS). Highly efficient seeding of the cells throughout the porous scaffold was attained with collagen gel. hMSC proliferated in CGIPS without contracting the collagen gel. Cells could migrate into CGIPS and mineralized the ECM when cultured in vitro under osteogenic differentiation conditions. CGIPS allowed the application of pressure and hMSC responded to mechanical force by a change in proliferation. hMSC xenotransplanted into immunocompetent rats survived for a month and expressed markers of osteogenic differentiation. While cells alone improved vascularization of the implants, they did not improve mineralization. Presence of collagen gel alone allowed for faster invasion of cells into the implanted TCP and improved radiodensity but did not affect vascularization. A combination of cell and gel within the TCP (CGIPS) was necessary to improve all the measured varialbes (tissue invasion, vascularization, mineralization and radioopacity). Thus biocompatibility, greater vascularization and enhanced mineralization of CGIPS implants established the foundation to proceed with large animal bone defect model studies utilizing CGIPS in the future. I established that CGIPS could deliver small molecules into the surrounding milieu by a process of simple diffusion. A rapid intital burst followed by a slow sustained release was observed when collagen gel containing EphrinB2-Fc clusters was incorporated ointo CGIPS. The released EphrinB2-Fc was physiologically functional and increased hMSC proliferation and chemotaxis. CGIPS inhibited the growth of Methicillin resistant Staphylococcus aureus when vancomycin was incorporated into the CGIPS. Thus the potential of CGIPS to serve as a drug delivery device was demonstrated. This work has provided the scientific foundation for use of CGIPS as bone graft substitute and 3-D model of osteogenesis. In this research study, a number of challenges were solved and questions answered, and the applications of the proposed strategy formulated. However, as is frequently the case many more avenues of future research have been exposed and a variety of new questions posed to be pursued and answered in future.studies.Item Histological Investigation of Human Glaucomatous Eyes: Extracellular Fibrotic Changes and Galectin3 Expression in the Trabecular Meshwork and Optic Nerve Head(2018-05) Belmares, Ricardo; Clark, Abbot F.; Rosales, Armando; Lovely, Rehana S.; Reeves, Rustin E.; Jung, Marianna; Yao, HaiGlaucoma is a leading cause of vision loss and is associated with fibrotic changes in two ocular tissues: the optic nerve head (ONH) and trabecular meshwork (TM). We investigated the differences of extracellular components of the two ocular tissues in human glaucomatous eyes to determine fibrotic changes. The extracellular components studied included: collagen, elastin, Galectin 3 (Gal3), and Transforming Growth Factor beta-2 Type II receptor (TGFβ-2 RII). We hypothesized that these components will be increased in glaucomatous eyes using chemical staining and immunohistochemistry. Chemical staining included: Masson's Trichrome and Sirius Red stains (collagen) and Vernhoeff-Van Giesen (elastin). Immunohistochemistry was used to determine expression of Gal3 and TGFβ-2 RII. Data was analyzed using Image J software to quantify expression of the extracellular components. The results from Image J analysis of extracellular components demonstrated an overall increase in glaucomatous tissue. TM studies showed an increase of collagen (P=0.0469), and Gal3 (P [less than] 0.0001), and TGFβ-2 RII (P=0.0005) in glaucomatous eyes. Collagen was apparently increased in ONH ((P=0.0517) and Galectin3 (P=0.041) was increased in myelin transition zone of the glaucomatous optic nerve. Vernhoeff-Van Giesen stain showed increased thickness and irregular arrangement of elastic fibers in ONH. Vernoeff-van Giesen and Sirius Red stains also showed increased staining in glaucomatous tissue, but were not quantifiable with Image J software. Analysis showed a correlation of TGFβ-2 RII with Gal3 in TM (P [less than ]0.0001) and myelin transition zone of optic nerve (P=0.0003). Analysis of extracellular components of TM and ONH showed that glaucomatous eyes demonstrate a fibrotic state. Increased collagen deposition and thickened elastic fibers are recognized features of fibrosis and both TM and ONH revealed these features through chemical staining. Galectin3, another known marker for fibrosis, was also elevated in TM and optic nerve. Moreover, Gal3 co-localization with TGFβ-2 RII suggests that it may be involved with the pro-fibrotic TGFβ-2 signaling pathway.Item Osteogenesis Imperfecta: An analysis of the inner ear development in Mus musculus (house mouse) with comments on hearing quality(2022-05) Huston, Lila A.; Gonzales, Lauren A.; Handler, Emma; Menegaz, Rachel A.; Millar, J. CameronOsteogenesis imperfecta (OI), a developmental disorder of type I collagen, is known to cause hearing loss in ~ 60% of the diseased population. Identified forms include conductive hearing loss (17.4% of OI patients), involving loss of function within the ossicular chain, and sensorineural hearing loss (25.8%), resulting from damage to the cochlea, with the most predominant form being mixed hearing loss (56.8%), involving damage to both the cochlea and ossicles. While OI-related pathologies have largely focused on the middle ear, the pathological appearance of the cochlea (the organ most often compromised in OI-related hearing loss) has gained little focus. In this study, we examine OI-related pathologies on the cochlea in a mouse model for the severe type III OI, to document 1) the morphological differences in the inner ear for adult wildtype mice compared to OI mice in order to determine the anatomy of the diseased state, and 2) intraindividual variation between cochlea of WT and OI mice to determine potential asymmetry in the etiology of the inner ear. We hypothesize that cochlea in mice with OI will have less consistent morphology overall than their WT counterparts due to abnormal growth of the bony capsule. 4 week and 16 week old OIM mice (B6C3Fe a/a-Col1a2oim/J) (n=25) were compared to unaffected wildtype (WT) littermates (n=29) with no known hearing defects. High-resolution micro-CT scans were created for all specimens and 3D models and volumes of the cochlea were generated using 3D Slicer software. Two-tailed Mann-Whitney U-tests were used to investigate differences between 1) right and left ears of the same mouse to examine intraindividual symmetry and 2) differences in volumes between WT and OI cochlea. No major morphologic differences between OI and WT were observed, except for minor areas of higher ossification at the base of the cochlea, mostly within the OI sample. Within WT specimens, we observed little intraindividual difference in the cochlear volume (0-3%). Within OI specimens, significant differences were observed in cochlear volume between right and left ears in the same animal, indicating potential unilateral effects (Mann-Whitney U, p<0.05). When average WT and OI volumes were compared, there was much overlap between the two samples although the OI volumes had a significantly larger range than the WT range (Mann-Whitney U, p=0.704 (w16), p=0.703 (w4)). Overall, our results indicate that mice with OI are much more likely to have evidence of unilateral cochlear volume losses, despite very little difference in overall shape appearance, possibly due to bony capsule encroachment. This find indicates an extremely high potential for sensorineural and mixed hearing loss in OI-bred mice and elucidates at least one mechanism behind how this type of hearing loss might be occurring. Little is known about the pathological appearance of the cochlea in OI, leading to difficulty in managing hearing loss. Further investigation of the etiology and progression of cochlear pathologies will allow for better outcomes in hearing for those patients afflicted with OI-related hearing loss.Item The Effect of Diet on Craniofacial Growth in Osteogenesis Imperfecta Mouse Model(2018-05) Ladd, Summer H.; Menegaz, Rachel A.; Maddux, Scott D.; Reeves, Rustin E.; Borejdo, JulianOsteogenesis imperfecta (OI, or "brittle bone disease") is a rare disorder that is caused by genetic point mutations (COL1A1/COL1A2) that affect type 1 collagen. In OI type III (severe) patients, limb bones are more susceptible to skeletal fractures and the bones of the craniofacial region are underdeveloped. Some OI type III patients also suffer from dental malocclusions or fractures (dentinogenesis imperfecta, DI). The goals of this project are 1) to describe the facial phenotype in an OI mouse model, to see if this model can be used to test potential behavioral and pharmaceutical interventions; and 2) to determine if diet and masticatory loading affect the development of the craniofacial region in the OI model. The homozygous OI murine (OIM-/-), a mouse strain with a nonlethal recessively inherited mutation of the COL1A2 gene, is a potential model for the human OI type III. OIM-/- and wild type (WT) littermates were raised from weaning (21 days) to adulthood (16 weeks). Digital 3D craniofacial landmarks were taken from in-vivo micro CT scans, and Kuskal-Wallis ANOVAs, along with Mann-Whitney tests, were used to compare centroid size and interlandmark distances among treatment groups. This practicum focuses on the Week 10 mice, with 3 treatment groups: OIMxM, WTxM, and WTxP. We acknowledge that the sample is incomplete due to factors beyond our immediate control, such as OIM-/- survivability. Adolescent OIM-/- mice (week 10) were found to have on average smaller cranial and mandibular centroid sizes compared to WT mice regardless of diet. Week 10 OIM-/- mice also show several morphological similarities to the OI type III human phenotype, such as shortened cranial vault height, shortened jaw length, and altered dental spacing secondary to a shortened tooth row. We conclude that the OIM mouse model shows potential for future investigations of the growth mechanisms underlying the craniofacial presentation of OI. Furthermore, preliminary results suggest that masticatory loading during the early growth period can be used to stimulate craniofacial bone growth and improve bone quality in the OIM mouse model. Future studies will continue to improve sample size by treatment and age groups. The significance of this project is that it will give a better understanding of the role of type 1 collagen and the biomechanical mechanics of craniofacial development, which are important in the search for a new treatment method in OI.