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Transgenic Mouse Model: Examination of Healing, Development and Mechanical Response of Cells

Chokalingam, Kumar
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1259076989

Abstract Details

2009, PhD, University of Cincinnati, Engineering : Biomedical Engineering.
Tracking real-time changes in Col1 and Col2 gene expression could speed up the FTE process and could lead to new strategies to create zonal insertions in repair tendons. Hence double transgenic mice with fluorescent promoters for Col1 and Col2 gene expression were created and used to examine natural healing of patellar tendons, development of knee structures and the mechanical response of primitive MSCs as well as specialized chondrocytes and fibroblasts.The natural healing study sought to determine how full thickness, mid-substance punch defect injuries affect spatial and temporal patterns of Col1 and Col2 gene expression and biomechanics compared to age-matched, normal patellar tendons. Col1 expression in injured tendons was highest at two weeks after injury. No detectable Col2 gene expression was seen in the mid-substance healing wound site at any time point. Natural healing resulted in inferior repair properties at both 4 weeks and 6 weeks post injury compared to contralateral sham and age-matched tendons. Monitoring fluorescent changes in murine knees from embryonic through early post-natal development indicates that Col2 expression precedes Col1 expression. There were temporal and spatial changes in the expression of two related growth factors, TGF-β1 and BMP-2, and their downstream markers, pSmad1,5,8 and pSmad2,3 respectively. Exposing specialized and primitive cell-scaffold constructs to dynamic compressive strains produced temporal changes in gene, protein expression and biomechanics. Compression of chondrocyte-agarose scaffolds significantly increased ECFP fluorescence, Col2 mRNA expression, type II collagen content and aggregate modulus. ECFP fluorescence and Col2 mRNA expression were found to be positively correlated. Compression of MSC-agarose constructs significantly increased ECFP fluorescence, type II collagen content and aggregate modulus. Finally, 28 days of compressive stimulation of fibroblast-agarose constructs produced significant decreases in GFP-T fluorescence as well as increases in aggregate modulus. The absence of ECFP expression in any of the fibroblast-agarose constructs indicates that cells were not upregulating the Col2 gene. In a similar way, tensile stimulation of specialized and primitive cells altered both gene expression and tensile stiffness. While tensile stimulation of MSC-collagen constructs increased GFP-T fluorescence, Col1 gene expression and linear stiffness, tensile stimulation of chondrocyte-collagen constructs decreased ECFP fluorescence and linear stiffness. Absence of GFP-T expressing cells in the chondrocyte-collagen constructs indicates that the cells were not upregulating the Col1 gene. In summary, natural healing of patellar tendon results in inferior tissue even after 6 weeks of healing. Murine knees exhibit temporal and spatial variations in Col1 and Col2 gene expression. In-vitro mechanical stimulation studies show that specialized cells respond more rapidly and to a greater extent than MSCs when stimulated with signals that they normally experience in-vivo. MSCs respond more rapidly and to a greater extent than specialized cells when they are stimulated with signals they do not normally experience in-vivo. Future studies must identify appropriate mechanical cues to control/modulate MSC, chondrocyte, and fibroblast differentiation in-vitro so that multiple cellular phenotypes can be spatially and temporally produced, resulting in the synthesis of functional extracellular matrices after surgery.
David Butler, PhD (Committee Chair)
Jason Shearn, PhD (Committee Member)
Marepalli Rao, PhD (Committee Member)
Shawn Hunter, PHD (Committee Member)
232 p.
Biomedical Research
Double Transgenic Mice; Fluorescent Proteins; Collagen Promoters; Mechanical Stimulation; Natural Healing of Tendon; Growth and Development of Knees

Recommended Citations

Citations

  • Chokalingam, K. (2009). Transgenic Mouse Model: Examination of Healing, Development and Mechanical Response of Cells [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1259076989

    APA Style (7th edition)

  • Chokalingam, Kumar. Transgenic Mouse Model: Examination of Healing, Development and Mechanical Response of Cells. 2009. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1259076989.

    MLA Style (8th edition)

  • Chokalingam, Kumar. "Transgenic Mouse Model: Examination of Healing, Development and Mechanical Response of Cells." Doctoral dissertation, University of Cincinnati, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1259076989

    Chicago Manual of Style (17th edition)

ucin1259076989
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© 2009, all rights reserved.
This open access ETD is published by University of Cincinnati and OhioLINK.