Browsing by Author "Schmidt, Tannin A."

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    Bioprocess Development for Large-Scale Production of Skin Derived Precursor Schwann Cells
    (2018-08-16) Walsh, Tylor Douglas; Kallos, Michael S.; Schmidt, Tannin A.; Biernaskie, Jeff A.
    Peripheral nerve and spinal cord injuries are debilitating, leading to lifelong complications and reduced quality of life. Cellular therapies have demonstrated beneficial outcomes when treating these injuries especially when using Schwann cells. However, there is currently no robust and reproducible method for producing Schwann cells at clinical scale. Bioprocesses that use bioreactors have significant advantages when scaling-up cellular therapies. Therefore, the research in this thesis was done to address this gap and develop methods, tools, and protocols to create a bioprocess for the large-scale expansion of Schwann cells. The hydrodynamics of the bioreactor were investigated by using CFD modeling, comparing velocity, shear rate, and energy dissipation rate at different agitation rates and their effect on cell expansion. The model that was generated can be used to scale up processes to larger, clinical and manufacturing scale, bioreactors. Upstream and downstream unit operations were then developed. Commercially available microcarriers were evaluated and tested in bioreactors to find the microcarrier that supported both inoculation and expansion of SKP-SCs. Different bioreactor platforms were evaluated, showing that controlling the process parameters increased cell densities. An in-depth DOE was conducted to find the best inoculation conditions, investigating which parameters had significant effects on cell attachment, distribution, and expansion. Detachment of SKP-SCs from microcarriers was investigated with different enzymes and agitation rates to develop an in-vessel passaging protocol that can easily be scale-up. After harvest, cryopreservation medium and cell density were investigated to ensure a quality product can be frozen and delivered to the patient. After the process was developed, 3 rat lines and 1 human line were tested. The process was reproducible and robust and easily adapted to human cells. Additional development is needed to use this process for nerve derived Schwann cells. This process was then integrated together and 150 x106 cells were produced from 3x106 in 7 days. Lastly, the expansion design space was investigated to determine the effects of pH, DO, and agitation on the expansion of SKP-SCs. All the tools and methods developed in this thesis can easily be adapted to nearly any bioprocess that utilize bioreactors for cellular therapies.
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    Cartilage boundary lubrication synergism is mediated by hyaluronan concentration and PRG4 concentration and structure
    (BioMed Central Musculoskeletal Disorders, 2015-12) Ludwig, Taryn E.; Hunter, Miles M.; Schmidt, Tannin A.
    Background Proteoglycan 4 (PRG4) and hyaluronan (HA) are key synovial fluid constituents that contribute synergistically to cartilage boundary lubrication; however, the effects of their concentrations as well as their structure, both of which can be altered in osteoarthritis, on this functional synergism are unknown. The objectives of this study were to evaluate cartilage boundary lubricating ability of 1) PRG4 + HA in solution at constant HA concentration in a range of PRG4 concentrations, 2) constant PRG4 concentration in a range of HA concentrations, 3) HA + reduced/alkylated (R/A) PRG4, and 4) hylan G-F 20 + PRG4. Methods Static and kinetic friction coefficients (μstatic,Neq, <μkinetic,Neq>) were measured using a previously characterized cartilage-cartilage boundary mode friction test for the following concentrations of purified PRG4 and HA: Test 1: HA (1.5 MDa, 3.3 mg/mL) + PRG4 from 4.5 – 1500 μg/mL; Test 2: PRG4 (450, 150, 45 μg/mL) + HA (1.5 MDa) from 0.3 – 3.3 mg/mL. Test 3: hylan G-F 20 (3. 3 mg/mL) + PRG4 (450 μg/mL). Test 4: HA (3.3 mg/mL) + R/A PRG4 (450 μg/mL). ANOVA was used to compare lubricants within (comparing 6 lubricants of interest) and between (comparing 3 lubricants of interest) test sequences, with Tukey and Fishers post-hoc testing respectively. Results This study demonstrates that both PRG4 and HA concentration, as well as PRG4 disulfide-bonded structure, can alter the cartilage boundary lubricating ability of PRG4 + HA solutions. The boundary lubricating ability of high MW HA + PRG4 solutions was limited by very low concentrations of PRG4. Decreased concentrations of high MW HA also limited the cartilage boundary lubricating ability of HA + PRG4 solutions, with the effect exacerbated by low PRG4 concentrations. The reduction of friction by addition of PRG4 to a cross-linked HA viscosupplement product, but not with addition of R/A PRG4 to HA, is consistent with a non-covalent mechanism of interaction where tertiary and quaternary PRG4 structure are important. Conclusions Collectively, these results demonstrate that deficiency of either or both PRG4 and HA, or alterations in PRG4 structure, may be detrimental to SF cartilage boundary lubricating function. This study provides further insight into the nature of cartilage boundary lubrication and advancement towards potential formulation of new intra-articular biotherapeutic treatments for osteoarthritis using PRG4 ± HA.
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    The Characterization of Proteoglycan 4 and Hyaluronan Composition in the Vitreous Humour and Their Potential Contributions to Steady Shear Viscosity
    (2020-01-27) Alarifi, Abdulaziz A.; Schmidt, Tannin A.; Hart, David A.; Krawetz, Roman J.; Stell, William K.; Moritz, Orson L.
    Background and aim: The vitreous humour is a connective tissue in the eye that contributes to several physiological and pathological processes. The full details of the complex vitreous humour composition and the extent of its functionality remain elusive. Proteoglycan 4 (PRG4), a mucin-like O-linked glycosylated protein, has recently been identified in human vitreous humour. PRG4 is known to act synergistically with hyaluronan (HA) in synovial fluids, where it functions as a boundary lubricant, contributes to viscosity, and more. Studying PRG4 in this newly discovered site might advance knowledge about its rheological properties (specifically within the eye) and whether or not PRG4 is involved. The aim of this thesis was to analyze the presence, source and potential function of PRG4 in vitreous humour. The objectives were: 1) to quantify the PRG4 and HA (including its molecular weight (MW) distribution) content, 2) to immunolocalize PRG4 in the eye globe, and examine its local synthesis, and 3) to characterize the viscosity of vitreous humour samples with varying PRG4 and HA concentrations. Methods: Human vitreous humour samples were collected from post-mortem human patients. Alpha amplified luminescent proximity homogeneous assay (AlphaLISA) and enzyme-linked immunosorbent assay (ELISA) tests were performed to measure the concentration of PRG4 and HA, respectively. The MW distribution of HA was determined by agarose gel electrophoresis. Vitreous humour cells were cultured from porcine to examine the synthesis of PRG4. PRG4 was immunolocalized in eye globes from wild type (WT) and PRG4-KO mice. Steady shear viscosity was characterized for human vitreous humour using a rheometer, before and after adding exogenous recombinant human PRG4 (rh PRG4). Results: The mean (± standard deviation) concentration of PRG4 in human vitreous humour (N=36) was 25 ± 32 µg/mL. For HA, the mean concentration in human vitreous humour was 344±317 µg/mL, and 71% of the overall HA in vitreous humour was below 459 kDa. Increasing age was positively correlated with concentration of PRG4. PRG4 was synthesized by cultured porcine vitreous humour cells. Using wild type and PRG4-KO (knockout) mice, PRG4 was immunolocalized in the ciliary body, cornea, sclera, vitreous cortex, and some parts of the retina. Viscosities for human vitreous humour samples showed minor variations, and were negatively correlated with HA MW. Conclusion: This is the first investigation of both the presence and potential function of PRG4 in vitreous humour. The findings serve to expand understanding of vitreous humour composition, and how that might be a factor in ocular pathology and potential therapies.
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    Mechanical fatigue of bovine cortical bone using ground reaction force waveforms in running
    (2018-01) Loundagin, Lindsay L.; Schmidt, Tannin A.; Edwards, William Brent
    Stress fractures are a common overuse injury among runners associated with the mechanical fatigue of bone. Several in vivo biomechanical studies have investigated specific characteristics of the vertical ground reaction force (vGRF) in heel-toe running and have observed an association between increased loading rate during impact and individuals with a history of stress fracture. The purpose of this study was to examine the fatigue behavior of cortical bone using vGRF-like loading profiles, including those that had been decomposed into their respective impact and active phase components. Thirty-eight cylindrical cortical bone samples were extracted from bovine tibiae and femora. Hydrated samples were fatigue tested at room temperature in zero compression under load control using either a raw (n = 10), active (n = 10), low impact (n = 10), or high impact (n = 8) vGRF profile. The number of cycles to failure was quantified and the test was terminated if the sample survived 105 cycles. Fatigue life was significantly greater for both impact groups compared to the active (p < 0.001) and raw (p < 0.001) groups, with all low impact samples and 6 of 8 high impact samples surviving 105 cycles. The mean (± SD) number of cycles to failure for the active and raw groups was 12,133±11,704 and 16,552±29,612, respectively. The results suggest that loading rates associated with the impact phase of a typical vGRF in running have little influence on the mechanical fatigue behavior of bone relative to loading magnitude, warranting further investigation of the mechanism by which increased loading rates are associated with stress fracture.
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    Muscular loading of joints triggers cellular secretion of PRG4 into the joint fluid
    (Journal of Biomechanics, 2013-04-26) Abusara, Ziad; Krawetz, Roman J.; Steele, Bridgett L.; DuVall, Michael M.; Schmidt, Tannin A.; Herzog, Walter
    We developed a novel testing system that allows quantification of joint loading and permits analysis of changes in total protein and PRG4 contents in joint fluid of intact knees in live mice. A sequence of 15 repeat, isometric muscular contractions of "low" intensity (less than 50% of the maximal isometric muscular force), and "high" intensity (greater than 55% of maximal) were applied repeatedly (up to five times with a 15 min rest between contractions) to the mouse knee. Increases in knee joint loading were accompanied with significant increases in total protein (p<0.0001) and PRG4 concentrations in the synovial fluid. Total protein and PRG4 concentrations decreased with repeated "high" intensity loading. However, the addition of cell secretion inhibitors to the knee prior to muscular loading resulted in PRG4 levels that remained below the detection limit for all loading conditions. These results suggest that changes in synovial fluid proteins and PRG4 concentrations upon joint loading are mediated by cells within the joint, and that these changes may be used as quantitative indicators for the intensity and duration of acute joint loading, and might serve as a powerful clinical tool to assess the effectiveness of rehabilitation and prevention exercise programs.
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    Proteoglycan-4 in Equine Joint Disease, Exercise, and in vitro Cartilage Repair
    (2020-05-25) Matheson, Austyn Reid; Schmidt, Tannin A.; Scott, W. Michael; Herzog, Walter; Matyas, John Robert
    Proteoglycan-4 (PRG4) and hyaluronan (HA) are biological macromolecules with varied and diverse functions distributed throughout the body. In synovial fluid (SF), PRG4 and HA provide independent and synergistic contributions to tissue health and cartilage boundary lubrication. The biological consequences of joint injury or disease such as osteoarthritis (OA) may include altered concentration, structure, and function of PRG4 and HA, leading to degraded SF quality and function, changes which are not fully understood. Furthermore, the effects of joint disease on circulating (serum) PRG4 and HA, both of unknown origin and function in blood, requires clarification. Monitoring changes to PRG4 and HA to elucidate the effects on SF and serum may facilitate the development of therapeutics, biomarkers, or novel biomaterials to restore joint health and function. The objectives of this thesis were to 1) investigate clinically relevant changes to PRG4 and HA composition in SF and serum, and SF biomechanical function from equine cases of joint disease and injury, 2) to investigate the effect of exercise on equine serum PRG4, and 3) to characterize the effect of recombinant human PRG4 (rhPRG4) integration on the biomechanical, architectural, and biological aspects of a collagen-based scaffold for cartilage repair. A combination of novel and previously characterized biochemical and biomechanical techniques were used to evaluate SF and serum composition, the lubricating ability of SF and tissue-engineered collagen-scaffolds, and the in vitro bioactivity of rhPRG4-integrated collagen-scaffolds. The composition of equine SF changed in acute joint injury compared to SF from normal horses. Both PRG4 concentration and HA molecular weight were altered, with decreased SF viscosity, yet no associated detectable effects on serum PRG4. The concentration of serum PRG4 in a group of racehorses decreased significantly five minutes post-exercise, perhaps clearing from the circulation. Hence, serum PRG4 and HA concentrations alone may not be useful biomarkers for equine joint disease. rhPRG4-integrated scaffolds had enhanced lubricating properties, a highly porous architecture, and supported cell infiltration and growth across most concentrations tested. Collectively these results indicate that PRG4 is an essential lubricant, an indicator for injury, and a promising therapeutic for integration within cartilage repair biomaterials.