Browsing by Subject "Tissue engineering"
Now showing 1 - 10 of 12
Results Per Page
Sort Options
Item Effects of interstitial fluid flow and cell compression in FAK and SRC activities in chondrocytes(2013-11-08) Cho, Eunhye; Na, Sungsoo; Yokota, Hiroki, 1955-; Li, JiliangArticular cartilage is subjected to dynamic mechanical loading during normal daily activities. This complex mechanical loading, including cell deformation and interstitial fluid flow, affects chondrocyte mechano-chemical signaling and subsequent cartilage homeostasis and remodeling. Focal adhesion kinase (FAK) and Src are known to be main mechanotransduction proteins, but little is known about the effect of mechanical loading on FAK and Src under its varying magnitudes and types. In this study, we addressed two questions using C28/I2 chondrocytes subjected to the different types and magnitudes of mechanical loading: Does a magnitude of the mechanical loading affect activities of FAK and Src? Does a type of the mechanical loading also affect their activities? Using fluorescence resonance energy transfer (FRET)-based FAK and Src biosensor in live C28/I2 chondrocytes, we monitored the effects of interstitial fluid flow and combined effects of cell deformation/interstitial fluid flow on FAK and Src activities. The results revealed that both FAK and Src activities in C28/I2 chondrocytes were dependent on the different magnitudes of the applied fluid flow. On the other hand, the type of mechanical loading differently affected FAK and Src activities. Although FAK and Src displayed similar activities in response to interstitial fluid flow only, simultaneous application of cell deformation and interstitial fluid flow induced differential FAK and Src activities possibly due to the additive effects of cell deformation and interstitial fluid flow on Src, but not on FAK. Collectively, the data suggest that the intensities and types of mechanical loading are critical in regulating FAK and Src activities in chondrocytes.Item Enhanced Viability of Endothelial Colony Forming Cells in Fibrin Microbeads for Sensor Vascularization(MDPI AG, 2015-09-18) Gandhi, Jarel K.; Zivkovic, Lada; Fisher, John P.; Yoder, Mervin C.; Brey, Eric M.; Department of Pediatrics, School of MedicineEnhanced vascularization at sensor interfaces can improve long-term function. Fibrin, a natural polymer, has shown promise as a biomaterial for sensor coating due to its ability to sustain endothelial cell growth and promote local vascularization. However, the culture of cells, particularly endothelial cells (EC), within 3D scaffolds for more than a few days is challenging due to rapid loss of EC viability. In this manuscript, a robust method for developing fibrin microbead scaffolds for long-term culture of encapsulated ECs is described. Fibrin microbeads are formed using sodium alginate as a structural template. The size, swelling and structural properties of the microbeads were varied with needle gauge and composition and concentration of the pre-gel solution. Endothelial colony-forming cells (ECFCs) were suspended in the fibrin beads and cultured within a perfusion bioreactor system. The perfusion bioreactor enhanced ECFCs viability and genome stability in fibrin beads relative to static culture. Perfusion bioreactors enable 3D culture of ECs within fibrin beads for potential application as a sensor coating.Item Exploring Chondrocyte Integrin Regulation of Growth Factor IGF-I Expression from a Transient pAAV Vector(2013-08-20) Ratley, Samantha Kay; Trippel, Stephen B.; Lin, Chien-Chi; Stocum, David L.Insulin-like Growth Factor I (IGF-I) is a growth factor that stimulates both mitogenic and anabolic responses in articular chondrocytes. While it has been shown that exogenous IGF-I can regulate chondrocyte integrins, little is known regarding regulatory effects of IGF-I produced from a transiently expressed plasmid based adeno-associated virus (pAAV) vector. Because chondrocytes are using cellular machinery to overexpress IGF-I, it is of interest to see whether or not pAAV IGF-I will significantly upregulate or downregulate chondrocyte integrins. Additionally, it is of interest to know whether chondrocyte adhesion through integrins will have any regulatory effects on the production of IGF-I from the transgene. Therefore, this study will ascertain if pAAV IGF-I will have similar effects that exogenous IGF-I has on integrin regulation and if integrin silencing mechanisms will affect the production of IGF-I from the transgene. To test these hypotheses, adult articular chondrocytes were doubly transfected with the pAAV vector for IGF-I and short interference ribonucleic acid (siRNA) for integrins beta 1 and alpha V. Gene products were monitored at the transcriptional levels using quantitative real time polymerase chain reactions (qPCR) and IGF-I protein production was monitored at the translational level using enzyme linked immunoabsorbant assays (ELISAs). Adult articular chondrocytes doubly transfected were encapsulated in a three dimensional hydrogel system to simulate an in vivo environment. Samples were collected for analysis at days 2, 4, and 6 post encapsulation. Results show that IGF-I treatment with the pAAV vector does not cause significant changes in the transcriptional regulation of the beta 1 integrin in a three dimensional hydrogel system. The pAAV IGF-I vector did not cause significant regulatory changes on integrin alpha V at any time point during the experiment. Additionally, by knocking down the expression levels of integrins by using siRNA, it was shown that integrin knockdown does not have a significant regulatory effect on transcriptional or translational expression levels of IGF-I from the pAAV vector.Item In Vitro Multitissue Interface Model Supports Rapid Vasculogenesis and Mechanistic Study of Vascularization across Tissue Compartments(ACS Publications, 2016-08-31) Bruno, Kevin P.; Chen, Xuemei; Weibel, Justin A.; Thiede, Stephanie N.; Garimella, Suresh V.; Yoder, Mervin C.; Voytik-Harbin, Sherry L.; Department of Pediatrics, IU School of MedicineA significant challenge facing tissue engineers is the design and development of complex multitissue systems, including vascularized tissue-tissue interfaces. While conventional in vitro models focus on either vasculogenesis (de novo formation of blood vessels) or angiogenesis (vessels sprouting from existing vessels or endothelial monolayers), successful therapeutic vascularization strategies will likely rely on coordinated integration of both processes. To address this challenge, we developed a novel in vitro multitissue interface model in which human endothelial colony forming cell (ECFC)-encapsulated tissue spheres are embedded within a surrounding tissue microenvironment. This highly reproducible approach exploits biphilic surfaces (nanostructured surfaces with distinct superhydrophobic and hydrophilic regions) to (i) support tissue compartments with user-specified matrix composition and physical properties as well as cell type and density and (ii) introduce boundary conditions that prevent the cell-mediated tissue contraction routinely observed with conventional three-dimensional monodispersion cultures. This multitissue interface model was applied to test the hypothesis that independent control of cell-extracellular matrix (ECM) and cell-cell interactions would affect vascularization within the tissue sphere as well as across the tissue-tissue interface. We found that high-cell-density tissue spheres containing 5 × 10(6) ECFCs/mL exhibit rapid and robust vasculogenesis, forming highly interconnected, stable (as indicated by type IV collagen deposition) vessel networks within only 3 days. Addition of adipose-derived stromal cells (ASCs) in the surrounding tissue further enhanced vasculogenesis within the sphere as well as angiogenic vessel elongation across the tissue-tissue boundary, with both effects being dependent on the ASC density. Overall, results show that the ECFC density and ECFC-ASC crosstalk, in terms of paracrine and mechanophysical signaling, are critical determinants of vascularization within a given tissue compartment and across tissue interfaces. This new in vitro multitissue interface model and the associated mechanistic insights it yields provide guiding principles for the design and optimization of multitissue vascularization strategies for research and clinical applications.Item Investigating pediatric disorders with induced pluripotent stem cells(Springer Nature, 2018-10) Durbin, Matthew D.; Cadar, Adrian G.; Chun, Young W.; Hong, Charles C.; Pediatrics, School of MedicineThe study of disease pathophysiology has long relied on model systems, including animal models and cultured cells. In 2006, Shinya Yamanaka achieved a breakthrough by reprogramming somatic cells into induced pluripotent stem cells (iPSCs). This revolutionary discovery provided new opportunities for disease modeling and therapeutic intervention. With established protocols, investigators can generate iPSC lines from patient blood, urine, and tissue samples. These iPSCs retain ability to differentiate into every human cell type. Advances in differentiation and organogenesis move cellular in vitro modeling to a multicellular model capable of recapitulating physiology and disease. Here, we discuss limitations of traditional animal and tissue culture models, as well as the application of iPSC models. We highlight various techniques, including reprogramming strategies, directed differentiation, tissue engineering, organoid developments, and genome editing. We extensively summarize current established iPSC disease models that utilize these techniques. Confluence of these technologies will advance our understanding of pediatric diseases and help usher in new personalized therapies for patients.Item Mouse and human islets survive and function after coating by biosilicification(American Physiological Society, 2013-11-15) Jaroch, David B.; Lu, Jing; Madangopal, Rajtarun; Stull, Natalie D.; Stensberg, Matthew; Shi, Jin; Kahn, Jennifer L.; Herrera-Perez, Ruth; Zeitchek, Michael; Sturgis, Jennifer; Robinson, J. Paul; Yoder, Mervin C.; Porterfield, D. Marshall; Mirmira, Raghavendra; Rickus, Jenna L.; Medicine, School of MedicineInorganic materials have properties that can be advantageous in bioencapsulation for cell transplantation. Our aim was to engineer a hybrid inorganic/soft tissue construct by inducing pancreatic islets to grow an inorganic shell. We created pancreatic islets surrounded by porous silica, which has potential application in the immunoprotection of islets in transplantation therapies for type 1 diabetes. The new method takes advantage of the islet capsule surface as a template for silica formation. Mouse and human islets were exposed to medium containing saturating silicic acid levels for 9-15 min. The resulting tissue constructs were then cultured for up to 4 wk under normal conditions. Scanning electron microscopy and energy dispersive X-ray spectroscopy was used to monitor the morphology and elemental composition of the material at the islet surface. A cytokine assay was used to assess biocompatibility with macrophages. Islet survival and function were assessed by confocal microscopy, glucose-stimulated insulin release assays, oxygen flux at the islet surface, expression of key genes by RT-PCR, and syngeneic transplant into diabetic mice.Item Oxygenation Profiles of Human Blood, Cell Culture Medium, and Water for Perfusion of 3D-Bioprinted Tissues using the FABRICA Bioreactor Platform(Nature Research, 2020-04-29) Chen, Angela M.; Lashmet, Matthew; Isidan, Abdulkadir; Sterner, Jane L.; Walsh, Julia; Koehler, Cutter; Li, Ping; Ekser, Burcin; Smith, Lester; Surgery, School of MedicinePersistent and saturated oxygen distribution from perfusion media (i.e., blood, or cell culture media) to cells within cell-dense, metabolically-active biofabricated tissues is required to keep them viable. Improper or poor oxygen supply to cells within the tissue bulk severely limits the tissue culturing potential of many bioreactors. We added an oxygenator module to our modular FABRICA bioreactor in order to provide stable oxygenation to biofabricated tissues during culture. In this proof of concept study of an oxygenated and perfused bioreactor, we characterized the oxygenation of water, cell culture medium, and human blood in the FABRICA as functions of augmenting vacuum (air inlet) pressure, perfusion (volumetric flow) rate, and tubing/oxygenator components. The mean oxygen levels for water and cell culture media were 27.7 ± 2.1% and 27.6 ± 4.1%, respectively. The mean oxygen level for human blood was 197.0 ± 90.0 mmHg, with near-physiologic levels achieved with low-permeability PharMed tubing alone (128.0 ± 14.0 mmHg). Hematologic values pre- and post-oxygenation, respectively were (median ± IQR): Red blood cell: 6.0 ± 0.5 (106/μL) and 6.5 ± 0.4 (106/μL); Hemoglobin: 17.5 ± 1.2 g/dL and 19.2 ± 3.0 g/dL; and Hematocrit: 56.7 ± 2.4% and 61.4 ± 7.5%. The relative stability of the hematologic parameters indicates that blood function and thus blood cell integrity were maintained throughout oxygenation. Already a versatile research tool, the now oxygenated FABRICA provides easy-to-implement, in vivo-like perfusion and stable oxygenation culture conditions in vitro semi-independently of one another, which means the bioreactor has the potential to serve as a platform for investigating the behavior of 3D tissue models (regardless of biofabrication method), performing drug toxicity-testing, and testing pharmaceutical efficacy/safety.Item Regenerative tissue filler for breast conserving surgery and other soft tissue restoration and reconstruction needs(Springer Nature, 2021-02-01) Puls, Theodore J.; Fisher, Carla S.; Cox, Abigail; Plantenga, Jeannie M.; McBride, Emma L.; Anderson, Jennifer L.; Goergen, Craig J.; Bible, Melissa; Moller, Tracy; Voytik‑Harbin, Sherry L.; Surgery, School of MedicineComplete removal of cancerous tissue and preservation of breast cosmesis with a single breast conserving surgery (BCS) is essential for surgeons. New and better options would allow them to more consistently achieve this goal and expand the number of women that receive this preferred therapy, while minimizing the need for re-excision and revision procedures or more aggressive surgical approaches (i.e., mastectomy). We have developed and evaluated a regenerative tissue filler that is applied as a liquid to defects during BCS prior to transitioning to a fibrillar collagen scaffold with soft tissue consistency. Using a porcine simulated BCS model, the collagen filler was shown to induce a regenerative healing response, characterized by rapid cellularization, vascularization, and progressive breast tissue neogenesis, including adipose tissue and mammary glands and ducts. Unlike conventional biomaterials, no foreign body response or inflammatory-mediated “active” biodegradation was observed. The collagen filler also did not compromise simulated surgical re-excision, radiography, or ultrasonography procedures, features that are important for clinical translation. When post-BCS radiation was applied, the collagen filler and its associated tissue response were largely similar to non-irradiated conditions; however, as expected, healing was modestly slower. This in situ scaffold-forming collagen is easy to apply, conforms to patient-specific defects, and regenerates complex soft tissues in the absence of inflammation. It has significant translational potential as the first regenerative tissue filler for BCS as well as other soft tissue restoration and reconstruction needs.Item Targeting acute phosphatase PTEN inhibition and investigation of a novel combination treatment with Schwann cell transplantation to promote spinal cord injury repair in rats(2014-04-02) Walker, Chandler L.; Xu, Xiao-Ming; Zhou, Feng C.; Jin, Xiao-Ming; Cummins, Theodore R.Human traumatic spinal cord injuries (SCI) are primarily incomplete contusion or compression injuries at the cervical spinal level, causing immediate local tissue damage and a range of potential functional deficits. Secondary damage exacerbates initial mechanical trauma and contributes to function loss through delayed cell death mechanisms such as apoptosis and autophagy. As such, understanding the dynamics of cervical SCI and related intracellular signaling and death mechanisms is essential. Through behavior, Western blot, and histological analyses, alterations in phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3-kinase (PI3K) signaling and the neuroprotective, functional, and mechanistic effects of administering the protein tyrosine phosphatase (PTP) inhibitor, potassium bisperoxo (picolinato) vanadium ([bpV[pic]) were analyzed following cervical spinal cord injury in rats. Furthermore, these studies investigated the combination of subacute Schwann cell transplantation with acute bpV(pic) treatment to identify any potential additive or synergistic benefits. Although spinal SC transplantation is well-studied, its use in combination with other therapies is necessary to complement its known protective and growth promoting characteristics. v The results showed 400 μg/kg/day bpV(pic) promoted significant tissue sparing, lesion reduction, and recovery of forelimb function post-SCI. To further clarify the mechanism of action of bpV(pic) on spinal neurons, we treated injured spinal neurons in vitro with 100 nM bpV(pic) and confirmed its neurprotection and action through inhibition of PTEN and promotion of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling. Following bpV(pic) treatment and green fluorescent protein (GFP)-SC transplantation, similar results in neuroprotective benefits were observed. GFP-SCs alone exhibited less robust effects in this regard, but promoted significant ingrowth of axons, as well as vasculature, over 10 weeks post-transplantation. All treatments showed similar effects in forelimb function recovery, although the bpV and combination treatments were the only to show statistical significance over non-treated injury. In the following chapters, the research presented contributes further understanding of cellular responses following cervical hemi-contusion SCI, and the beneficial effects of bpV(pic) and SC transplantation therapies alone and in combination. In conclusion, this work provides a thorough overview of pathology and cell- and signal-specific mechanisms of survival and repair in a clinically relevant rodent SCI model.Item Use of autologous adipose-derived mesenchymal stem cells for creation of laryngeal cartilage(Wiley, 2018-04) Zhang, Hongji; Voytik-Harbin, Sherry; Brookes, Sarah; Zhang, Lujuan; Wallace, Joseph; Parker, Noah; Halum, Stacey; Biomedical Engineering, School of Engineering and TechnologyOBJECTIVES/HYPOTHESIS: Adipose-derived mesenchymal stem cells (ASCs) are an exciting potential cell source for tissue engineering because cells can be derived from the simple excision of autologous fat. This study introduces a novel approach for tissue-engineering cartilage from ASCs and a customized collagen oligomer solution, and demonstrates that the resultant cartilage can be used for laryngeal cartilage reconstruction in an animal model. STUDY DESIGN: Basic science experimental design. METHODS: ASCs were isolated from F344 rats, seeded in a customized collagen matrix, and cultured in chondrogenic differentiation medium for 1, 2, and 4 weeks until demonstrating cartilage-like characteristics in vitro. Large laryngeal cartilage defects were created in the F344 rat model, with the engineered cartilage used to replace the cartilage defects, and the rats followed for 1 to 3 months. Staining examined cellular morphology and cartilage-specific features. RESULTS: In vitro histological staining revealed rounded chondrocyte-appearing cells evenly residing throughout the customized collagen scaffold, with positive staining for cartilage-specific markers. The cartilage was used to successfully repair large cartilaginous defects in the rat model, with excellent functional results. CONCLUSIONS: This study is the first study to demonstrate, in an animal model, that ASCs cultured in a unique form of collagen oligomer can create functional cartilage-like grafts that can be successfully used for partial laryngeal cartilage replacement.