Browsing by Author "Witt, Robert L."

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    A novel in vivo model for evaluating functional restoration of a tissue-engineered salivary gland
    (Wiley, 2014) Pradhan-Bhatt, Swati; Harrington, Daniel Anton; Duncan, Randall L.; Farach-Carson, Mary C.; Jia, Xinqiao; Witt, Robert L.
    Objectives/Hypothesis: To create a novel model for development of a tissue-engineered salivary gland from human salivary gland cells that retains progenitor cell markers useful for treatment of radiation-induced xerostomia. Study Design: A three-dimensional (3D) hyaluronic acid (HA)-based hydrogel scaffold was used to encapsulate primary human salivary gland cells and to obtain organized acini-like spheroids. Hydrogels were implanted into rat models, and cell viability and receptor expression were evaluated. Methods: A parotid gland surgical resection model for xenografting was developed. Salivary cells loaded in HA hydrogels formed spheroids and in vitro were implanted in the three-fourths resected parotid bed of athymic rats. Implants were removed after 1 week and analyzed for spheroid viability and phenotype retention. Results: Spheroids in 3D stained positive for HA receptors CD168/RHAMM and CD44, which is also a progenitor cell marker. The parotid gland three-fourths resection model was well-tolerated by rodent hosts, and the salivary cell/hydrogel scaffolds were adherent to the remaining parotid gland, with no obvious signs of inflammation. A majority of human cells in the extracted hydrogels demonstrated robust expression of CD44. Conclusions: A 3D HA-based hydrogel scaffold that supported long-term culture of salivary gland cells into organized spheroids was established. An in vivo salivary gland resection model was developed that allowed for integration of the 3D HA hydrogel scaffold with the existing glandular parenchyma. The expression of CD44 among salivary cultures may partially explain their regenerative potential, and the expression of CD168/RHAMM along with CD44 may aid the development of these 3D spheroids into regenerated salivary glands.
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    Dynamic Assembly of Human Salivary Stem/Progenitor Microstructures Requires Coordinated α1β1 Integrin-Mediated Motility
    (Frontiers, 2019) Wu, Danielle; Witt, Robert L.; Harrington, Daniel Anton; Farach-Carson, Mary C.
    A tissue engineering approach can provide replacement salivary gland structures to patients with hyposalivation disorders and xerostomia. Salivary human stem/progenitor cells (hS/PCs) were isolated from healthy regions of parotid glands of head and neck surgery patients, expanded, then encapsulated in biocompatible hyaluronate (HA)-based hydrogels. These bioactive hydrogels provide a surrogate territorial matrix suitable for the dynamic assembly, growth and reorganization of salivary gland components. This study examined the dynamics of salivary microstructure formation, growth, and reorganization using time-lapse imaging over 15 h. Immunofluorescence detection monitored production of individual basement membrane components forming around developing microstructures, and Ki67 assessed proliferation. Dynamic movements in hydrogels were quantified by measuring angular velocity (ω) of rotating salivary microstructures and changes in basement membrane architecture during microstructure growth. Integrin involvement in the dynamic reassembly was assessed using knockdown and inhibitor approaches. Single hS/PCs expanded over 5 days into spherical microstructures typically containing 3–10 cells. In larger macrostructures, proliferation occurred near the peripheral basement membrane that underwent growth-associated cycles of thinning and collapse. De novo secretion of laminin/collagen IV from reorganizing hS/PCs preceded that of perlecan/HSPG2. Microstructures routinely expressed β1 integrin-containing complexes at basement membrane-associated regions and exhibited spontaneous and coordinated rotation during basement membrane maturation. β1 integrin siRNA knockdown at the single-cell state prevented hS/PC microstructure growth. After microstructure formation, β1 integrin knockdown reduced rotation and mean ω by 84%. Blockade of the α1 integrin subunit (CD49a) that associates with β1 reduced mean ω by 66%. Studies presented here show that initial hS/PC structure growth and basement membrane maturation depends on α1β1-integrin mediated signaling. Coordinated cellular motility during neotissue reorganization reminiscent of salivary gland acini was critically dependent both on hS/PC-secretion of laminin,collagen type-IV, and perlecan/HSPG2 and the force-driven interactions of α1β1-integrin activation. We conclude that α1β1-integrin plays a critical role in establishing human salivary gland coordinated structure and function, and that its activation in tissue engineered systems is essential to tissue assembly.
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    Hyaluronan: A simple polysaccharide with diverse biological functions
    (Elsevier, 2014) Dicker, Kevin T.; Gurski, Lisa A.; Pradhan-Bhatt, Swati; Witt, Robert L.; Farach-Carson, Mary C.; Jia, Xinqiao
    Hyaluronan (HA) is a linear polysaccharide with disaccharide repeats of d-glucuronic acid and N-acetyl-d-glucosamine. It is evolutionarily conserved and abundantly expressed in the extracellular matrix (ECM), on the cell surface and even inside cells. Being a simple polysaccharide, HA exhibits an astonishing array of biological functions. HA interacts with various proteins or proteoglycans to organize the ECM and to maintain tissue homeostasis. The unique physical and mechanical properties of HA contribute to the maintenance of tissue hydration, the mediation of solute diffusion through the extracellular space and the lubrication of certain tissues. The diverse biological functions of HA are manifested through its complex interactions with matrix components and resident cells. Binding of HA with cell surface receptors activates various signaling pathways, which regulate cell function, tissue development, inflammation, wound healing and tumor progression and metastasis. Taking advantage of the inherent biocompatibility and biodegradability of HA, as well as its susceptibility to chemical modification, researchers have developed various HA-based biomaterials and tissue constructs with promising and broad clinical potential. This paper illustrates the properties of HA from a matrix biology perspective by first introducing the principles underlying the biosynthesis and biodegradation of HA, as well as the interactions of HA with various proteins and proteoglycans. It next highlights the roles of HA in physiological and pathological states, including morphogenesis, wound healing and tumor metastasis. A deeper understanding of the mechanisms underlying the roles of HA in various physiological processes can provide new insights and tools for the engineering of complex tissues and tissue models.
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    Implantable modular hydrogel for salivary gland restoration
    (2017-05-09) Witt, Robert L.; Jia, Xinqiao; Bhatt, Swati Pradham; Farach-Carson, Mary C.; Harrington, Daniel A.; Rice University; United States Patent and Trademark Office
    Implantable modular hydrogels to aid in salivary gland restoration and associated methods are provided. In one embodiment, the present disclosure provides for a hydrogel network comprising: a hyaluronic acid macromer crosslinked with a multiblock copolymer.
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    Primary Salivary Human Stem/Progenitor Cells Undergo Microenvironment-Driven Acinar-Like Differentiation in Hyaluronate Hydrogel Culture
    (AlphaMed Press, 2016) Srinivasan, Padma Pradeepa; Patel, Vaishali N.; Liu, Shuang; Harrington, Daniel Anton; Hoffman, Matthew P.; Jia, Xinqiao; Witt, Robert L.; Farach-Carson, Mary C.; Pradhan-Bhatt, Swati
    Radiotherapy for head and neck cancer often has undesirable effects on salivary glands that lead to xerostomia or severe dry mouth, which can increase oral infections. Our goal is to engineer functional, three-dimensional (3D) salivary gland neotissue for autologous implantation to provide permanent relief. An immediate need exists to obtain autologous adult progenitor cells as the use of embryonic and induced pluripotent stem cells potentially pose serious risks such as teratogenicity and immunogenic rejection. Here, we report an expandable population of primary salivary human stem/progenitor cells (hS/PCs) that can be reproducibly and scalably isolated and propagated from tissue biopsies. These cells have increased expression of progenitor markers (K5, K14, MYC, ETV4, ETV5) compared with differentiation markers of the parotid gland (acinar: MIST1/BHLHA15 and AMY1A; ductal: K19 and TFCP2L1). Isolated hS/PCs grown in suspension formed primary and secondary spheres and could be maintained in long-term 3D hydrogel culture. When grown in a customized 3D modular hyaluronate-based hydrogel system modified with bioactive basement membrane-derived peptides, levels of progenitor markers, indices of proliferation, and viability of hS/PCs were enhanced. When appropriate microenvironmental cues were provided in a controlled manner in 3D, such as stimulation with β-adrenergic and cholinergic agonists, hS/PCs differentiated into an acinar-like lineage, needed for saliva production. We conclude that the stem/progenitor potential of adult hS/PCs isolated without antigenic sorting or clonal expansion in suspension, combined with their ability to differentiate into specialized salivary cell lineages in a human-compatible culture system, makes them ideal for use in 3D bioengineered salivary gland applications.
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    Tuning Hydrogel Properties to Promote the Assembly of Salivary Gland Spheroids in 3D
    (American Chemical Society, 2016) Ozdemir, Tugba; Fowler, Eric W.; Liu, Shuang; Harrington, Daniel Anton; Witt, Robert L.; Farach-Carson, Mary C.; Pradhan-Bhatt, Swati; Jia, Xinqiao
    Current treatments for chronic xerostomia, or “dry mouth”, do not offer long-term therapeutic benefits for head and neck cancer survivors previously treated with curative radiation. Towards the goal of creating tissue-engineered constructs for the restoration of salivary gland functions, we developed new hyaluronic acid (HA)-based hydrogels using thiolated HA (HA-SH) and acrylated HA (HA-AES) with a significant molecular weight mismatch. Four hydrogel formulations with varying HA concentration, (1–2.4 wt%) and thiol/acrylate ratios (2/1 to 36/1) and elastic moduli (G’: 35 to 1897 Pa, 2 h post-mixing) were investigated. In our system, thiol/acrylate reaction was initiated rapidly upon mixing of HA-SH/HA-AES to establish thioether crosslinks with neighboring ester groups, and spontaneous sulfhydryl oxidation occurred slowly over several days to install a secondary network. The concurrent reactions cooperatively create a cell-permissive network to allow for cell expansion and aggregation. Multicellular spheroids formed readily from a robust ductal epithelial cell line (Madin-Darby Canine Kidney, MDCK cells) in all hydrogel formulations investigated. Primary salivary human stem/progenitor cells (hS/PCs), on the other hand, are sensitive to the synthetic extracellular environment, and organized acini-like structures with an average diameter of 50 µm were obtained only in gels with G’ ≤ 216 Pa and a thiol/acrylate ratio ≥18. The spheroid size and size distribution were dependent on the HA content in the hydrogel. Cells in hS/PC spheroids formed tight junctions (occludin), remained viable and proliferative, secreted structural proteins (collagen IV and laminin) found in the basement membrane and maintained key stem/progenitor markers. We conclude that incorporation of time-dependent, dynamic features into a covalently crosslinked HA network produces an adaptable hydrogel framework that promotes hS/PC assembly and supports early aspects of salivary morphogenesis, key to reconstitution of a fully functional implantable salivary gland.