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Salivary gland tissue engineering offers an attractive alternative for the treatment of radiation-induced xerostomia. Key to the success of this approach is the maintenance and expansion of secretory acinar cells in vitro. However, recent studies revealed that in vitro culture of primary salivary gland epithelial cells led to undesirable upregulation of the expression of keratin-7 (K7), a marker of ductal phenotype and frequently associated with cellular stress. We have previously shown that hyaluronic acid (HA)-based, RGDSP-decorated hydrogels support the 3D growth and assembly of primary human salivary gland stem/progenitor cells (hS/PCs). Here, we investigate whether the RGDSP culture also promotes K7 expression, and if so, what factors govern the K7 expression. Compared to hS/PCs maintained in blank HA gels, those grown in RGDSP cultures expressed a significantly higher level of K7. In other tissues, various transforming growth factor-β (TGF-β) superfamily members are reported to regulate K7 expression. Similarly, our immunoblot array and ELISA experiments confirmed the increased expression of TGF-β1 and growth/differentiation factor-15 (GDF-15) in RGDSP cultures. However, 2D model studies show that only TGF-β1 is required to induce K7 expression in hS/PCs. Immunocytochemical analysis of the intracellular effectors of TGF-β signaling, SMAD 2/3, further confirmed the elevated TGF-β signaling in RGDSP cultures. To maximize the regenerative potential of h/SPCs, cultures were treated with a pharmacological inhibitor of TGF-β receptor, A83-01. Our results show that A83-01 treatment can repress K7 expression not only in 3D RGDSP cultures but also under 2D conditions with exogenous TGF-β1. Collectively, we provide a link between TGF-β signaling and K7 expression in hS/PC cultures and demonstrate the effectiveness of TGF-β inhibition to repress K7 expression while maintaining the ability of RGDSP-conjugated HA gels to facilitate the rapid development of amylase expressing spheroids. These findings represent an important step towards regenerating salivary function with a tissue-engineered salivary gland.

Adventitial fibroblasts (AFs) are critical mediators of vascular remodeling. However, the contributions of AFs towards development of vasculature and the specific mechanisms by which these cells regulate physiological expansion of the vasa vasorum, the specialized microvasculature that supplies nutrients to the vascular wall, are not well understood. To determine the regulatory role of AFs in microvascular endothelial cell (MVEC) neovasculogenesis and to investigate the regulatory pathways utilized for communication between the two cell types, AFs and MVECs were cultured together in poly(ethylene glycol)-based hydrogels. Following preliminary evaluation of a set of cell adhesion peptides (AG10, AG73, A2G78, YIGSR, RGD), 7.5wt% hydrogels containing 3 mM RGD were selected as these substrates did not initiate primitive tubule structures in 3D MVEC monocultures, thus providing a passive platform to study AF-MVEC interaction. The addition of AFs to hydrogels promoted MVEC viability; however, increasing AF density within hydrogels stimulated MVEC proliferation, increased microvessel density and size, and enhanced deposition of basement membrane proteins, collagen IV and laminin. Importantly, AF-MVEC communication through the transforming growth factor beta (TGF-β)/activin receptor-like kinase 5 (ALK5) signaling pathway was observed to mediate microvessel formation, as inhibition of ALK5 significantly decreased MVEC proliferation, microvessel formation, mural cell recruitment, and basement membrane production. These data indicate that AFs regulate MVEC neovasculogenesis and suggest that therapeutics targeting the TGF-β/ALK5 pathway may be useful for regulation of vasculogenic and anti-vasculogenic responses.

Radiation treatment in patients with head and neck tumors commonly results in hyposalivation and xerostomia due to the loss of fluid-secreting salivary acinar cells. Patients develop susceptibility to oral infections, dental caries, impaired speech and swallowing, reducing the quality of life. Clinical management is largely unsatisfactory. The development of a tissue-engineered, implantable salivary gland will greatly benefit patients suffering from xerostomia. This report compares the ability of a 2.5-dimensional (2.5D) and a three-dimensional (3D) hyaluronic acid (HA)-based culture system to support functional salivary units capable of producing fluid and phenotypic proteins. Parotid cells seeded on 2.5D, as well as those encapsulated in 3D HA hydrogels, self-assembled into acini-like structures and expressed functional neurotransmitter receptors. Structures in 3D hydrogels merged to form organized 50 μm spheroids that could be maintained in culture for over 100 days and merged to form structures over 500 μm in size. Treatment of acini-like structures with the β-adrenergic agonists norepinephrine or isoproterenol increased granule production and α-amylase staining in treated structures, demonstrating regain of protein secretion. Upon treatment with the M3 muscarinic agonist acetylcholine, acini-like structures activated the fluid production pathway by increasing intracellular calcium levels. The increase in intracellular calcium seen in structures in the 3D hydrogel culture system was more robust and prolonged than that in 2.5D. To compare the long-term survival and retention of acini-like structures in vivo , cell-seeded 2.5D and 3D hydrogels were implanted into an athymic rat model. Cells in 2.5D failed to maintain organized acini-like structures and dispersed in the surrounding tissue. Encapsulated cells in 3D retained their spheroid structure and structural integrity, along with the salivary biomarkers and maintained viability for over 3 weeks in vivo . This report identifies a novel hydrogel culture system capable of creating and maintaining functional 3D salivary spheroid structures for long periods in vitro that regain both fluid and protein secreting functions and are suitable for tissue restoration.

Successful engineering of functional salivary glands necessitates the creation of cell‐instructive environments for ex vivo expansion and lineage specification of primary human salivary gland stem cells (hS/PCs). Herein, basement membrane mimetic hydrogels are prepared using hyaluronic acid, cell adhesive peptides, and hyperbranched polyglycerol (HPG), with or without sulfate groups, to produce “hyperGel+” or “hyperGel”, respectively. Differential scanning fluorescence experiments confirm the ability of the sulfated HPG precursor to stabilize fibroblast growth factor 10. The hydrogels are nanoporous, cytocompatible, and cell‐permissive, enabling the development of multicellular hS/PC spheroids in 14 days. The incorporation of sulfated HPG species in the hydrogel enhances cell proliferation. Culture of hS/PCs in hyperGel+ in the presence of a Rho kinase inhibitor Y‐27632 (Y‐27) leads to the development of spheroids with a central lumen, increases the expression of acinar marker aquaporin‐3 at the transcript level ( AQP3 ), and decreases the expression of ductal marker keratin 7 at both the transcript ( KRT7 ) and the protein levels (K7). Reduced expression of transforming growth factor beta (TGF‐β) targets SMAD2/3 is also observed in Y27‐treated cultures, suggesting attenuation of TGF‐β signaling. Thus, hyperGel+ cooperates with the Rho‐associated protein kinase inhibitor to promote the development of lumened spheroids with enhanced expression of acinar markers.

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. Stem Cells Translational Medicine 2017;6:110–120

To study the individual functions of hyaluronan interacting proteins in prostate cancer (PCa) motility through connective tissues, we developed a novel three-dimensional (3D) hyaluronic acid (HA) hydrogel assay that provides a flexible, quantifiable, and physiologically relevant alternative to current methods. Invasion in this system reflects the prevalence of HA in connective tissues and its role in the promotion of cancer cell motility and tissue invasion, making the system ideal to study invasion through bone marrow or other HA-rich connective tissues. The bio-compatible cross-linking process we used allows for direct encapsulation of cancer cells within the gel where they adopt a distinct, cluster-like morphology. Metastatic PCa cells in these hydrogels develop fingerlike structures, \"invadopodia\", consistent with their invasive properties. The number of invadopodia, as well as cluster size, shape, and convergence, can provide a quantifiable measure of invasive potential. Among candidate hyaluronan interacting proteins that could be responsible for the behavior we observed, we found that culture in the HA hydrogel triggers invasive PCa cells to differentially express and localize receptor for hyaluronan mediated motility (RHAMM)/CD168 which, in the absence of CD44, appears to contribute to PCa motility and invasion by interacting with the HA hydrogel components. PCa cell invasion through the HA hydrogel also was found to depend on the activity of hyaluronidases. Studies shown here reveal that while hyaluronidase activity is necessary for invadopodia and inter-connecting cluster formation, activity alone is not sufficient for acquisition of invasiveness to occur. We therefore suggest that development of invasive behavior in 3D HA-based systems requires development of additional cellular features, such as activation of motility associated pathways that regulate formation of invadopodia. Thus, we report development of a 3D system amenable to dissection of biological processes associated with cancer cell motility through HA-rich connective tissues.

Background Osteoarthritis (OA) of the knee involves degeneration of articular cartilage of the diarthrodial joints. Current treatment options temporarily relieve the joint pain but do not restore the lost cartilage. We recently designed a novel bone morphogenetic protein receptor type I (BMPRI) mimetic peptide, CK2.1, that activates BMPRIa signaling in the absence of bone morphogenetic protein (BMP). Our previous research demonstrated that CK2.1 induced chondrogenesis in vitro and in vivo; however, it is unknown if CK2.1 restores damaged articular cartilage in vivo. In this study, we demonstrate that CK2.1 induced articular cartilage (AC) repair in an OA mouse model. Methods We designed hyaluronic acid (HA)-based hydrogel particles (HGPs) that slowly release CK2.1. HGP-CK2.1 particles were tested for chondrogenic potency on pluripotent mesenchymal stem cells (C3H10T1/2 cells) and locally injected into the intra-articular capsule in mice with cartilage defects. C57BL/6J mice were operated on to destabilize the medial meniscus and these mice were kept for 6 weeks after surgery to sustain OA-like damage. Mice were then injected via the intra-articular capsule with HGP-CK2.1; 4 weeks after injection the mice were sacrificed and their femurs were analyzed for cartilage defects. Results Immunohistochemical analysis of the cartilage demonstrated complete repair of the AC compared to sham-operated mice. Immunofluorescence analysis revealed collagen type IX production along with collagen type II in the AC of mice injected with HGP-CK2.1. Mice injected with phosphate-buffered saline (PBS) and HGP alone had greater collagen type X and osteocalcin production, in sharp contrast to those injected with HGP-CK2.1, indicating increased chondrocyte hypertrophy. Conclusions Our results demonstrate that the slow release HGP-CK2.1 drives cartilage repair without the induction of chondrocyte hypertrophy. The peptide CK2.1 could be a powerful tool in understanding the signaling pathways contributing to the repair process, and also may be used as a potential therapeutic for treating degenerative cartilage diseases such as OA.

The biomechanical function of the vocal folds (VFs) depends on their viscoelastic properties. Many conditions can lead to VF scarring that compromises voice function and quality. To identify candidate replacement materials, the structure, composition, and mechanical properties of native tissues need to be understood at phonation frequencies. Previously, the authors developed the torsional wave experiment (TWE), a stress-wave-based experiment to determine the linear viscoelastic shear properties of small, soft samples. Here, the viscoelastic properties of porcine and human VFs were measured over a frequency range of 10–200 Hz. The TWE utilizes resonance phenomena to determine viscoelastic properties; therefore, the specimen test frequency is determined by the sample size and material properties. Viscoelastic moduli are reported at resonance frequencies. Structure and composition of the tissues were determined by histology and immunochemistry. Porcine data from the TWE are separated into two groups: a young group, consisting of fetal and newborn pigs, and an adult group, consisting of 6–9-month olds and 2+-year olds. Adult tissues had an average storage modulus of 2309±1394 Pa and a loss tangent of 0.38±0.10 at frequencies of 36–200 Hz. The VFs of young pigs were significantly more compliant, with a storage modulus of 394±142 Pa and a loss tangent of 0.40±0.14 between 14 and 30 Hz. No gender dependence was observed. Histological staining showed that adult porcine tissues had a more organized, layered structure than the fetal tissues, with a thicker epithelium and a more structured lamina propria. Elastin fibers in fetal VF tissues were immature compared to those in adult tissues. Together, these structural changes in the tissues most likely contributed to the change in viscoelastic properties. Adult human VF tissues, recovered postmortem from adult patients with a history of smoking or disease, had an average storage modulus of 756±439 Pa and a loss tangent of 0.42±0.10. Contrary to the results of some other investigators, no significant frequency dependence was observed. This lack of observable frequency dependence may be due to the modest frequency range of the experiments and the wide range of stiffnesses observed within nominally similar sample types.

Vocal fold diseases and disorders are difficult to treat surgically or therapeutically. Tissue engineering offers an alternative strategy for the restoration of functional vocal folds. As a first step toward vocal fold tissue engineering, we investigated the responses of primary vocal fold fibroblasts (PVFFs) to two types of collagen and hyaluronic acid (HA)-based hydrogels that are compositionally similar, but structurally variable and mechanically different. Type A hydrogels were composed of mature collagen fibers reinforced by oxidized HA, whereas type B hydrogels contained immature collagen fibrils interpenetrated in an amorphous, covalently cross-linked HA matrix. PVFFs encapsulated in either matrix adopted a fibroblastic morphology and expressed genes related to important extracellular matrix proteins. DNA analysis indicated a linear growth profile for cells encapsulated in type B gels from day 0 to 21, in contrast to an initial dormant, nonproliferative period from day 0 to 3 experienced by cells in type A gels. At the end of the culture, similar DNA content was detected in both types of constructs. A reduction in collagen content was observed for both types of constructs after 28 days of culture, with type A constructs generally retaining higher amounts of collagen than type B constructs. The HA content in the constructs decreased steadily throughout the culture, with type A constructs consistently exhibiting less HA than type B constructs. Using the torsional wave analysis, we found that the elastic moduli for type A constructs decreased sharply during the first week of culture, followed by 2 weeks of matrix stabilization without significant changes in matrix stiffness. Conversely, the elastic modulus for type B constructs increased moderately over time. It is postulated that PVFFs residing in gels alter the matrix organization, chemical compositions, and viscoelasticity through cell-mediated remodeling processes.

Described herein is a protocol for producing a synthetic extracellular matrix that can be modified in situ during 3D cell culture. The hydrogel platform is established using modular building blocks employing bioorthogonal tetrazine (Tz) ligation with slow (norbornene, Nb) and fast (trans-cyclooctene, TCO) dienophiles. A cell-laden gel construct is created via the slow, off-stoichiometric Tz/Nb reaction. After a few days of culture, matrix properties can be altered by supplementing the cell culture media with TCO-tagged molecules through the rapid reaction with the remaining tetrazine groups in the network at the gel-liquid interface. Because Tz/TCO reaction is faster than molecular diffusion, matrix properties can be modified in a spatiotemporal fashion simply by altering the identity of the diffusive species and the diffusion time/path. Our strategy does not interfere with native biochemical processes, nor does it require external triggers or a second, independent chemistry. The biomimetic 3D cultures can be analyzed by standard molecular and cellular techniques and visualized by confocal microscopy. We have previously used this method to demonstrate how in situ modulation of matrix properties induces epithelial-to-mesenchymal transition, elicits fibroblast transition from mesenchymal stem cells, and regulates myofibroblast differentiation. Following the detailed procedures, individuals with bachelor's in science and engineering fields can successfully complete the protocol in four to five weeks. This protocol can be applied to model tissue morphogenesis and disease progression; it can also be used to establish engineered constructs with tissue-like anisotropy and tissue-specific functions. This protocol describes a bioorthogonal method for dynamically altering the adhesiveness or stiffness of the synthetic extracellular matrix during 3D culture in a spatiotemporal manner to induce phenotypic changes and to produce functional tissues. Bioorthogonal tuning of matrix properties during 3D cell culture to induce morphological and phenotypic changes. Tuning matrix properties during 3D cell culture