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

The goal of this study was to use bioengineered injectable microgels to enhance the action of bone morphogenetic protein 2 (BMP2) and stimulate cartilage matrix repair in a reversible animal model of osteoarthritis (OA). A module of perlecan (PlnD1) bearing heparan sulfate (HS) chains was covalently immobilized to hyaluronic acid (HA) microgels for the controlled release of BMP2 in vivo. Articular cartilage damage was induced in mice using a reversible model of experimental OA and was treated by intra-articular injection of PlnD1-HA particles with BMP2 bound to HS. Control injections consisted of BMP2 free PlnD1-HA particles, HA particles, free BMP2 or saline. Knees dissected following these injections were analyzed using histological, immunostaining and gene expression approaches. Our results show that knees treated with PlnD1-HA/BMP2 had lesser OA-like damage compared to control knees. In addition, the PlnD1-HA/BMP2-treated knees had higher mRNA levels encoding for type II collagen, proteoglycans, and xylosyltransferase 1, a rate-limiting anabolic enzyme involved in the biosynthesis of glycosaminoglycan chains, relative to control knees (PlnD1-HA). This finding was paralleled by enhanced levels of aggrecan in the articular cartilage of PlnD1-HA/BMP2 treated knees. Additionally, decreases in the mRNA levels encoding for cartilage-degrading enzymes and type X collagen were seen relative to controls. In conclusion, PlnD1-HA microgels constitute a formulation improvement compared to HA for efficient in vivo delivery and stimulation of proteoglycan and cartilage matrix synthesis in mouse articular cartilage. Ultimately, PlnD1-HA/BMP2 may serve as an injectable therapeutic agent for slowing or inhibiting the onset of OA after knee injury.

Myoepithelial cells are flat, stellate cells present in exocrine tissues including the salivary glands. While myoepithelial cells have been studied extensively in mammary and lacrimal gland tissues, less is known of the function of myoepithelial cells derived from human salivary glands. Several groups have isolated tumorigenic myoepithelial cells from cancer specimens, however, only one report has demonstrated isolation of normal human salivary myoepithelial cells needed for use in salivary gland tissue engineering applications. Establishing a functional organoid model consisting of myoepithelial and secretory acinar cells is therefore necessary for understanding the coordinated action of these two cell types in unidirectional fluid secretion. Here, we developed a bottom-up approach for generating salivary gland microtissues using primary human salivary myoepithelial cells (hSMECs) and stem/progenitor cells (hS/PCs) isolated from normal salivary gland tissues. Phenotypic characterization of isolated hSMECs confirmed that a myoepithelial cell phenotype consistent with that from other exocrine tissues was maintained over multiple passages of tissue culture. Additionally, hSMECs secreted basement membrane proteins, expressed adrenergic and cholinergic neurotransmitter receptors, and released intracellular calcium [Ca2+i] in response to parasympathetic agonists. In a collagen I contractility assay, activation of contractile machinery was observed in isolated hSMECs treated with parasympathetic agonists. Recombination of hSMECs with assembled hS/PC spheroids in a microwell system was used to create microtissues resembling secretory complexes of the salivary gland. We conclude that the engineered salivary gland microtissue complexes provide a physiologically relevant model for both mechanistic studies and as a building block for the successful engineering of the salivary gland for restoration of salivary function in patients suffering from hyposalivation.

Voltage-sensitive calcium channels (VSCC) regulate cellular calcium influx, one of the earliest responses to mechanical stimulation in osteoblasts. Here, we postulate that T-type VSCCs play an essential role in bone mechanical response to load and participate in events leading to the pathology of load-induced OA. Repetitive mechanical insult was used to induce OA in Cav3.2 T-VSCC null and wild-type control mouse knees. Osteoblasts (MC3T3-E1) and chondrocytes were treated with a selective T-VSCC inhibitor and subjected to fluid shear stress to determine how blocking of T-VSCCs alters the expression profile of each cell type upon mechanical stimulation. Conditioned-media (CM) obtained from static and sheared MC3T3-E1 was used to assess the effect of osteoblast-derived factors on the chondrocyte phenotype. T-VSCC null knees exhibited significantly lower focal articular cartilage damage than age-matched controls. In vitro inhibition of T-VSCC significantly reduced the expression of both early and late mechanoresponsive genes in osteoblasts but had no effect on gene expression in chondrocytes. Furthermore, treatment of chondrocytes with CM obtained from sheared osteoblasts induced expression of markers of hypertrophy in chondrocytes and this was nearly abolished when osteoblasts were pre-treated with the T-VSCC-specific inhibitor. These results indicate that T-VSCC plays a role in signaling events associated with induction of OA and is essential to the release of osteoblast-derived factors that promote an early OA phenotype in chondrocytes. Further, these findings suggest that local inhibition of T-VSCC may serve as a therapy for blocking load-induced bone formation that results in cartilage degeneration.

BackgroundOsteoarthritis (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.MethodsWe 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.ResultsImmunohistochemical 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.ConclusionsOur 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.

Increased proteoglycan (PG) synthesis is essential for the stimulation of cartilage repair processes that take place during the reversible phase of osteoarthritis (OA). In articular cartilage, xylosyltransferase 1 (Xylt1) is the key enzyme that initiates glycosaminoglycan (GAG) chain synthesis by transferring the first sugar residue to the PG core protein. Biological activity of PGs is closely linked to GAG biosynthesis since their polyanionic nature directly contributes to the proper hydration and elastic properties of the cartilage tissue present at the articular interface. The aim of this study was to investigate whether variations in the level of Xylt1 present in serum can be used to predict OA disease progression. The influence of bone forming activity on the systemic release of this enzyme was addressed by experimentally-inducing OA in mice of two different genetic backgrounds that were previously characterized for their distinct bone metabolism: C57BL/6J (B6, high bone remodelers) or C3H/HeJ (C3H, high bone formers). Serum was collected after medial meniscectomy or sham surgeries in young adult mice of these two strains over a period of 3.5 months at which point knee histopathology was assessed. A significant increase in serum Xylt1 levels observed shortly after meniscectomy positively correlated with severe cartilage damage evaluated by histological assessment at later time points in mice of the C3H background. In contrast, no temporal regulation of Xylt1 level was found between meniscectomies and control surgeries in B6 mice, which developed OA at a slower rate. Additionally, longitudinal evaluation of the serum levels of other markers of cartilage/bone metabolism (C1,2C, osteocalcin) did not reveal any association with late knee damages. Our results strongly support the idea that serum Xylt1 has a clinical value for monitoring risk of OA progression in young adults with high bone forming potential. Ultimately, the understanding of posttraumatic mechanisms regulating PG synthesis and their modification by GAG will be essential so that interventions that stimulate cartilage regrowth can be undertaken prior to irreversible destruction of the joint tissue.

Distal appendages play essential roles in ciliary membrane anchoring and ciliary protein recruitment. Previously we have revealed a hierarchical relationship among distal appendage proteins, including CEP83, CEP89, SCLT1, CEP164, and FBF1. Despite their importance, their relative placement on distal appendages remains largely unknown due to the small length scale beyond the diffraction limit. Using dual-color direct stochastic optical reconstruction microscopy (dSTORM), here we revealed a staggered arrangement of CEP83, CEP89, SCLT1, CEP164, and FBF1, with surprising alternating arrangement of CEP164 and FBF1. We also found the cell cycle-dependent distribution of TTBK2 on distal appendages, illustrating the coupling between cell cycle and ciliogenesis. Together, we have determined the hierarchical architecture of the distal appendages, suggesting their potential membrane interaction site and kinase recruiting site.

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