Padma P. Srinivasan

Injectable perlecan domain 1-hyaluronan microgels potentiate the cartilage repair effect of BMP2 in a murine model of early osteoarthritis.

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.

Primary Salivary Human Stem/Progenitor Cells Undergo Microenvironment-Driven Acinar-Like Differentiation in Hyaluronate Hydrogel Culture

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

Inhibition of T-Type Voltage Sensitive Calcium Channel Reduces Load-Induced OA in Mice and Suppresses the Catabolic Effect of Bone Mechanical Stress on Chondrocytes

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.

Bottom-up assembly of salivary gland microtissues for assessing myoepithelial cell function

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 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.

Leptin inhibitor as a novel therapeutic for osteoarthritis

Osteoarthritis (OA) is a progressive disorder characterized by articular cartilage attrition and metabolic changes in the subchondral bone and the synovium. Recently, leptin was shown to be elevated in synovial fluid of patients with OA. Here, we propose to use leptin inhibitor as intraarticular injections to slow/prevent OA progression.

171 PERLECAN-HA BASED BIOMATRICES PROLONG THE ANABOLIC EFFECT OF BMP2 IN A MOUSE MODEL OF EARLY KNEE OSTEOARTHRITIS

Purpose: Stem cell therapy has emerged as a promising approach for orthopaedic treatment because of their potential ability of regenerating damaged tissues. One of the challenges is the ability to track implanted cells noninvasively following implantation. Several papers have addressed the topic of visualizing and tracking transplanted cells non-invasively using superparamagnetic iron oxide (SPIO) in magnetic resonance imaging (MRI). The aim of this study is to determine if SPIO internalization interferes with the chondrogenic differentiation ability of human mesenchymal stem cells (MSCs) seeded in collagen sponge biomaterials. Methods: Human bone marrow-derived mesenchymal stem cells (hMSCs) were expanded in monolayer and are first labeled with different SPIO concentrations between 0 and 200 μg/ml Fe. The endorem-labeled hMSCs were cultured into collagen sponge during 28 days with or without TGF-β1 to induce chondrogenic differentiation. Chondrogenic gene expression (coll 2, coll 1, coll 10, aggrecan, versican, COMP, sox 9, osteocalcin, alkaline phosphatase) were investigated by using real-time quantitative RT-PCR. Synthesized extracellular matrix inside sponge was assessed histologically (proteoglycan and collagen content, SPIO detection) and immunohistochemically (coll 1, coll 2). Results: TGF-β1 induced an increase of chondrogenic gene expression (col 2, aggrecan, Sox 9, COMP) and an abundant synthesized extracellular matrix inside sponge. SPIO labeling of MSC interfered with chondrogenesis: a dose-dependent decrease of gene expression such as aggrecan, Coll 1, coll 2, Sox 9, COMP was observed in SPIO labeled cells. Histologically, the quality and the quantity of synthesized matrix were affected by SPIO labeling in a dose dependant manner. The low concentration 12.5 μg/ml Fe induced a small decrease of proteoglycan and collagen 2 content. From 50 μg/ml Fe, a marked inhibition of chondrogenesis was observed: extracellular matrix was poor in proteoglycan and collagen contents. Conclusions: We observed a dose-dependent deleterious effect of SPIO concentration on the chondrogenic differentiation of human MSCs. The use of SPIO labeling to monitor cell-based therapies in MSC driven articular cartilage engineering requires very low SPIO concentration to minimize significant influence cell metabolism.