Developing Fibrous Biomaterials to Modulate Epithelial-to-Mesenchymal Transition.

Despite their critical roles in tissue repair and pathological processes such as fibrosis, tumor invasion, and metastasis, the origins of mesenchymal cells remain poorly understood. Among the likely routes, epithelial-to-mesenchymal transitions (EMTs) emerge as important source of these cells. EMTs manifest themselves as a phenotypic transition in terminally differentiated epithelial cells into mesenchymal cells which are closely related to embryogenesis and organ development as well as in chronically inflamed tissues and neoplasia. There exists a potential for successful engineering of biomimetic environments that closely reflects and reciprocates the dynamic changes in the cellular microenvironment during EMT and relies on integrating the mechanical sensing mechanisms found in the native tissues into the synthetic scaffolds to understand cellular plasticity. Extracellular matrix (ECM) has complex structures composed of a collection of extracellular molecules including fibrous proteins and glycoproteins in a hydrated mixture of glycosaminoglycans and proteoglycans. Therefore, fibrous materials have been increasingly applied in tissue engineering applications since biomaterials need to restore ECM structures to provide physical, biochemical, and biomechanical signals to define cellular behaviors and tissue functions. This review summarizes materials used for fibrous scaffolds including natural and synthetic materials, highlights recent development of fabrication techniques, characteristic architectures, and properties and different applications of fibrous scaffolds in tissue engineering. The prospects and challenges about fibrous materials in tissue engineering applications are also discussed. Finally, we summarized relevant bioengineering approaches to modulate each type of EMT as potential avenues to consider toward future biomaterials design.

In Vitro and In Vivo Assessment of the Infection Resistance and Biocompatibility of Small-Molecule-Modified Polyurethane Biomaterials.

Bacterial intracellular nucleotide second messenger signaling is involved in biofilm formation and regulates biofilm development. Interference with the bacterial nucleotide second messenger signaling provides a novel approach to control biofilm formation and limit microbial infection in medical devices. In this study, we tethered small-molecule derivatives of 4-arylazo-3,5-diamino-1H-pyrazole on polyurethane biomaterial surfaces and measured the biofilm resistance and initial biocompatibility of modified biomaterials in in vitro and in vivo settings. Results showed that small-molecule-modified surfaces significantly reduced the Staphylococcal epidermidis biofilm formation compared to unmodified surfaces and decreased the nucleotide levels of c-di-AMP in biofilm cells, suggesting that the tethered small molecules interfere with intracellular nucleotide signaling and inhibit biofilm formation. The hemocompatibility assay showed that the modified polyurethane films did not induce platelet activation or red blood cell hemolysis but significantly reduced plasma coagulation and platelet adhesion. The cytocompatibility assay with fibroblast cells showed that small-molecule-modified surfaces were noncytotoxic and cells appeared to be proliferating and growing on modified surfaces. In a 7-day subcutaneous infection rat model, the polymer samples were implanted in Wistar rats and inoculated with bacteria or PBS. Results show that modified polyurethane significantly reduced bacteria by ∼2.5 log units over unmodified films, and the modified polymers did not lead to additional irritation/toxicity to the animal tissues. Taken together, the results demonstrated that small molecules tethered on polymer surfaces remain active, and the modified polymers are biocompatible and resistant to microbial infection in vitro and in vivo.

Improving mechanical and antibacterial properties of PMMA via polyblend electrospinning with silk fibroin and polyethyleneimine towards dental applications.

Poly(methylmethacrylate) (PMMA) is a widely used material in dental applications, particularly as denture resins. Due to thermally unstable and wet oral cavity, the implanted PMMA based resins occasionally deform and grow bacterial biofilms at the interface between oral cavity and the biomaterial. Several strategies attempted earlier to improve the bacterial resistance and mechanical performance of PMMA. Poly(ethyleneimine) (PEI) is a hyperbranched cationic polymer shown earlier to improve antibacterial activity of resins but do not improve mechanical properties of the resins alone, while silk fibroin (SF) is a natural biopolymer with unique material properties. In this study, we combined SF and PEI towards development of antibacterial and mechanically superior PMMA based materials towards overcoming its drawbacks. Using polyblend electrospinning to combine SF, PEI and PMMA, we successfully developed intrinsically antibacterial and mechanically reinforced nanofiber mats. We propose that the resulting nanofiber mats have the potential to be incorporated into PMMA based denture resin materials to overcome the problems of patients and improve their quality of life.

Identification of Key Signaling Pathways Orchestrating Substrate Topography Directed Osteogenic Differentiation Through High-Throughput siRNA Screening.

Fibrous scaffolds are used for bone tissue engineering purposes with great success across a variety of polymers with different physical and chemical properties. It is now evident that the correct degree of curvature promotes increased cytoskeletal tension on osteoprogenitors leading to osteogenic differentiation. However, the mechanotransductive pathways involved in this phenomenon are not fully understood. To achieve a reproducible and specific cellular response, an increased mechanistic understanding of the molecular mechanisms driving the fibrous scaffold mediated bone regeneration must be understood. High throughput siRNA mediated screening technology has been utilized for dissecting molecular targets that are important in certain cellular phenotypes. In this study, we used siRNA mediated gene silencing to understand the osteogenic differentiation observed on fibrous scaffolds. A high-throughput siRNA screen was conducted using a library collection of 863 genes including important human kinase and phosphatase targets on pre-osteoblast SaOS-2 cells. The cells were grown on electrospun poly(methyl methacrylate) (PMMA) scaffolds with a diameter of 0.938 ± 0.304 µm and a flat surface control. The osteogenic transcription factor RUNX2 was quantified with an in-cell western (ICW) assay for the primary screen and significant targets were selected via two sample t-test. After selecting the significant targets, a secondary screen was performed to identify osteoinductive markers that also effect cell shape on fibrous topography. Finally, we report the most physiologically relevant molecular signaling mechanisms that are involved in growth factor free, fibrous topography mediated osteoinduction. We identified GTPases, membrane channel proteins, and microtubule associated targets that promote an osteoinductive cell shape on fibrous scaffolds.

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.

Regulation of Epithelial-to-Mesenchymal Transition Using Biomimetic Fibrous Scaffolds.

Epithelial-to-mesenchymal transition (EMT) is a well-studied biological process that takes place during embryogenesis, carcinogenesis, and tissue fibrosis. During EMT, the polarized epithelial cells with a cuboidal architecture adopt an elongated fibroblast-like morphology. This process is accompanied by the expression of many EMT-specific molecular markers. Although the molecular mechanism leading to EMT has been well-established, the effects of matrix topography and microstructure have not been clearly elucidated. Synthetic scaffolds mimicking the meshlike structure of the basement membrane with an average fiber diameter of 0.5 and 5 µm were produced via the electrospinning of poly(ε-caprolactone) (PCL) and were used to test the significance of fiber diameter on EMT. Cell-adhesive peptide motifs were conjugated to the fiber surface to facilitate cell attachment. Madin-Darby Canine Kidney (MDCK) cells grown on these substrates showed distinct phenotypes. On 0.5 µm substrates, cells grew as compact colonies with an epithelial phenotype. On 5 µm scaffolds, cells were more individually dispersed and appeared more fibroblastic. Upon the addition of hepatocyte growth factor (HGF), an EMT inducer, cells grown on the 0.5 µm scaffold underwent pronounced scattering, as evidenced by the alteration of cell morphology, localization of focal adhesion complex, weakening of cell-cell adhesion, and up-regulation of mesenchymal markers. In contrast, HGF did not induce a pronounced scattering of MDCK cells cultured on the 5.0 µm scaffold. Collectively, our results show that the alteration of the fiber diameter of proteins found in the basement membrane may create enough disturbances in epithelial organization and scattering that might have important implications in disease progression.

Tuning Hydrogel Properties to Promote the Assembly of Salivary Gland Spheroids in 3D.

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.

Biomaterials-based strategies for salivary gland tissue regeneration.

The salivary gland is a complex, secretory tissue that produces saliva and maintains oral homeostasis. Radiation induced salivary gland atrophy, manifested as "dry mouth" or xerostomia, poses a significant clinical challenge. Tissue engineering recently has emerged as an alternative, long-term treatment strategy for xerostomia. In this review, we summarize recent efforts towards the development of functional and implantable salivary glands utilizing designed polymeric substrates or synthetic matrices/scaffolds. Although the in vitro engineering of a complex implantable salivary gland is technically challenging, opportunities exist for multidisciplinary teams to assemble implantable and secretory tissue modules by combining stem/progenitor cells found in the adult glands with biomimetic and cell-instructive materials.

Molecular mechanisms orchestrating the stem cell response to translational scaffolds.

A 2013 Perspective in Science titled "Deconstructing Dimensionality" noted the importance of fiber morphology on cell phenotype, concluding with the statement "Identifying the mechanisms by which cells assess the nature of their environment will advance basic cell biology and facilitate the development of synthetic matrices for specific tissue engineering applications." Nanofibers have revolutionized scaffold-based approaches for musculoskeletal tissues; demonstrating surprising efficacy over promoting mesenchymal stem cell, MSC, differentiation down multiple musculoskeletal lineages. Understanding the fundamental mechanisms involved will allow the future design of nanofiber-based scaffolds to target a lineage with specificity. This article focuses on how three geometry sensors: focal adhesions, membrane associated vesicle stabilizing and trafficking proteins, and adherens junctions; potentially regulate MSC lineage commitment in response to bio-instructive nanofibers.

Osteoinductive biomaterial geometries for bone regenerative engineering.

Worldwide, more than 2.2 million patients undergo bone graft procedures annually. In each of these procedures an interface is formed between the host tissue and the graft material. Synthetic implants exhibit an interface with the host tissue and the formation of a homogenous interface consisting of bone and void of intervening soft tissue is desired (osseointegration); recent developments have highlighted the benefit of incorporating nanostructures at that interface. Autograft and allograft bone are frequently used for bone loss, nonunion fractures, and spinal fusions; however, both are plagued with complications either due to supply or inadequate graft properties. In contrast to bone tissue engineering, which uses a top-down approach to repair bone defects, bone regenerative engineering uses a bottom-up approach focused on strategies incorporating stem cells, biomaterials, and growth factors alone or in combination to generate or regenerate bone tissue. Early constructs developed for bone regenerative engineering utilized polymeric microstructures, presenting surface features with characteristic dimensions similar to that of a cell (1µm - 1000µm). These microstructures were typically biodegradable and demonstrated an excellent ability to match the mechanics of native bone tissue. They were also osteoconductive-capable of promoting osteoblast growth. On the other hand, the osteoinductive abilities of these microstructures were lacking. Osteoinduction, or the ability to promote the progression of a preosteoblastic cell to a mature osteoblast, historically was achieved in two ways: via the addition of nanoscale ceramics to the microstructures or via an external stimulus such as the addition of bone morphogenetic proteins (BMPs). More recent developments in bone regenerative engineering have utilized polymeric nanostructures (less than 1µm) with characteristic dimensions an order of magnitude or more less than that of a cell to stimulate and drive an osteoinductive response in the absence of growth factors. Despite strong literature evidence supporting the nanostructures' ability to be both osteoconductive and osteoinductive, there is still disparity regarding how nanostructures regulate the progression towards an osteoblastic phenotype. This review will explore unique micro- and nano-architectures, how they initiate osteoinductive signals through pathways similar to BMPs, and how these unique geometries can be translated to the clinic.

Substrate curvature sensing through Myosin IIa upregulates early osteogenesis.

Topographical cues mimicking the extracellular matrix (ECM) have demonstrated control over a diverse range of cellular behaviours including: initial adhesion, migration, cell growth, differentiation and death. How cells sense, and in turn translate, the topographical cues remains to be answered, but likely involves interactions through interfacial forces that influence cytoskeletal structure and integrin clustering, leading to the downstream activity of intracellular signalling cascades. Electrospun fibers have shown significant success as a biomimetic topography for bone tissue engineering applications, but mechanisms by which osteoprogenitor cells translate the fiber geometry into intracellular signalling activity is only recently being examined. We hypothesized that increased cellular differentiation observed on fibrous topography is due to acto-myosin contractility and cellular stiffness via the small GTPase RhoA. In order to evaluate this hypothesis, MC3T3-E1 osteoprogenitor cells were grown on poly(methyl methacrylate) (PMMA) fibers of 1.153 ± 0.310 µm diameter. The elastic modulus of the cell surface was measured by atomic force microscopy (AFM) with a colloidal probe. Overall cellular stiffness was found to increase more than three-fold in osteoprogenitors adhered to a fiber, as opposed to those grown on a flat substrate. Pharmacological inhibition of RhoA signalling activity decreased cellular stiffness and cytoskeletal integrity of osteoprogenitors growing on fibrous substrates. Finally, we demonstrated not only RhoA activity through its effector Rho-associated coiled coil kinase II (ROCKII), but also Myosin IIa promotes early osteogenic differentiation, as shown by alkaline phosphatase (ALP) staining. Previous studies have demonstrated the importance of ROCKII on early differentiation. Our results shed light on mechanisms underlying geometry sensing by highlighting the role of Myosin IIa in addition to ROCKII and could ultimately contribute to scaffold design strategies.