Evaluation of focal adhesion mediated subcellular curvature sensing in response to engineered extracellular matrix.

Fibril curvature is bioinstructive to attached cells. Similar to natural healthy tissues, an engineered extracellular matrix can be designed to stimulate cells to adopt desired phenotypes. To take full advantage of the curvature control in biomaterial fabrication methodologies, an understanding of the response to fibril subcellular curvature is required. In this work, we examined morphology, signaling, and function of human cells attached to electrospun nanofibers. We controlled curvature across an order of magnitude using nondegradable poly(methyl methacrylate) (PMMA) attached to a stiff substrate with flat PMMA as a control. Focal adhesion length and the distance of maximum intensity from the geographic center of the vinculin positive focal adhesion both peaked at a fiber curvature of 2.5 µm-1 (both ∼2× the flat surface control). Vinculin experienced slightly less tension when attached to nanofiber substrates. Vinculin expression was also more affected by a subcellular curvature than structural proteins α-tubulin or α-actinin. Among the phosphorylation sites we examined (FAK397, 576/577, 925, and Src416), FAK925 exhibited the most dependance on the nanofiber curvature. A RhoA/ROCK dependance of migration velocity across curvatures combined with an observation of cell membrane wrapping around nanofibers suggested a hybrid of migration modes for cells attached to fibers as has been observed in 3D matrices. Careful selection of nanofiber curvature for regenerative engineering scaffolds and substrates used to study cell biology is required to maximize the potential of these techniques for scientific exploration and ultimately improvement of human health.

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.

Polyphosphazene/nano-hydroxyapatite composite microsphere scaffolds for bone tissue engineering.

The nontoxic, neutral degradation products of amino acid ester polyphosphazenes make them ideal candidates for in vivo orthopedic applications. The quest for new osteocompatible materials for load bearing tissue engineering applications has led us to investigate mechanically competent amino acid ester substituted polyphosphazenes. In this study, we have synthesized three biodegradable polyphosphazenes substituted with side groups, namely, leucine, valine, and phenylalanine ethyl esters. Of these polymers, the phenylalanine ethyl ester substituted polyphosphazene showed the highest glass transition temperature (41.6 degrees C) and, hence, was chosen as a candidate material for forming composite microspheres with 100 nm sized hydroxyapatite (nHAp). The fabricated composite microspheres were sintered into a three-dimensional (3-D) porous scaffold by adopting a dynamic solvent sintering approach. The composite microsphere scaffolds showed compressive moduli of 46-81 MPa with mean pore diameters in the range of 86-145 microm. The 3-D polyphosphazene-nHAp composite microsphere scaffolds showed good osteoblast cell adhesion, proliferation, and alkaline phosphatase expression and are potential suitors for bone tissue engineering applications.

3D Near-Field Electrospinning of Biomaterial Microfibers with Potential for Blended Microfiber-Cell-Loaded Gel Composite Structures.

This paper describes the development of a novel low-cost and efficient method, 3D near-field electrospinning, to fabricate high-resolution, and repeatable 3D polymeric fiber patterns on nonconductive materials with potential use in tissue engineering. This technology is based on readily available hobbyist grade 3D printers. The result is exquisite control of the deposition of single fibers in an automated manner. Additionally, the fabrication of various fiber patterns, which are subsequently translated to unique cellular patterns, is demonstrated. Finally, poly(methyl methacrylate) fibers are printed within 3D collagen gels loaded with cells to introduce anisotropic properties of polymeric fibers within the cell-loaded gels.

Polymeric nanoparticles: the future of nanomedicine.

Polymeric nanoparticles (NPs) are one of the most studied organic strategies for nanomedicine. Intense interest lies in the potential of polymeric NPs to revolutionize modern medicine. To determine the ideal nanosystem for more effective and distinctly targeted delivery of therapeutic applications, particle size, morphology, material choice, and processing techniques are all research areas of interest. Utilizations of polymeric NPs include drug delivery techniques such as conjugation and entrapment of drugs, prodrugs, stimuli-responsive systems, imaging modalities, and theranostics. Cancer, neurodegenerative disorders, and cardiovascular diseases are fields impacted by NP technologies that push scientific boundaries to the leading edge of transformative advances for nanomedicine.

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.

Synthesis and characterization of biomimetic citrate-based biodegradable composites.

Natural bone apatite crystals, which mediate the development and regulate the load-bearing function of bone, have recently been associated with strongly bound citrate molecules. However, such understanding has not been translated into bone biomaterial design and osteoblast cell culture. In this work, we have developed a new class of biodegradable, mechanically strong, and biocompatible citrate-based polymer blends (CBPBs), which offer enhanced hydroxyapatite binding to produce more biomimetic composites (CBPBHAs) for orthopedic applications. CBPBHAs consist of the newly developed osteoconductive citrate-presenting biodegradable polymers, crosslinked urethane-doped polyester and poly (octanediol citrate), which can be composited with up to 65 wt % hydroxyapatite. CBPBHA networks produced materials with a compressive strength of 116.23 ± 5.37 MPa comparable to human cortical bone (100-230 MPa), and increased C2C12 osterix gene and alkaline phosphatase gene expression in vitro. The promising results above prompted an investigation on the role of citrate supplementation in culture medium for osteoblast culture, which showed that exogenous citrate supplemented into media accelerated the in vitro phenotype progression of MG-63 osteoblasts. After 6 weeks of implantation in a rabbit lateral femoral condyle defect model, CBPBHA composites elicited minimal fibrous tissue encapsulation and were well integrated with the surrounding bone tissues. The development of citrate-presenting CBPBHA biomaterials and preliminary studies revealing the effects of free exogenous citrate on osteoblast culture shows the potential of citrate biomaterials to bridge the gap in orthopedic biomaterial design and osteoblast cell culture in that the role of citrate molecules has previously been overlooked.

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.

Composite scaffolds: bridging nanofiber and microsphere architectures to improve bioactivity of mechanically competent constructs.

Tissue engineering often benefits from the use of composites to produce an ideal scaffold. We present the focused development of a novel structure that combines the biomimetic properties of nanofibers with the robust mechanical aspects of the sintered microsphere scaffold to produce a composite scaffold that demonstrates an ability to mimic the mechanical environment of trabecular bone while also promoting the phenotype progression of osteoblast progenitor cells. These composite nanofiber/microsphere scaffolds exhibited a mechanical modulus and compressive strength similar to trabecular bone and exhibited degradation resulting in a mass loss of 30% after 24 weeks. The nanofiber portion of these scaffolds was sufficiently porous to allow cell migration throughout the fibrous portion of the scaffold and promoted phenotype progression through focal adhesion kinase-mediated activation of the transcription factor Runx2, control scaffolds not containing nanofibers did not demonstrate extensive cell migration or phenotype progression. Ultimately, the focal adhesion kinase activity on the composite nanofiber/microsphere scaffolds demonstrated causality over the production of the mature osteoblast marker, osteocalcin, and the development of a calcified matrix.

Biomimetic, bioactive etheric polyphosphazene-poly(lactide-co-glycolide) blends for bone tissue engineering.

The long-term goal of this work is to develop biomimetic polymer-based systems for bone regeneration that both allow for neutral pH degradation products and have the ability to nucleate bonelike apatite. In this study, the etheric biodegradable polyphosphazene, poly[(50%ethyl glycinato)(50%methoxyethoxyethoxy)phosphazene] (PNEG(50)MEEP(50)) was blended with poly(lactide-co-glycolide) PLAGA and studied their ability to produce high-strength degradable biomaterials with bioactivity. Accordingly, two blends with weight ratios of PNEG(50)MEEP(50) to PLAGA 25:75 (BLEND25) and 50:50 (BLEND50) were fabricated using a mutual solvent approach. Increases in PNEG(50)MEEP(50) content in the blend system resulted in decreased elastic modulus of 779 MPa when compared with 1684 MPa (PLAGA) as well as tensile strength 7.9 MPa when compared with 25.7 MPa (PLAGA). However, the higher PNEG(50)MEEP(50) content in the blend system resulted in higher Ca/P atomic ratio of the apatite layer 1.35 (BLEND50) when compared with 0.69 (BLEND25) indicating improved biomimicry. Furthermore, these blends supported primary rat osteoblast adhesion and proliferation with an enhanced phenotypic expression when compared with PLAGA. These findings establish the suitability of PNEG(50)MEEP(50)-PLAGA biodegradable blends as promising bioactive materials for orthopedic applications.

Solvent/non-solvent sintering: a novel route to create porous microsphere scaffolds for tissue regeneration.

Solvent/non-solvent sintering creates porous polymeric microsphere scaffolds suitable for tissue engineering purposes with control over the resulting porosity, average pore diameter, and mechanical properties. Five different biodegradable biocompatible polyphosphazenes exhibiting glass transition temperatures from -8 to 41 degrees C and poly (lactide-co-glycolide), (PLAGA) a degradable polymer used in a number of biomedical settings, were examined to study the versatility of the process and benchmark the process to heat sintering. Parameters such as: solvent/non-solvent sintering solution composition and submersion time effect the sintering process. PLAGA microsphere scaffolds fabricated with solvent/non-solvent sintering exhibited an interconnected porosity and pore size of 31.9% and 179.1 mum, respectively which was analogous to that of conventional heat sintered PLAGA microsphere scaffolds. Biodegradable polyphosphazene microsphere scaffolds exhibited a maximum interconnected porosity of 37.6% and a maximum compressive modulus of 94.3 MPa. Solvent/non-solvent sintering is an effective strategy for sintering polymeric microspheres, with a broad spectrum of glass transition temperatures, under ambient conditions making it an excellent fabrication route for developing tissue engineering scaffolds and drug delivery vehicles.