The influence of nanotopographical structures on osteoblast adhesion formation and the functional response of mesenchymal stem cell populations

It is predicted that the percentage of persons over 50 years of age affected by bone diseases will double by 2020 (Navarro et al., 2008). Clearly this represents a need for permanent, temporary or biodegradable orthopaedic devices that are designed to substitute or guide bone repair. Polymeric medical devices are widely used in orthopaedic surgery and play a key role in fracture fixation and in areas of orthopaedic implant design. Initial uncertainty regarding the adequacy of polymeric materials to withstand functional stresses obliged clinicians to implement these biomaterials in non-load-bearing applications such as fixation of the maxillofacial skeleton. Strategies to guide bone repair, have included topographical modification of these devices in an attempt to regulate cellular adhesion, a process fundamental in the initiation of osteoinduction and osteogenesis. Advances in fabrication techniques have evolved the field of surface modification and, in particular, nanotechnology has allowed the development of experimental nanoscale substrates for the investigation into cell-nanofeature interactions. This thesis is concerned with the study of nanotopographical structures on osteoblast adhesion and mesenchymal stem cell (MSC) function, with an aim to improving the functionality of orthopaedic craniomaxillofacial devices. In this study primary human osteoblast (HOBs) were cultured on nanoscale topographies fabricated by lithographic and phase separation techniques in poly(methyl methacrylate) (pMMA). Adhesion subtypes in HOBs were quantified by immunofluorescent microscopy and cell-substrate interactions investigated via immunocytochemistry with scanning electron microscopy. To investigate the effects of these substrates on cellular function 1.7 K microarray analysis was employed to study the changes in gene profiles of enriched MSC populations cultured on these nanotopographies. Nanotopography differentially affected the formation of adhesions in HOBs and induced significant changes in genetic expression of MSCs on experimental substrates. Nanopit type topographies fabricated by electron beam lithography were shown to inhibit directly the formation of large adhesion complexes in HOBs and induce significant down-regulation of canonical signalling and functional pathways in MSCs. Nanocrater and nanoisland type topographies fabricated by polymer demixing however reduced adhesion formation and induced up-regulation of osteospecific pathways. Nanogrooved topographies fabricated by photolithography influenced HOB adhesion formation and MSC osteospecific function in a manner dependant on the groove width. The findings of this study indicate that nanotopographical modification significantly modulates both osteoblast adhesion and MSC function, implicating topographical modification as a viable strategy to enhance orthopaedic device functionality

Sensing the difference: the influence of anisotropic cues on cell behavior

From tissue morphogenesis to homeostasis, cells continuously experience and respond to physical, chemical and biological cues commonly presented in gradients. In this article we focus our discussion on the importance of nano/micro topographic cues on cell activity, and the role of anisotropic milieus play on cell behavior, mostly adhesion and migration. We present the need to study physiological gradients in vitro. To do this, we review different cell migration mechanisms and how adherent cells react to the presence of complex tissue-like environments and cell-surface stimulation in 2D and 3D (e.g. ventral/dorsal anisotropy)

Advances in functional assemblies for regenerative medicine

The ability to synthesise bioresponsive systems and selectively active biochemistries using polymer-based materials with supramolecular features has led to a surge in research interest directed towards their development as next generation biomaterials for drug delivery, medical device design and tissue engineering

Responsive biomaterials:advances in materials based on shape-memory polymers

Shape-memory polymers (SMPs) are morphologically responsive materials with potential for a variety of biomedical applications, particularly as devices for minimally invasive surgery and the delivery of therapeutics and cells for tissue engineering. A brief introduction to SMPs is followed by a discussion of the current progress toward the development of SMP-based biomaterials for clinically relevant biomedical applications

Osteogenic lineage restriction by osteoprogenitors cultured on nanometric grooved surfaces – the role of focal adhesion maturation

The differentiation of progenitor cells is dependent on more than biochemical signalling. Topographical cues in natural bone extracellular matrix guide cellular differentiation through the formation of focal adhesions, contact guidance, cytoskeletal rearrangement and ultimately gene expression. Osteoarthritis and a number of bone disorders present as growing challenges for our society. Hence, there is a need for next generation implantable devices to substitute for, or guide, bone repair in vivo. Cellular responses to nanometric topographical cues need to be better understood in vitro in order to ensure the effective and efficient integration and performance of these orthopaedic devices. In this study, the FDA approved plastic polycaprolactone, was embossed with nanometric grooves and the response of primary and immortalised osteoprogenitor cells observed. Nanometric groove dimensions were 240 nm or 540 nm deep and 12.5 μm wide. Cells cultured on test surfaces followed contact guidance along the length of groove edges, elongated along their major axis and showed nuclear distortion, they formed more focal complexes and a lower proportions of mature adhesions relative to planar controls. Down-regulation of the osteoblast marker genes RUNX2 and BMPR2 in primary and immortalised cells was observed on grooved substrates. Down-regulation appeared to directly correlate with focal adhesion maturation, indicating the involvement of ERK 1/2 negative feedback pathways following integrin mediated FAK activation

Enhanced osteoconductivity on electrically charged titanium implants treated by physicochemical surface modifications methods

Biomimetic design is a key tenet of orthopedic device technology, and in particular the development of responsive surfaces that promote ion exchange with interfacing tissues, facilitating the ionic events that occur naturally during bone repair, hold promise in orthopedic fixation strategies. Non-bioactive nanostructured titanium implants treated by shot-blasting and acid-etching (AE) induced higher bone implant contact (BIC=52% and 65%) compared to shot-blasted treated (SB) implants (BIC=46% and 47%) at weeks 4 and 8, respectively. However, bioactive charged implants produced by plasma (PL) or thermochemical (BIO) processes exhibited enhanced osteoconductivity through specific ionic surface-tissue exchange (PL, BIC= 69% and 77% and BIO, BIC= 85% and 87% at weeks 4 and 8 respectively). Furthermore, bioactive surfaces (PL and BIO) showed functional mechanical stability (resonance frequency analyses) as early as 4 weeks post implantation via increased total bone area (BAT=56% and 59%) ingrowth compared to SB (BAT=35%) and AE (BAT=35%) surfaces.Peer ReviewedPostprint (author's final draft

A flexible strain-responsive sensor fabricated from a biocompatible electronic ink via an additive-manufacturing process

Biosensor technologies are of great interest for applications in wearable electronics, soft robotics and implantable biomedical devices. To accelerate the adoption of electronics for chronic recording of physiological parameters in health and disease, there is a demand for biocompatible, conductive & flexible materials that can integrate with various tissues while remaining biologically inert. Conventional techniques used to fabricate biosensors, such as mask lithography and laser cutting, lack the versatility to produce easily customisable, micro-fabricated biosensors in an efficient, cost-effective manner. In this paper, we describe the development and characterisation of an electronic ink made from an environmentally sustainable copolymer - x-pentadecalactone-co-e-decalactone, (PDL) incorporating silver nanowires (AgNW), which are known for their antimicrobial and conductive properties. The composites were shown to possess a low percolation threshold (1% w/w of AgNW to PDL), achieve a low electrical resistance (320 +/- 9 O/sq) and a high electrical capacitance (2.06 +/- 0.06 mF/cm2). PDL nanocomposites were biocompatible, demonstrated in vitro through the promotion of neural adhesion and prevention of astrocyte activation. An optimised ink formulation was subsequently used to fabricate strain-responsive biosensors with high spatial resolution (sub-100 mm) using a direct write additive manufacturing process. Using a customized in vitro set-up, the sensitivity of these biosensors to biologically-relevant strains was assessed under simulated physiological conditions for 21 days. Critically, these 3D printed biosensors have applications in chronic prophylactic monitoring of pressure changes within the body and related pathologies.This publication has emanated from research conducted with the financial support of the Science Foundation Ireland (SFI) Technology Innovation Development Programme, grant no. 15/TIDA/2992 and was co-funded under the European Regional Development Fund under Grant Number 13/RC/2073 and the Hardiman PhD Research Scholarship from the National University of Ireland, Galway. This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 713690. The authors acknowledge the facilities and scientific and technical assistance of the Centre for Microscopy & Imaging at the National University of Ireland Galway, a facility that is funded by NUIG and the Irish Government's Programme for Research in Third Level Institutions, Cycles 4 and 5, National Development Plan 20072013.r The Basque Government GV/EJ (Department of Education, Linguistic Politics and Culture) is also acknowledged for financial support to the consolidated research groups project IT927-16 (UPV/EHU, GIC/152)

Benefits of Polydopamine as Particle/Matrix Interface in Polylactide/PD-BaSO4 Scaffolds

This work reports the versatility of polydopamine (PD) when applied as a particle coating in a composite of polylactide (PLA). Polydopamine was observed to increase the particle–matrix interface strength and facilitate the adsorption of drugs to the material surface. Here, barium sulfate radiopaque particles were functionalized with polydopamine and integrated into a polylactide matrix, leading to the formulation of a biodegradable and X-ray opaque material with enhanced mechanical properties. Polydopamine functionalized barium sulfate particles also facilitated the adsorption and release of the antibiotic levofloxacin. Analysis of the antibacterial capacity of these composites and the metabolic activity and proliferation of human dermal fibroblasts in vitro demonstrated that these materials are non-cytotoxic and can be 3D printed to formulate complex biocompatible materials for bone fixation devices.The authors express thanks for technical and human support provided by SGIker of UPV/EHU and European funding: European Regional Development Fund (ERDF) and European Social Fund (ESF)

Surface-modified piezoelectric copolymer poly(vinylidene fluoride–trifluoroethylene) supporting physiological extracellular matrixes to enhance mesenchymal stem cell adhesion for nanoscale mechanical stimulation

There is an unmet clinical need to provide viable bone grafts for clinical use. Autologous bone, one of the most commonly transplanted tissues, is often used but is associated with donor site morbidity. Tissue engineering strategies to differentiate an autologous cell source, such as mesenchymal stromal cells (MSCs), into a potential bone-graft material could help to fulfill clinical demand. However, osteogenesis of MSCs can typically require long culture periods that are impractical in a clinical setting and can lead to significant cost. Investigation into strategies that optimize cell production is essential. Here, we use the piezoelectric copolymer poly(vinylidene fluoride–trifluoroethylene) (PVDF-TrFE), functionalized with a poly(ethyl acrylate) (PEA) coating that drives fibronectin network formation, to enhance MSC adhesion and to present growth factors in the solid phase. Dynamic electrical cues are then incorporated, via a nanovibrational bioreactor, and the MSC response to electromechanical stimulation is investigated

Stimulation of 3D osteogenesis by mesenchymal stem cells using a nanovibrational bioreactor

Bone grafts are one of the most commonly transplanted tissues. However, autologous grafts are in short supply, and can be associated with pain and donor-site morbidity. The creation of tissue-engineered bone grafts could help to fulfil clinical demand and provide a crucial resource for drug screening. Here, we show that vibrations of nanoscale amplitude provided by a newly developed bioreactor can differentiate a potential autologous cell source, mesenchymal stem cells (MSCs), into mineralized tissue in 3D. We demonstrate that nanoscale mechanotransduction can stimulate osteogenesis independently of other environmental factors, such as matrix rigidity. We show this by generating mineralized matrix from MSCs seeded in collagen gels with stiffness an order of magnitude below the stiffness of gels needed to induce bone formation in vitro. Our approach is scalable and can be compatible with 3D scaffolds