Fabrication and Characterisation of Stimuli Responsive Piezoelectric PVDF and Hydroxyapatite-Filled PVDF Fibrous Membranes.

Poly(vinylidene fluoride) has attracted interest from the biomaterials community owing to its stimuli responsive piezoelectric property and promising results for application in the field of tissue engineering. Here, solution blow spinning and electrospinning were employed to fabricate PVDF fibres and the variation in resultant fibre properties assessed. The proportion of piezoelectric ß-phase in the solution blow spun fibres was higher than electrospun fibres. Fibre production rate was circa three times higher for solution blow spinning compared to electrospinning for the conditions explored. However, the solution blow spinning method resulted in higher fibre variability between fabricated batches. Fibrous membranes are capable of generating different cellular response depending on fibre diameter. For this reason, electrospun fibres with micron and sub-micron diameters were fabricated, along with successful inclusion of hydroxyapatite particles to fabricate stimuli responsive bioactive fibres.

State of the art composites comprising electrospun fibres coupled with hydrogels: a review.

Research into scaffolds tailored for specific tissue engineering and biomaterial applications continues to develop as these structures are commonly impeded by their limitations. For example, electrospun fibres and hydrogels are commonly exploited because of their ability to mimic natural tissues; however, their clinical use remains restricted due to negligible cellular infiltration and poor mechanical properties, respectively. A small number of research groups are beginning to investigate composite scaffolds based on electrospun fibres and hydrogels in an attempt to overcome their individual shortcomings. This review paper discusses the various methodologies and approaches currently undertaken to create these novel composite structures and their intended applications. The combination of these two commonly used scaffold architectures to create synergistically superior structures is showing potential with regards to therapeutic use within the tissue engineering community. FROM THE CLINICAL EDITOR: This review discusses methodologies to create novel electrospun nanofibers and hydrogels, and their intended applications. The combination of these two scaffold architectures has important future clinical applications, although their use is currently limited to the experimental tissue engineering community.

Instructive electroactive electrospun silk fibroin-based biomaterials for peripheral nerve tissue engineering.

Aligned sub-micron fibres are an outstanding surface for orienting and promoting neurite outgrowth; therefore, attractive features to include in peripheral nerve tissue scaffolds. A new generation of peripheral nerve tissue scaffolds is under development incorporating electroactive materials and electrical regimes as instructive cues in order to facilitate fully functional regeneration. Herein, electroactive fibres composed of silk fibroin (SF) and poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) were developed as a novel peripheral nerve tissue scaffold. Mats of SF with sub-micron fibre diameters of 190 ± 50 nm were fabricated by double layer electrospinning with thicknesses of ∼100 µm (∼70-80 µm random fibres and ∼20-30 µm aligned fibres). Electrospun SF mats were modified with interpenetrating polymer networks (IPN) of PEDOT:PSS in various ratios of PSS/EDOT (α) and the polymerisation was assessed by hard X-ray photoelectron spectroscopy (HAXPES). The mechanical properties of electrospun SF and IPNs mats were characterised in the wet state tensile and the electrical properties were examined by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The cytotoxicity and biocompatibility of the optimal IPNs (α = 2.3 and 3.3) mats were ascertained via the growth and neurite extension of mouse neuroblastoma x rat glioma hybrid cells (NG108-15) for 7 days. The longest neurite outgrowth of 300 µm was observed in the parallel direction of fibre alignment on laminin-coated electrospun SF and IPN (α = 2.3) mats which is the material with the lowest electron transfer resistance (Ret, ca. 330 Ω). These electrically conductive composites with aligned sub-micron fibres exhibit promise for axon guidance and also have the potential to be combined with electrical stimulation treatment as a further step for the effective regeneration of nerves.

Engineering a cell-hydrogel-fibre composite to mimic the structure and function of the tendon synovial sheath.

The repair of tendon injuries is often compromised by post-operative peritendinous adhesions. Placing a physical barrier at the interface between the tendon and the surrounding tissue could potentially solve this problem by reducing adhesion formation. At present, no such system is available for routine use in clinical practice. Here, we propose the development of a bilayer membrane combining a nanofibrous poly(ε-caprolactone) (PCL) electrospun mesh with a layer of self-assembling peptide hydrogel (SAPH) laden with type-B synoviocytes. This bilayer membrane would act as an anti-adhesion system capable of restoring tendon lubrication, while assisting with synovial sheath regeneration. The PCL mesh showed adequate mechanical properties (Young's modulus=19±4 MPa, ultimate tensile stress=9.6±1.7 MPa, failure load=0.5±0.1 N), indicating that the membrane is easy to handle and capable to withstand the frictional forces generated on the tendon's surface during movement (~0.3 N). Morphological analysis confirmed the generation of a mesh with nanosized PCL fibres and small pores (< 3 µm), which prevented fibroblast infiltration to impede extrinsic healing but still allowing diffusion of nutrients and waste. Rheological tests showed that incorporation of SAPH layer allows good lubrication properties when the membrane is articulated against porcine tendon or hypodermis, suggesting that restoration of tendon gliding is possible upon implantation. Moreover, viability and metabolic activity tests indicated that the SAPH was conducive to rabbit synoviocyte growth and proliferation over 28 days of 3D culture, sustaining cell production of specific matrix components, particularly hyaluronic acid. Synoviocyte-laden peptide hydrogel promoted a sustained endogenous production of hyaluronic acid, providing an anti-friction layer that potentially restores the tendon gliding environment.

Piezoelectric materials as stimulatory biomedical materials and scaffolds for bone repair.

The process of bone repair and regeneration requires multiple physiological cues including biochemical, electrical and mechanical - that act together to ensure functional recovery. Myriad materials have been explored as bioactive scaffolds to deliver these cues locally to the damage site, amongst these piezoelectric materials have demonstrated significant potential for tissue engineering and regeneration, especially for bone repair. Piezoelectric materials have been widely explored for power generation and harvesting, structural health monitoring, and use in biomedical devices. They have the ability to deform with physiological movements and consequently deliver electrical stimulation to cells or damaged tissue without the need of an external power source. Bone itself is piezoelectric and the charges/potentials it generates in response to mechanical activity are capable of enhancing bone growth. Piezoelectric materials are capable of stimulating the physiological electrical microenvironment, and can play a vital role to stimulate regeneration and repair. This review gives an overview of the association of piezoelectric effect with bone repair, and focuses on state-of-the-art piezoelectric materials (polymers, ceramics and their composites), the fabrication routes to produce piezoelectric scaffolds, and their application in bone repair. Important characteristics of these materials from the perspective of bone tissue engineering are highlighted. Promising upcoming strategies and new piezoelectric materials for this application are presented. STATEMENT OF SIGNIFICANCE: Electrical stimulation/electrical microenvironment are known effect the process of bone regeneration by altering the cellular response and are crucial in maintaining tissue functionality. Piezoelectric materials, owing to their capability of generating charges/potentials in response to mechanical deformations, have displayed great potential for fabricating smart stimulatory scaffolds for bone tissue engineering. The growing interest of the scientific community and compelling results of the published research articles has been the motivation of this review article. This article summarizes the significant progress in the field with a focus on the fabrication aspects of piezoelectric materials. The review of both material and cellular aspects on this topic ensures that this paper appeals to both material scientists and tissue engineers.

Electroactive biomaterials: Vehicles for controlled delivery of therapeutic agents for drug delivery and tissue regeneration.

Electrical stimulation for delivery of biochemical agents such as genes, proteins and RNA molecules amongst others, holds great potential for controlled therapeutic delivery and in promoting tissue regeneration. Electroactive biomaterials have the capability of delivering these agents in a localized, controlled, responsive and efficient manner. These systems have also been combined for the delivery of both physical and biochemical cues and can be programmed to achieve enhanced effects on healing by establishing control over the microenvironment. This review focuses on current state-of-the-art research in electroactive-based materials towards the delivery of drugs and other therapeutic signalling agents for wound care treatment. Future directions and current challenges for developing effective electroactive approach based therapies for wound care are discussed.

Four-Dimensional Imaging of Soft Tissue and Implanted Biomaterial Mechanics: A Barbed Suture Case Study for Tendon Repair.

Timely, recent developments in X-ray microcomputed tomography (XµCT) imaging such as increased resolution and improved sample preparation enable nondestructive time-lapse imaging of polymeric biomaterials when implanted in soft tissue, which we demonstrate herein. Imaging the full three-dimensional (3D) structure of an implanted biomaterial provides new opportunities to assess the micromechanics of the interface between the implant and tissues and how this changes over time as force is applied in load-bearing musculoskeletal applications. In this paper, we present a case study demonstrating in situ XµCT and finite element analysis, using a dynamically loaded barbed suture repair for its novel use in tendon tissue. The aim of this study was to identify the distribution of stress in the suture and tendon as load is applied. The data gained demonstrate a clear 3D visualization of microscale features in both the tissue and implant in wet conditions. XµCT imaging has revealed, for the first time, pores around the suture, preventing full engagement of all the barbs with the tendon tissue. Subsequent finite element analysis reveals the localized stress and strain, which are not evenly distributed along the suture, or throughout the tissue. This case study demonstrates for the first time a powerful in situ mechanical imaging tool, which could be readily adapted by other laboratories to interrogate and optimize the interface between the implanted biomaterials and the soft tissue.

In vitro evaluation of poly (lactic-co-glycolic acid)/polyisoprene fibers for soft tissue engineering.

The polymeric blend of poly (lactic-co-glycolic acid) (PLGA) and polyisoprene (PI) has recently been explored for application as stents for tracheal stenosis and spring for the treatment of craniosynostosis. From the positive results presented in other biomedical applications comes the possibility of investigating the application of this material as scaffold for tissue engineering (TE), acquiring a deeper knowledge about the polymeric blend by exploring a new processing technique while attending to the most fundamental demands of TE scaffolds. PLGA/PI was processed into randomly oriented microfibers through the dripping technique and submitted to physical-chemical and in vitro characterization. The production process of fibers did not show an effect over the polymer's chemical composition, despite the fact that PLGA and PI were observed to be immiscible. Mechanical assays reinforce the suitability of these scaffolds for soft tissue applications. Skeletal muscle cells demonstrated increases in metabolic activity and proliferation to the same levels of the control group. Human dermal fibroblasts didn't show the same behaviour, but presented cell growth with the same development profile as presented in the control group. It is plausible to believe that PLGA/PI fibrous three-dimensional scaffolds are suitable for applications in soft tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2581-2591, 2017.

Cell response to sterilized electrospun poly(ɛ-caprolactone) scaffolds to aid tendon regeneration in vivo.

The functional replacement of tendon represents an unmet clinical need in situations of tendon rupture, tendon grafting, and complex tendon reconstruction, as usually there is a finite source of healthy tendon to use as donors. The microfibrous architecture of tendon is critical to the function of tendon. This study investigates the use of electrospun poly(ɛ-caprolactone) scaffolds as potential biomaterial substitutes for tendon grafts. We assessed the performance of two electrospinning manufacturers (small- and large-scale) and the effect of two sterilization techniques-gamma irradiation and ethanol submersion-on cell response to these electrospun scaffolds after their implantation into a murine tendon model. Cell infiltration and proliferation analyses were undertaken to determine the effect on cell response within the implant over a 6-week period. Immunohistochemical analysis was performed to characterize inflammatory response and healing characteristics (proliferation, collagen deposition, myofibroblast activity, and apoptosis). Neither the sterilization techniques nor the manufacturer was observed to significantly affect the cell response to the scaffold. At each time point, cell response was similar to the autograft control. This suggests that ethanol submersion can be used for research purposes and that the scaffold can be easily reproduced by a large-scale manufacturer. These results further imply that this electrospun scaffold may provide an alternative to autograft, thus eliminating the need for sourcing healthy tendon tissue from a secondary site. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 389-397, 2017.

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(ε-caprolactone) Electrospun Yarns.

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(ε-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37 (o)C, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged - the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

Microarchitectural and mechanical characterization of oriented porous polymer scaffolds.

Biodegradable porous polymer scaffolds are widely used in tissue engineering to provide a structural template for cell seeding and extracellular matrix formation. Scaffolds must often possess sufficient structural integrity to temporarily withstand functional loading in vivo or cell traction forces in vitro. Both the mechanical and biological properties of porous scaffolds are determined in part by the local microarchitecture. Quantification of scaffold structure-function relationships is therefore critical for optimizing mechanical and biological performance. In this study, porous poly(L-lactide-co-DL-lactide) scaffolds with axially oriented macroporosity and random microporosity were produced using a solution coating and porogen decomposition method. Microarchitectural parameters were quantified as a function of porogen concentration using microcomputed tomography (micro-CT) analysis and related to compressive mechanical properties. With increasing porogen concentration, volume fraction decreased consistently due to microarchitectural changes in average strut thickness, spacing, and density. The three-dimensional interconnectivity of the scaffold porosity was greater than 99% for all porogen concentration levels tested. Over a porosity range of 58-80%, the average compressive modulus and ultimate strength of the scaffolds ranged from 43.5-168.3 MPa and 2.7-11.0 MPa, respectively. Thus, biodegradable porous polymer scaffolds have been produced with oriented microarchitectural features designed to facilitate vascular invasion and cellular attachment and with initial mechanical properties comparable to those of trabecular bone.

Effects of medium perfusion rate on cell-seeded three-dimensional bone constructs in vitro.

Cellular activity at the center of tissue-engineered constructs in static culture is typically decreased relative to the construct periphery because of transport limitations. We have designed a tissue culture system that perfuses culture medium through three-dimensional (3D) porous cellular constructs to improve nutrient delivery and waste removal within the constructs. This study examined the effects of medium perfusion rate on cell viability, proliferation, and gene expression within cell-seeded 3D bone scaffolds. Human trabecular bone scaffolds were seeded with MC3T3-E1 osteoblast-like cells and perfused for 1 week at flow rates of 0.01, 0.1, 0.2, and 1.0 mL/min. Confocal microscopy after 1 week of culture indicated that a flow rate of 1.0 mL/min resulted in substantial cell death throughout the constructs whereas lowering the flow rate led to an increasing proportion of viable cells, particularly at the center of the constructs. DNA analysis showed increases in cell proliferation at a flow rate of 0.01 mL/min relative to 0.2 mL/min and static controls. Conversely, mRNA expressions of Runx2, osteocalcin, and alkaline phosphatase were upregulated at 0.2 mL/min compared with lower flow rates as quantified by real-time RT-PCR. These data suggest that medium perfusion may benefit the development of 3-D tissues in vitro by enhancing transport of nutrients and waste within the constructs and providing flow-mediated mechanical stimuli.

Conductive polymers: towards a smart biomaterial for tissue engineering.

Developing stimulus-responsive biomaterials with easy-to-tailor properties is a highly desired goal of the tissue engineering community. A novel type of electroactive biomaterial, the conductive polymer, promises to become one such material. Conductive polymers are already used in fuel cells, computer displays and microsurgical tools, and are now finding applications in the field of biomaterials. These versatile polymers can be synthesised alone, as hydrogels, combined into composites or electrospun into microfibres. They can be created to be biocompatible and biodegradable. Their physical properties can easily be optimized for a specific application through binding biologically important molecules into the polymer using one of the many available methods for their functionalization. Their conductive nature allows cells or tissue cultured upon them to be stimulated, the polymers' own physical properties to be influenced post-synthesis and the drugs bound in them released, through the application of an electrical signal. It is thus little wonder that these polymers are becoming very important materials for biosensors, neural implants, drug delivery devices and tissue engineering scaffolds. Focusing mainly on polypyrrole, polyaniline and poly(3,4-ethylenedioxythiophene), we review conductive polymers from the perspective of tissue engineering. The basic properties of conductive polymers, their chemical and electrochemical synthesis, the phenomena underlying their conductivity and the ways to tailor their properties (functionalization, composites, etc.) are discussed.

Osteoblast: osteoclast co-cultures on silk fibroin, chitosan and PLLA films.

This study investigates the growth of a co-culture of osteoblasts and osteoclasts on four different types of degradable biomaterials with bone tissue engineering potential. Single or co-cultures of osteoblasts and osteoclasts (used at a ratio of 1:100 osteoblast:osteoclasts) were cultured on vapour stabilised silk fibroin, methanol stabilised silk fibroin, chitosan and poly (l lactic acid) (PLLA) films for 10 days. Osteoclast differentiation was determined by tartrate resistant acid phosphatase (TRAP) staining, total cell number by a picogreen DNA assay, cell morphology by scanning electron microscopy (SEM) and the material topography by atomic force microscopy (AFM). Samples were also monitored for degradation by differential scanning calorimetry (DSC) and fourier transform infrared (FTIR). Results demonstrated that vapour stabilised silk fibroin, methanol stabilised silk fibroin and chitosan all support the growth of osteoblasts and osteoclasts in both single and co-cultures. PLLA showed poor osteoclast differentiation in both single and co-cultures but supported osteoblast attachment and proliferation. Both silk fibroin materials showed sign of early degradation in the ten-day period, but very little change was seen in chitosan and PLLA samples. This study indicates that this novel co-culture approach for bone tissue engineering may be possible if scaffolds are created from silk fibroin or chitosan.