Treena Livingston Arinzeh

Biphasic Calcium Phosphate Ceramics for Bone Regeneration and Tissue Engineering Applications

Biphasic calcium phosphates (BCP) have been sought after as biomaterials for the reconstruction of bone defects in maxillofacial, dental and orthopaedic applications. They have demonstrated proven biocompatibility, osteoconductivity, safety and predictability in in vitro, in vivo and clinical models. More recently, in vitro and in vivo studies have shown that BCP can be osteoinductive. In the field of tissue engineering, they represent promising scaffolds capable of carrying and modulating the behavior of stem cells. This review article will highlight the latest advancements in the use of BCP and the characteristics that create a unique microenvironment that favors bone regeneration.

Neurite extension of primary neurons on electrospun piezoelectric scaffolds

Neural tissue engineering may be a promising option for neural repair treatment, for which a well-designed scaffold is essential. Smart materials that can stimulate neurite extension and outgrowth have been investigated as potential scaffolding materials. A piezoelectric polymer polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE) was used to fabricate electrospun aligned and random scaffolds having nano- or micron-sized fiber dimensions. The advantage of using a piezoelectric polymer is its intrinsic electrical properties. The piezoelectric characteristics of PVDF-TrFE scaffolds were shown to be enhanced by annealing. Dorsal root ganglion (DRG) neurons attached to all fibrous scaffolds. Neurites extended radially on random scaffolds, whereas aligned scaffolds directed neurite outgrowth for all fiber dimensions. Neurite extension was greatest on aligned, annealed PVDF-TrFE having micron-sized fiber dimensions in comparison with annealed and as-spun random PVDF-TrFE scaffolds. DRG on micron-sized aligned, as-spun and annealed PVDF-TrFE also had the lowest aspect ratio amongst all scaffolds, including non-piezoelectric PVDF and collagen-coated substrates. Findings from this study demonstrate the potential use of a piezoelectric fibrous scaffold for neural repair applications.

Characterization and in vitro cytocompatibility of piezoelectric electrospun scaffolds.

Previous studies have shown that electrical charges influence cell behavior (e.g. enhancement of nerve regeneration, cell adhesion, cell morphology). Thus, piezoelectric scaffolds might be useful for various tissue engineering applications. Fibrous scaffolds were successfully fabricated from permanent piezoelectric poly(vinylidene fluoride-trifluoroethylene) (PVDF-TrFE) by the electrospinning technique. Scanning electron microscopy and capillary flow analyses verified that the fiber mats had an average fiber diameter of 970 +/- 480 nm and a mean pore diameter of 1.7 microm, respectively. Thermally stimulated depolarization current spectroscopy measurements confirmed the piezoelectric property of the PVDF-TrFE fibrous scaffolds by the generation of a spontaneous current with the increase in temperature in the absence of an electric field, which was not detected in the unprocessed PVDF-TrFE powder. Differential scanning calorimetry, thermogravimetric analysis, X-ray diffraction and Fourier transform infrared spectroscopy results showed that the electrospinning process increased the crystallinity and presence of the polar, beta-phase crystal compared with the unprocessed powder. Confocal fluorescence microscopy and a cell proliferation assay demonstrated spreading and increased cell numbers (human skin fibroblasts) over time on PVDF-TrFE scaffolds, which was comparable with tissue culture polystyrene. The relative quantity of gene expression for focal adhesion proteins (measured by real-time RT-PCR) increased in the following order: paxillin < vinculin < focal adhesion kinase < talin. However, no differences could be seen among the TCPS surface and the fibrous scaffolds. Future studies will focus on possible applications of these cytocompatible PVDF-TrFE scaffolds in the field of regenerative medicine.

Piezoelectric materials for tissue regeneration: A review

UNLABELLED The discovery of piezoelectricity, endogenous electric fields and transmembrane potentials in biological tissues raised the question whether or not electric fields play an important role in cell function. It has kindled research and the development of technologies in emulating biological electricity for tissue regeneration. Promising effects of electrical stimulation on cell growth and differentiation and tissue growth has led to interest in using piezoelectric scaffolds for tissue repair. Piezoelectric materials can generate electrical activity when deformed. Hence, an external source to apply electrical stimulation or implantation of electrodes is not needed. Various piezoelectric materials have been employed for different tissue repair applications, particularly in bone repair, where charges induced by mechanical stress can enhance bone formation; and in neural tissue engineering, in which electric pulses can stimulate neurite directional outgrowth to fill gaps in nervous tissue injuries. In this review, a summary of piezoelectricity in different biological tissues, mechanisms through which electrical stimulation may affect cellular response, and recent advances in the fabrication and application of piezoelectric scaffolds will be discussed. STATEMENT OF SIGNIFICANCE The discovery of piezoelectricity, endogenous electric fields and transmembrane potentials in biological tissues has kindled research and the development of technologies using electrical stimulation for tissue regeneration. Piezoelectric materials generate electrical activity in response to deformations and allow for the delivery of an electrical stimulus without the need for an external power source. As a scaffold for tissue engineering, growing interest exists due to its potential of providing electrical stimulation to cells to promote tissue formation. In this review, we cover the discovery of piezoelectricity in biological tissues, its connection to streaming potentials, biological response to electrical stimulation and commonly used piezoelectric materials for tissue regeneration. This review summarizes their potential as a promising scaffold in the tissue engineering field.

Structural changes in PVDF fibers due to electrospinning and its effect on biological function.

Polyvinylidine fluoride (PVDF) is being investigated as a potential scaffold for bone tissue engineering because of its proven biocompatibility and piezoelectric property, wherein it can generate electrical activity when mechanically deformed. In this study, PVDF scaffolds were prepared by electrospinning using different voltages (12-30 kV), evaluated for the presence of the piezoelectric β-crystal phase and its effect on biological function. Electrospun PVDF was compared with unprocessed/raw PVDF, films and melt-spun fibers for the presence of the piezoelectric β-phase using differential scanning calorimetry, Fourier transform infrared spectroscopy and x-ray diffraction. The osteogenic differentiation of human mesenchymal stem cells (MSCs) was evaluated on scaffolds electrospun at 12 and 25 kV (PVDF-12 kV and PVDF-25 kV, respectively) and compared to tissue culture polystyrene (TCP). Electrospinning PVDF resulted in the formation of the piezoelectric β-phase with the highest β-phase fraction of 72% for electrospun PVDF at 25 kV. MSCs cultured on both the scaffolds were well attached as indicated by a spread morphology. Cells on PVDF-25 kV scaffolds had the greatest alkaline phosphatase activity and early mineralization by day 10 as compared to TCP and PVDF-12 kV. The results demonstrate the potential for the use of PVDF scaffolds for bone tissue engineering applications.

Examining the formulation of emulsion electrospinning for improving the release of bioactive proteins from electrospun fibers

Emulsion electrospinning has been sought as a method to prepare fibrous materials/scaffolds for growth factor delivery. Emulsion conditions, specifically sonication and the addition of a surfactant, were evaluated to determine their effect on the release and bioactivity of proteins from electrospun scaffolds. Polycaprolactone (PCL) and poly(ethylene oxide) (PEO/PCL) blends were evaluated where PEO, a hydrophilic polymer, was shown to enhance the incorporation of proteins. Electrospun scaffolds prepared with the addition of the nonionic surfactant Span® 80 at a concentration greater than the critical micelle concentration followed by mild sonication (10% amplitude) released lysozyme, the model protein, with a higher level of bioactivity as compared with other surfactant and sonication conditions. These conditions were then used to prepare emulsions of platelet-derived growth factor-BB (PDGF-BB) in PEO/PCL blends. Electrospun mats prepared by emulsions consisting of PDGF-BB incorporated with Span® 80 and sonicated at 10% amplitude exhibited a controlled release of PDGF-BB over 96 h as compared with a more rapid release from solutions that were not emulsified (Direct Addition) or emulsions that did not receive Span® 80 or sonication. Bioactive PDGF-BB incorporated in electrospun scaffolds enhanced the osteogenic differentiation of human mesenchymal stem cells as evidenced by increased alkaline phosphatase activity, improved cell attachment and reorganized cytoskeletal filaments. The findings in this study provide improved methods for achieving controlled release of bioactive proteins from electrospun materials.

Mesenchymal stem cells accelerate bone allograft incorporation in the presence of diabetes mellitus

Allograft (Allo) incorporation in the presence of a systemic disease like diabetes mellitus (DM) is becoming a major issue in the orthopedic community. Mesenchymal stem cells (MSC) are multipotent stem cells that may be derived from adult, whole bone marrow and have been shown to induce bone formation in segmental defects when combined with the appropriate carrier/scaffold. The objectives of this study were to analyze the effect of DM upon Allo incorporation in a segmental rat femoral defect and to also investigate MSC augmentation of Allo incorporation. Segmental (5 mm) femoral defects were created in non‐DM and DM rats and treated with Allo containing demineralized bone matrix (DBM) or DBM with MSC augmentation. Histological scoring at 4 weeks demonstrated less mature bone in the DM/DBM group compared to its non‐DM counterpart (p < 0.001). However, there was significantly more mature bone in the DM/MSC group when compared to the DM/DBM group at both 4 and 8 weeks (p < 0.001 and p = 0.004). Furthermore, significantly more bone formation was observed in the DM/MSC group compared to the DM/DBM group at the 4‐week time point (p < 0.001). The results of this study suggest that MSC are a potential adjunct for bone regeneration when implanted in an orthotopic site in the presence of DM.

Osteogenic Differentiation of Human Mesenchymal Stem Cells on Poly(ethylene glycol)-Variant Biomaterials

This study evaluated the osteogenic differentiation of human mesenchymal stem cells (MSCs), on tyrosine-derived polycarbonates copolymerized with poly(ethylene glycol) (PEG) to determine their potential as a scaffold for bone tissue engineering applications. The addition of PEG in the backbone of polycarbonates has been shown to alter mechanical properties, degradation rates, degree of protein adsorption, and subsequent cell adhesion and motility in mature cell phenotypes. Its effect on MSC behavior is unknown. MSC morphology, motility, proliferation, and osteogenic differentiation were evaluated on polycarbonates containing 0-5% PEG over a 14 day culture. MSCs on polycarbonates containing 0% or 3% PEG content upregulated the expression of osteogenic markers as demonstrated by alkaline phosphatase activity and osteocalcin expression although at different stages in the 14 day culture. Cells on polycarbonates containing no PEG were characterized as having early onset of cell spreading and osteogenic differentiation. Cells on 3% PEG surfaces were delayed in cell spreading and osteogenic differentiation, but had the highest motility as compared with cells on substrates containing no PEG and substrates containing 5% PEG at early time points. Throughout the culture, cells on polycarbonates containing 5% PEG had the lowest levels of osteogenic markers, displayed poor cell-substrate adhesion, and established cell-cell aggregates. Thus, designing substrates with minute variations in PEG may serve as a tool to guide MSC adhesion and motility accompanying osteogenic differentiation, and may be beneficial for abundant bone tissue formation in vivo.

Evaluating apatite formation and osteogenic activity of electrospun composites for bone tissue engineering

Significant interest has been in examining calcium phosphate ceramics, specifically β‐tricalcium phosphate (β‐TCP) (Ca3(PO4)2) and synthetic hydroxyapatite (HA) (Ca10(PO4)6(OH)2), in composites and more recently, in fibrous composites formed using the electrospinning technique for bone tissue engineering applications. Calcium phosphate ceramics are sought because they can be bone bioactive, which means an apatite forms on their surface that facilitates bonding to bone tissue, and are osteoconductive. However, studies examining the bioactivity of electrospun composites containing calcium phosphates and their corresponding osteogenic activity have been limited. In this study, electrospun composites consisting of (20/80) HA/TCP nanoceramics and poly (ϵ‐caprolactone) (PCL) were fabricated. Solvent and solvent combinations were evaluated to form scaffolds with a maximum concentration and dispersion of ceramic and pore sizes large enough for cell infiltration and tissue growth. PCL was dissolved in either methylene chloride (Composite‐MC) or a combination of methylene chloride (80%) and dimethylformamide (20%; Composite‐MC + DMF). Composites were evaluated in vitro for degradation, apatite formation, and osteogenic differentiation of human mesenchymal stem cells (MSCs) with an emphasis on temporal gene expression of osteogenic markers and the pluripotent gene Sox‐2. Apatite formation and the osteogenic differentiation was the greatest for Composite‐MC as determined by gene expression, protein production and biochemical markers, even without the presence of osteoinductive factors in the media, in comparison to Composite‐MC + DMF and unfilled PCL mats. Sox‐2 levels also reduced over time. The results of this study demonstrate that the solvent or solvent combination used in preparing the electrospun composite mats plays a critical role in determining their bioactivity which may, in turn, affect cell behavior. Biotechnol. Biotechnol. Bioeng. 2014;111: 1000–1017.

The effect of PVDF-TrFE scaffolds on stem cell derived cardiovascular cells.

Recently, electrospun polyvinylidene fluoride (PVDF) and polyvinylidene fluoride‐trifluoroethylene (PVDF‐TrFE) scaffolds have been developed for tissue engineering applications. These materials have piezoelectric activity, wherein they can generate electric charge with minute mechanical deformations. Since the myocardium is an electroactive tissue, the unique feature of a piezoelectric scaffold is attractive for cardiovascular tissue engineering applications. In this study, we examined the cytocompatibility and function of pluripotent stem cell derived cardiovascular cells including mouse embryonic stem cell‐derived cardiomyocytes (mES‐CM) and endothelial cells (mES‐EC) on PVDF‐TrFE scaffolds. MES‐CM and mES‐EC adhered well to PVDF‐TrFE and became highly aligned along the fibers. When cultured on scaffolds, mES‐CM spontaneously contracted, exhibited well‐registered sarcomeres and expressed classic cardiac specific markers such as myosin heavy chain, cardiac troponin T, and connexin43. Moreover, mES‐CM cultured on PVDF‐TrFE scaffolds responded to exogenous electrical pacing and exhibited intracellular calcium handling behavior similar to that of mES‐CM cultured in 2D. Similar to cardiomyocytes, mES‐EC also demonstrated high viability and maintained a mature phenotype through uptake of low‐density lipoprotein and expression of classic endothelial cell markers including platelet endothelial cell adhesion molecule, endothelial nitric oxide synthase, and the arterial specific marker, Notch‐1. This study demonstrates the feasibility of PVDF‐TrFE scaffold as a candidate material for developing engineered cardiovascular tissues utilizing stem cell‐derived cells. Biotechnol. Bioeng. 2016;113: 1577–1585.