In situ miRNA delivery from a hydrogel promotes osteogenesis of encapsulated mesenchymal stromal cells.

Hydrogels are attractive candidates for use in tissue-engineering and the encapsulation and subsequent differentiation of mesenchymal stem/stromal cells (MSCs) is a strategy that holds great promise for the repair and regeneration of bone and cartilage. However, MSCs are well-known for their sensitivity to mechanical cues, particularly substrate stiffness, and so the inherent softness of hydrogels is poorly matched to the mechanical cues that drive efficient osteogenesis. One approach to overcome this limitation is to harness mechanotransductive signalling pathways and override the signals cells receive from their environment. Previous reports demonstrate that mechanosensitive miRNAs, miR-100-5p and miR-143-3p can enhance MSC osteogenesis, using a complex multi-step procedure to transfect, encapsulate and differentiate the cells. In this study, we develop and characterise a facile system for in situ transfection of MSCs encapsulated within a light-crosslinkable gelatin-PEG hydrogel. Comparing the influence of different transfection agents and hydrogel compositions, we show that particle size, charge, and hydrogel mechanical properties all influence the diffusion of embedded transfection agent complexes. By incorporating both MSCs and transfection agents into the hydrogels we demonstrate successful in situ transfection of encapsulated MSCs. Comparing the efficacy of pre- and in situ transfection of miR-100-5p/miR-143-3p on the osteogenic capacity of hydrogel-encapsulated MSCs, our data demonstrates superior mineralisation and osteogenic gene expression following in situ transfections. Overall, we demonstrate a simple, one-pot system for in situ transfection of miRNAs to enhance MSC osteogenic potential and thus demonstrates significant promise to improve the efficiency of MSC differentiation in hydrogels for bone tissue-engineering applications. STATEMENT OF SIGNIFICANCE: Mesenchymal stromal cells (MSCs) are sensitive to cues from their surrounding microenvironment. Osteogenesis is enhanced in MSCs grown on stiffer substrates, but this is limited when using hydrogels for bone tissue-engineering. Modulating pro-osteogenic genes with mechanosensitive microRNAs (miRNAs) represents a potential tool to overcome this challenge. Here we report a hydrogel platform to deliver miRNAs to encapsulated MSCs. We characterise effects of hydrogel composition and transfection agent type on their mobility and transfection efficiency, demonstrating successful in situ transfection of MSCs and showing that miRNAs can significantly enhance osteogenic mineral deposition and marker gene expression. This system was simpler and more effective than conventional 2D transfection prior to encapsulation and therefore holds promise to improve MSC differentiation in bone tissue-engineering.

Polyethylene glycol-gelatin hydrogels with tuneable stiffness prepared by horseradish peroxidase-activated tetrazine-norbornene ligation.

Tetrazine-norbornene ligation has previously been applied in bioorthognal polymer crosslinking to form hydrogels suitable for 3D cell culture. However, the tetrazine group is prone to reduction by the free thiol in a biological environment, reducing the crosslinking efficiency and shortening the storage of tetrazine containing linkers. Here, we introduce a method to form a tetrazine group in situ by catalytic oxidation of the dihydrogen tetrazine using horse radish peroxidase (HRP). Enzymatic oxidation is highly efficient at a low HRP concentration and does not require hydrogen peroxide, allowing for rapid gelation when HRP was added to an aqueous solution of 4-arm PEG dihydrogentetrazine and gelatin norbornene. The storage modulus of the resultant gels can be varied by changing the concentration of the crosslinker, which is in the range of 1.2-3.8 kPa. Human mesenchymal stem cells encapsulated within these gels, with varying stiffness, display varied interactions and morphologies and can be maintained with prolonged culture periods of at least 32 days of 3D culture. The enzymatic activation of tetrazine-norbornene is therefore an attractive addition to the tetrazine ligation that is highly suitable for cell related studies in tissue engineering.

A self-assembling ß-peptide hydrogel for neural tissue engineering.

We report a new class of ß-peptide based hydrogel for neural tissue engineering. Our ß-peptide forms a network of nanofibres in aqueous solution, resulting in a stable hydrogel at physiological conditions. The hydrogel shows excellent compatibility with neural cells and provides a suitable environment for cells to adhere and proliferate.

Chirality effects at each amino acid position on tripeptide self-assembly into hydrogel biomaterials.

Hydrogels formed by ultrashort peptides are emerging as cost-effective materials for cell culture. However, L-peptides are labile to proteases, while their D-isomers are thought to not support cell growth as well. In contrast, the self-assembly behaviour and biological performance of heterochiral peptides (i.e., made of both d and l amino acids) are largely unknown. In this study, we evaluate the effects of amino acid chirality on tripeptide self-assembly and hydrogelation at physiological pH, and cytocompatibility in fibroblast cell culture. A series of uncapped hydrophobic tripeptides with all combinations of d, l amino acids was prepared, tested for self-assembly under physiological conditions, and analysed by circular dichroism, FT-IR, cryo-TEM, AFM, and Thioflavin T fluorescence imaging. Amino acid chirality has a profound effect on the peptides' supramolecular behaviour. Only selected isomers form hydrogels, and of amyloid structure, as confirmed by rheology and XRD. Importantly, they are able to maintain the viability and proliferation of fibroblasts in vitro. This study identifies two heterochiral gels that perform well in cell culture and will assist in the design of innovative and cost-effective peptide gel biomaterials.

Biofunctionalisation of polymeric scaffolds for neural tissue engineering.

Patients who experience injury to the central or peripheral nervous systems invariably suffer from a range of dysfunctions due to the limited ability for repair and reconstruction of damaged neural tissue. Whilst some treatment strategies can provide symptomatic improvement of motor and cognitive function, they fail to repair the injured circuits and rarely offer long-term disease modification. To this end, the biological molecules, used in combination with neural tissue engineering scaffolds, may provide feasible means to repair damaged neural pathways. This review will focus on three promising classes of neural tissue engineering scaffolds, namely hydrogels, electrospun nanofibres and self-assembling peptides. Additionally, the importance and methods for presenting biologically relevant molecules such as, neurotrophins, extracellular matrix proteins and protein-derived sequences that promote neuronal survival, proliferation and neurite outgrowth into the lesion will be discussed.

Biological responses of human osteoblasts and osteoclasts to flame-sprayed coatings of hydroxyapatite and fluorapatite blends.

The aim of this study was to determine how the activities of human osteoblastic cells and osteoclasts respond to substrates of thermal-sprayed mechanical blends of hydroxyapatite and fluorapatite with a view of determining an optimal blend ratio for osseointegration. Human osteoblastic cells and osteoclasts were grown on titanium alloy discs coated with blends of hydroxyapatite and fluorapatite, with concentrations ranging from 0 to 100% fluorapatite. Human osteoblastic cells attached in greater numbers and proliferated at a greater rate on blends containing 40% fluorapatite. Human osteoblastic cells grown on blends containing 40% fluorapatite for 7 days also expressed the highest levels of mRNA for several proteins involved with regulating bone metabolism (osteoprotegerin and receptor activator nuclear factor kappa B ligand), and bone formation (osteopontin, osteonectin and bone sialoprotein 1). Osteoclasts resorbed the dentine but poorly resorbed the hydroxyapatite-fluorapatite blends, particularly at high levels of fluorapatite. This in vitro study demonstrates that thermal-sprayed hydroxyapatitecoatings containing 40% fluorapatite may promote optimal bone growth and improve osseointegration of implants.

Enhancing neurite outgrowth from primary neurones and neural stem cells using thermoresponsive hydrogel scaffolds for the repair of spinal cord injury.

In this study, thermoresponsive xyloglucan hydrogel scaffolds were investigated as candidates for neural tissue engineering of the spinal cord. The hydrogels were optimized to provide similar mechanical properties to that of native spinal cord, although also being functionalized through the immobilization of poly-D-lysine to promote neurone adhesion and neurite outgrowth. Under 2D and 3D culture conditions, xyloglucan scaffolds supported the differentiation of primary cortical neurones. Furthermore, functionalization provided a means of controlling and optimizing the cell diameter, number, migration and the neurite density, and the direction of growth. The interaction of neural stem cells (NSCs) was also investigated on the xyloglucan scaffolds in vitro. The survival of the NSCs and the axonal extensions on the scaffolds were similar to that of the primary cortical neurones. These findings suggest that xyloglucan-based materials are suitable for providing a neurotrophic milieu.

Review paper: a review of the cellular response on electrospun nanofibers for tissue engineering.

Electrospinning has been employed extensively in tissue engineering to generate nanofibrous scaffolds from either natural or synthetic biodegradable polymers to simulate the cellular microenvironment. Electrospinning rapidly produces fibers of the nanolength scale and the process offers many opportunities to tailor the physical, chemical, and biological properties of a material for specific applications and cellular environments. There is growing evidence that nanofibers amplify certain biological responses such as contact guidance and differentiation, however this has not been fully exploited in tissue engineering. This review addresses the cellular interactions with electrospun scaffolds, with particular focus on neural, bone, cartilage, and vascular tissue regeneration. Some aspects of scaffold design, including architectural properties, surface functionalization and materials selection are also addressed.

Characterization of neural stem cells on electrospun poly(epsilon-caprolactone) submicron scaffolds: evaluating their potential in neural tissue engineering.

Development of biomaterials with specific characteristics to influence cell behaviour has played an important role in exploiting strategies to promote nerve regeneration. The effect of three-dimensional (3D) non-woven electrospun poly(epsilon-caprolactone) (PCL) scaffolds on the behaviour of rat brain-derived neural stem cells (NSCs) is reported. The interaction of NSCs on the randomly orientated submicron (PCL) fibrous scaffolds, with an average fibre diameter of 750 +/- 100 nm, was investigated. The PCL scaffolds were modified with ethylenediamine (ED) to determine if amino functionalisation and changes in surface tension of the fibrous scaffolds affected the proliferation and differentiation characteristics of NSCs. Surface tension of the fibrous scaffold increased upon treatment with ED which was attributed to amine moieties present on the surface of the fibres. Although surface treatment did not change the differentiation of the NSCs, the modified scaffolds were more hydrophilic, resulting in a significant increase in the number of adhered cells, and increased spreading throughout the entirety of the scaffold. When the NSCs were seeded on the PCL scaffolds in the presence of 10% FBS, the stem cells differentiated primarily into oligodendrocytes, indicating that electrospun PCL has the capacity to direct the differentiation of NSCs towards a specific lineage. The data presented here is useful for the development of electrospun biomaterial scaffolds for neural tissue engineering, to regulate the proliferation and differentiation of NSCs.

Interaction of embryonic cortical neurons on nanofibrous scaffolds for neural tissue engineering.

The interaction of murine embryonic cortical neurons on randomly orientated electrospun scaffolds of poly(L-lactide) (P(L)LA) and poly(lactide-co-glycolide) (PLGA) is investigated in this study. The scaffolds were surface treated with different concentrations of KOH to partially hydrolyze the surface and therefore change the surface tension. Hydrophilicity did not significantly influence the number of primary and secondary branches; however, it had a considerable effect on neurite extension. For scaffolds with surface tensions of 40-47 dyn cm(-1) there was a significantly greater overall neurite length for both the primary and secondary branches compared with more hydrophilic scaffolds. Another major finding of this work was that the interfibre distance influenced how the neurites extended. When the interfibre distance was greater than approximately 15 microm the neurites followed the fibres and avoided regions of very high fibre density. At interfibre distances less than approximately 15 microm, the neurites traversed between the fibres. Therefore, this study provided little evidence that contact guidance was the dominating cue in directing neurite extension, instead inferring that chemical cues, possibly from adjacent neurons had induced directional change.

Polylysine-functionalised thermoresponsive chitosan hydrogel for neural tissue engineering.

Foetal mouse cortical cells were cultured on 2D films and within 3D thermally responsive chitosan/glycerophosphate salt (GP) hydrogels. The biocompatibility of chitosan/GP 2D films was assessed in terms of cell number and neurites per cell. Osmolarity of the hydrogel was a critical factor in promoting cell survival with isotonic GP concentrations providing optimal conditions. To improve cell adhesion and neurite outgrowth, poly-D-lysine (PDL) was immobilised onto chitosan via azidoaniline photocoupling. Increase in PDL concentrations did not alter cell survival in 2D cultures but neurite outgrowth was significantly inhibited. Neurons exhibited a star-like morphology typical of 2D culture systems. The effects of PDL attachment on cell number, cell morphology and neurite outgrowth were more distinct in 3D culture conditions. Neurones exhibited larger cell bodies and sent out single neurites within the macroporous gel. Immobilised PDL improved cell survival up to an optimum concentration of 0.1%, however, further increases resulted in drops in cell number and neurite outgrowth. This was attributed to a higher cell interaction with PDL within a 3D hydrogel compared to the corresponding 2D surface. The results show that thermally responsive chitosan/GP hydrogels provide a suitable 3D scaffolding environment for neural tissue engineering.

Inflammatory response on injection of chitosan/GP to the brain.

Chitosan is a well-known biomaterial that, with the addition of glycerophosphate salt (GP), gels at physiological temperatures and therefore is useful for tissue engineering purposes. This study examines the procedure of injecting chitosan/ GP to the brain in order to form a gel track. The gel system and surgical technique were successful in this endeavour; however, on examining the inflammatory response to the material it was found that the chitosan/GP was wholly engulfed by macrophages within 7 days. This was determined by staining for both the gel and the macrophages, an important technique for localising injected material. The chitosan/GP-containing macrophages formed a neat tract at the lesion site, but after 45 days no chitosan/GP was found. It was concluded that, although chitosan/GP is present after implantation, it is not available for direct scaffolding in the brain.

The effect of surface hydrophilicity on the behavior of embryonic cortical neurons.

The aim of this study was to investigate the interaction of mouse embryonic cortical neurons on P(L)LA and PLGA substrates, which were partially hydrolysed using potassium hydroxide (KOH). The chemical and topographical properties of the surfaces were characterized, and it was discovered that there was a decrease in the hydrophilicity for the P(L)LA with increasing concentration of KOH. This was due to chemical modifications to the surfaces of the substrates. Alternatively for the PLGA substrate, only the 0.1 M KOH treated sample had a significantly different hydrophilicity highlighting that surface erosion resulted at higher concentrations. The morphology of the neurons grown on the two substrates were compared to poly(D)lysine (positive control). The neurons formed colonies on all of the substrates, but were dramatically reduced in size in the case of the 0.1 M KOH treated substrates. This finding was attributed to the increases in cell spreading and the size of the cells, as they were larger, more elongated and bipolar like those on the positive control. However, there was a significant decrease in the total number of live cells per unit area. Therefore, on these materials when there was increased cellular spreading there was significantly higher cell death. Furthermore, unlike the 0, 0.2, and 0.4 M KOH treated substrates, there was an absence of large bundles of axons that extended between colonies on the 0.1 M sample, instead exhibiting short axons that grew in free space.

Morphology and gelation of thermosensitive xyloglucan hydrogels.

Galactose modified xyloglucan is a thermally reversible hydrogel that is increasingly used in the biomedical field due to the ease of altering the gelation time and temperature by modifying the galactose removal ratio. However there is little information concerning the morphology and rheological properties of the hydrogel under physiological conditions. Differential scanning microcalorimetry (DSmicroC) showed the thermal gelation process to occur over a broad temperature range (5-50 degrees C). The rheological properties of the hydrogels were investigated as a function of concentration, temperature and ionic strength. The final elastic moduli of the hydrogels increased with increases in concentration. Isothermal rheology suggests that the gelation occurred in two distinct stages, which was influenced by the solution media. Scanning electron microscopy (SEM) was used to characterize the morphology of the xyloglucan which were thermally gelled at 37 degrees C. The resultant morphology was strongly dependent on the concentration of the hydrogel. Strong hydrogels were only obtained at 3 wt.% at 37 degrees C, and the morphology characterized by an open 3-dimensional network, comprised of thin membranes. It is proposed that the first stage of the isothermal gelation is the formation and growth of the thin membranes, followed by the formation of a three dimensional network.

Morphology and gelation of thermosensitive chitosan hydrogels.

The morphology of physical hydrogels is often difficult to examine due to the delicate nature of the system and therefore has not been studied in detail. Chitosan/GP (glycerophosphate salt) is a significant hydrogel in the biomedical and cosmetic fields as it is thermosensitive and contains less than 5% polysaccharide. The morphology of this system was examined with laser scanning confocal microscopy (LSCM) to image the gel morphology. The images indicate that the gel is quite heterogeneous, and power spectra reveal a fractal-like morphology. A study of composition found that increasing chitosan concentration increased the amount of polymer-rich phase present in the gel, and that the smallest aggregates decreased in size.

Colonization and maintenance of murine embryonic stem cells on poly(alpha-hydroxy esters).

The aim of this study was to determine the ability of various poly(alpha-hydroxy esters) to support the in vitro propagation of murine embryonic stem (ES) cells in an undifferentiated state. To this end, ES cell colonization, growth and Oct-4 immunoreactivity following a 48 h culture period upon poly((D,L)-lactide), poly((L)-lactide), poly(glycolide) and poly((D,L)-lactide-co-glycolide) (PLGA) were assessed. By the analysis of live and dead cell number indices and Oct-4 immunoreactivity, ES cell colonization rate during a 48 h culture period was found to be significantly greater on PLGA compared to all the other unmodified poly(alpha-hydroxy esters) tested. Surface treatment of all polymers with 0.1m potassium hydroxide revealed a significant increase in ES cell live numbers when compared to all unmodified polymers, thus revealing a correlation between polymer content, hydrophilicity and colonization rate. These data suggest that surface treated poly(alpha-hydroxy esters) may be employed for ES cell scale up procedures and in tissue engineering applications requiring the colonization of scaffolds by ES cells in an undifferentiated state. According to such applications, once the designated scaffold has been colonized, ES cell directed differentiation into the desired and fully differentiated, functional adult tissue may then be effected.

Sintered hydroxyfluorapatites--IV: The effect of fluoride substitutions upon colonisation of hydroxyapatites by mouse embryonic stem cells.

Biodegradable scaffolds serve a central role for tissue engineering scaffolds and guiding tissue regeneration. Some of these scaffolds, including apatites, display a significant effect upon cell adhesion and cell proliferation. The incorporation of scaffold technology with the developing embryonic stem (ES) cell field and the capacity of ES cells for self-renewal and differentiation are believed to hold enormous potential for applications in biomedical research and regenerative medicine. The purpose of this work was to determine the effect of hydroxyapatite (HAP) and fluoride substitutions of HAP upon ES cell growth and colonisation. Sintered hydroxyfluorapatite discs were found to support cellular proliferation and colonisation, and the ES cells displayed a tendency for differentiation on the apatite surface as determined by reductions in colony Oct4 immunoreactivity. Fluoride-containing HAPs were found to provide equivalent support to gelatin in terms of cell numbers, yet superior support for cellular colonisation when compared to HAP. This study indicates that fluoride substitutions of HAP may represent a viable strategy for the development of certain engineered tissue replacements and tissue regeneration systems using ES cells.