Scott Alexander Irvine

Micro‐/Nano‐engineered Cellular Responses for Soft Tissue Engineering and Biomedical Applications

The development of biomedical devices and reconstruction of functional ex vivo tissues often requires the need to fabricate biomimetic surfaces with features of sub-micrometer precision. This can be achieved with the advancements in micro-/nano-engineering techniques, allowing researchers to manipulate a plethora of cellular behaviors at the cell-biomaterial interface. Systematic studies conducted on these 2D engineered surfaces have unraveled numerous novel findings that can potentially be integrated as part of the design consideration for future 2D and 3D biomaterials and will no doubt greatly benefit tissue engineering. In this review, recent developments detailing the use of micro-/nano-engineering techniques to direct cellular orientation and function pertinent to soft tissue engineering will be highlighted. Particularly, this article aims to provide valuable insights into distinctive cell interactions and reactions to controlled surfaces, which can be exploited to understand the mechanisms of cell growth on micro-/nano-engineered interfaces, and to harness this knowledge to optimize the performance of 3D artificial soft tissue grafts and biomedical applications.

From Soft Self-Healing Gels to Stiff Films in Suckerin- Based Materials Through Modulation of Crosslink Density and β-Sheet Content

Suckerins are block-copolymer-like structural proteins constituting the building blocks of the strong squid sucker-ring teeth. Here, recombinant suckerin-19 is processed into biomaterials spanning a wide range of elasticity, from very soft hydrogels to stiff films with elastic modulus in the gigapascal range. The elasticity is controlled by the interplay between the β-sheet content and induced di-tyrosine crosslinking.

Bioprinting and Differentiation of Stem Cells

The 3D bioprinting of stem cells directly into scaffolds offers great potential for the development of regenerative therapies; in particular for the fabrication of organ and tissue substitutes. For this to be achieved; the lineage fate of bioprinted stem cell must be controllable. Bioprinting can be neutral; allowing culture conditions to trigger differentiation or alternatively; the technique can be designed to be stimulatory. Such factors as the particular bioprinting technique; bioink polymers; polymer cross-linking mechanism; bioink additives; and mechanical properties are considered. In addition; it is discussed that the stimulation of stem cell differentiation by bioprinting may lead to the remodeling and modification of the scaffold over time matching the concept of 4D bioprinting. The ability to tune bioprinting properties as an approach to fabricate stem cell bearing scaffolds and to also harness the benefits of the cells multipotency is of considerable relevance to the field of biomaterials and bioengineering.

Novel gradient casting method provides high-throughput assessment of blended polyester poly(lactic-co-glycolic acid) thin films for parameter optimization

Pure polymer films cannot meet the diverse range of controlled release and material properties demanded for the fabrication of medical implants or other devices. Additives are added to modulate and optimize thin films for the desired qualities. To characterize the property trends that depend on additive concentration, an assay was designed which involved casting a single polyester poly(lactic-co-glycolic acid) (PLGA) film that blends a linear gradient of any PLGA-soluble additive desired. Four gradient PLGA films were produced by blending polyethylene glycol or the more hydrophobic polypropylene glycol. The films were made using a custom glass gradient maker in conjunction with a 180 cm film applicator. These films were characterized in terms of thickness, percent additive, total polymer (PLGA+additive), and controlled drug release using drug-like fluorescent molecules such as coumarin 6 (COU) or fluorescein diacetate (FDAc). Material properties of elongation and modulus were also accessed. Linear gradients of additives were readily generated, with phase separation being the limiting factor. Additive concentration had a Pearsons correlation factor (R) of >0.93 with respect to the per cent total release after 30 days for all gradients characterized. Release of COU had a near zero-order release over the same time period, suggesting that coumarin analogs may be suitable for use in PLGA/polyethylene glycol or PLGA/polypropylene glycol matrices, with each having unique material properties while allowing tuneable drug release. The gradient casting method described has considerable potential in offering higher throughput for optimizing film or coating material properties for medical implants or other devices.

A novel 3D printing method for cell alignment and differentiation

The application of bioprinting allows precision deposition of biological materials for bioengineering applications. Here we propose a 2 stage methodology for bioprinting using a back pressure-driven, automated robotic dispensing system. This apparatus can prepare topographic guidance features for cell orientation and then bioprint cells directly onto them. Topographic guidance features generate cues that influence adhered cell morphology and phenotype. The robotic dispensing system was modified to include a sharpened stylus that etched on a polystyrene surface. The same computer-aided design (CAD) software was used for both precision control of etching and bioink deposition. Various etched groove patterns such as linear, concentric circles, and sinusoidal wave patterns were possible. Fibroblasts and mesenchymal stem cells (MSC) were able to sense the grooves, as shown by their elongation and orientation in the direction of the features. The orientated MSCs displayed indications of lineage commitment as detected by fluorescence-activated cell sorting (FACS) analysis. A 2% gelatin bioink was then used to dispense cells onto the etched features using identical, programmed co-ordinates. The bioink allows the cells to contact sense the pattern while containing their deposition within the printed pattern.The application of bioprinting allows the precision deposition of biological material for bioengineering applications. Here we propose a 2 stage methodology for bioprinting using a back pressure driven automated robotic dispensing system. This apparatus can prepare topographic guidance features for cell orientation and then bioprint cells directly to them. Topographic guidance features generate cues that influence adhered cell morphology and phenotype. The robotic dispensing system was modified to include a sharpened stylus that etched a polystyrene surface. The same CAD software was used for both precision control of etching and bioink deposition. Various etched groove patterns were possible, such as linear, concentric circles and sinusoidal wave patterns. Fibroblasts and mesenchymal stem cells (MSC) could sense the grooves, elongating and orientating themselves in the direction of the features, with the MSCs displaying indications of lineage commitment. A 2% gelatin bioink was then used to dispense cells onto the etched features using identical programmed co-ordinates. The bioink allows the cells to contact sense the pattern while containing their deposition within the printed pattern.

3D patterned substrates for bioartificial blood vessels – The effect of hydrogels on aligned cells on a biomaterial surface

UNLABELLED The optimal bio-artificial blood vessel construct is one that has a compliant tubular core with circumferentially aligned smooth muscle cells (SMCs). Obtaining this well-aligned pattern of SMCs on a scaffold is highly beneficial as this cellular orientation preserves the SMC contractile phenotype. We used 3D patterning to create channels on a polycaprolactone (PCL) scaffold; SMCs were then found to be aligned within the microchannels. To preserve this alignment, and to provide a protective coating that could further incorporate cells, we evaluated the use of two hydrogels, one based on poly(ethylene glycol) diacrylate (PEGDA) and the other based on gelatin. Hydrogels were either physically coated on the PCL surfaces or covalently linked via suitable surface modification of PCL. For covalent immobilization of PEGDA hydrogel, alkene groups were introduced on PCL, while for gelatin covalent linkage, serum proteins were introduced. It is, however, crucial that the hydrogel coating does not disrupt the cellular patterning and distribution. We show in this work that both the process of coating as well as the nature of the coating are critical to preservation of the aligned SMCs. The covalent coating methods involving the crosslinking of hydrogels with the surface of PCL films promoted hydrogel retention time on the film as compared with physical deposition. Furthermore, subsequent hydrogel degradation is affected by the components of the cell culture medium, hinting at a possible route to in vivo biodegradation. STATEMENT OF SIGNIFICANCE Surface features control cellular orientation and subsequently influence their functionality, a useful effect for cellularized biomedical devices. Such devices also can benefit from protective and cell friendly hydrogel coatings. However, literature is lacking on the fate of cells that have endured hydrogel coating whilst orientated on a biomaterial surface. In particular, elucidation of the cells ability to remain adherent and orientated post hydrogel addition. Coating requires two procedures that may be deleterious to the orientated cells: the surface pretreatment for gel binding and the hydrogel crosslinking reaction. We compare transglutaminase gelatin crosslinking and UV initiated PEGDA crosslinking, coated onto smooth muscle cells orientated on patterned PCL surfaces. This original study will be of considerable use to the wider biomaterials community.

Contact guidance for cardiac tissue engineering using 3D bioprinted gelatin patterned hydrogel

Here, we have developed a 3D bioprinted microchanneled gelatin hydrogel that promotes human mesenchymal stem cell (hMSC) myocardial commitment and supports native cardiomyocytes (CMs) contractile functionality. Firstly, we studied the effect of bioprinted microchanneled hydrogel on the alignment, elongation, and differentiation of hMSC. Notably, the cells displayed well defined F-actin anisotropy and elongated morphology on the microchanneled hydrogel, hence showing the effects of topographical control over cell behavior. Furthermore, the aligned stem cells showed myocardial lineage commitment, as detected using mature cardiac markers. The fluorescence-activated cell sorting analysis also confirmed a significant increase in the commitment towards myocardial tissue lineage. Moreover, seeded CMs were found to be more aligned and demonstrated synchronized beating on microchanneled hydrogel as compared to the unpatterned hydrogel. Overall, our study proved that microchanneled hydrogel scaffold produced by 3D bioprinting induces myocardial differentiation of stem cells as well as supports CMs growth and contractility. Applications of this approach may be beneficial for generating in vitro cardiac model systems to physiological and cardiotoxicity studies as well as in vivo generating custom designed cell impregnated constructs for tissue engineering and regenerative medicine applications.

Smooth Muscle Cell Alignment and Phenotype Control by Melt Spun Polycaprolactone Fibers for Seeding of Tissue Engineered Blood Vessels

A method has been developed to induce and retain a contractile phenotype for vascular smooth muscle cells, as the first step towards the development of a biomimetic blood vessel construct with minimal compliance mismatch. Melt spun PCL fibers were deposited on a mandrel to form aligned fibers of 10 μm in diameter. The fibers were bonded into aligned arrangement through dip coating in chitosan solution. This formed a surface of parallel grooves, 10 μm deep by 10 μm across, presenting a surface layer of chitosan to promote cell surface interactions. The aligned fiber surface was used to culture cells present in the vascular wall, in particular fibroblasts and smooth muscle cells. This topography induced “surface guidance” over the orientation of the cells, which adopted an elongated spindle-like morphology, whereas cells on the unpatterned control surface did not show such orientation, assuming more rhomboid shapes. The preservation of VSMC contractile phenotype on the aligned scaffold was demonstrated by the retention of α-SMA expression after several days of culture. The effect was assessed on a prototype vascular graft prosthesis fabricated from polylactide caprolactone; VSMCs aligned longitudinally along a fiberless tube, whereas, for the aligned fiber coated tubes, the VSMCs aligned in the required circumferential orientation.

Quantification of aldehyde terminated heparin by SEC-MALLS–UV for the surface functionalization of polycaprolactone biomaterials

A straight forward strategy of heparin surface grafting employs a terminal reactive-aldehyde group introduced through nitrous acid depolymerization. An advanced method that allows simultaneously monitoring of both heparin molar mass and monomer/aldehyde ratio by size exclusion chromatography, multi-angle laser light scattering and UV-absorbance (SEC-MALLS-UV) has been developed to improve upon heparin surface grafting. Advancements over older methods allow quantitative characterization by direct (aldehyde absorbance) and indirect (Schiff-based absorbance) evaluation of terminal functional aldehydes. The indirect quantitation of functional aldehydes through labeling with aniline (and the formation of a Schiff-base) allows independent quantitation of both polymer mass and terminal functional groups with the applicable UV mass extinction coefficients determined. The protocol was subsequently used to synthesize an optimized heparin-aldehyde that had minimal polydispersity (PDI<2) and high reaction yields (yield >60% by mass). The 8 kDa weight averaged molar mass heparin-aldehyde was then grafted on polycaprolactone (PCL), a common implant material. This optimized heparin-aldehyde retained its antithrombin activity, assessed in freshly drawn blood or surface immobilized on PCL films. Anticoagulant activity was equal to or better than the 24 kDa unmodified heparin it was fragmented from.

Bioprinted gelatin hydrogel platform promotes smooth muscle cell contractile phenotype maintenance

Three dimensional (3D) bioprinting has been proposed as a method for fabricating tissue engineered small diameter vascular prostheses. This technique not only involves constructing the structural features to obtain a desired pattern but the morphology of the pattern may also be used to influence the behavior of seeded cells. Herein, we 3D bioprinted a gelatin hydrogel microchannel construct to promote and preserve the contractile phenotype of vascular smooth muscle cells (vSMCs), which is crucial for vasoresponsiveness. The microchanneled surface of a gelatin hydrogel facilitated vSMC attachment and an elongated alignment along the microchannel direction. The cells displayed distinct F-actin anisotropy in the direction of the channel. The vSMC contractile phenotype was confirmed by the positive detection of contractile marker gene proteins (α-smooth muscle actin (α-SMA) and smooth muscle-myosin heavy chain (SM-MHC)). Having demonstrated the effectiveness of the hydrogel channels bioprinted on a film, the bioprinting was applied radially to the surface of a 3D tubular construct by integrating a rotating mandrel into the 3D bioprinter. The hydrogel microchannels printed on the 3D tubular vascular construct also orientated the vSMCs and strongly promoted the contractile phenotype. Together, our study demonstrated that microchannels bioprinted using a transglutaminase crosslinked gelatin hydrogel, could successfully promote and preserve vSMC contractile phenotype. Furthermore, the hydrogel bioink could be retained on the surface of a rotating polymer tube to print radial cell guiding channels onto a vascular graft construct.