Development of functional biomaterials with micro- and nanoscale technologies for tissue engineering and drug delivery applications.

Micro- and nanotechnologies have emerged as potentially effective fabrication tools for addressing the challenges faced in tissue engineering and drug delivery. The ability to control and manipulate polymeric biomaterials at the micron and nanometre scale with these fabrication techniques has allowed for the creation of controlled cellular environments, engineering of functional tissues and development of better drug delivery systems. In tissue engineering, micro- and nanotechnologies have enabled the recapitulation of the micro- and nanoscale detail of the cell's environment through controlling the surface chemistry and topography of materials, generating 3D cellular scaffolds and regulating cell-cell interactions. Furthermore, these technologies have led to advances in high-throughput screening (HTS), enabling rapid and efficient discovery of a library of materials and screening of drugs that induce cell-specific responses. In drug delivery, controlling the size and geometry of drug carriers with micro- and nanotechnologies have allowed for the modulation of parametres such as bioavailability, pharmacodynamics and cell-specific targeting. In this review, we introduce recent developments in micro- and nanoscale engineering of polymeric biomaterials, with an emphasis on lithographic techniques, and present an overview of their applications in tissue engineering, HTS and drug delivery.

Microfabricated biomaterials for engineering 3D tissues.

Mimicking natural tissue structure is crucial for engineered tissues with intended applications ranging from regenerative medicine to biorobotics. Native tissues are highly organized at the microscale, thus making these natural characteristics an integral part of creating effective biomimetic tissue structures. There exists a growing appreciation that the incorporation of similar highly organized microscale structures in tissue engineering may yield a remedy for problems ranging from vascularization to cell function control/determination. In this review, we highlight the recent progress in the field of microscale tissue engineering and discuss the use of various biomaterials for generating engineered tissue structures with microscale features. In particular, we will discuss the use of microscale approaches to engineer the architecture of scaffolds, generate artificial vasculature, and control cellular orientation and differentiation. In addition, the emergence of microfabricated tissue units and the modular assembly to emulate hierarchical tissues will be discussed.

Microfabrication of complex porous tissue engineering scaffolds using 3D projection stereolithography.

The success of tissue engineering will rely on the ability to generate complex, cell seeded three-dimensional (3D) structures. Therefore, methods that can be used to precisely engineer the architecture and topography of scaffolding materials will represent a critical aspect of functional tissue engineering. Previous approaches for 3D scaffold fabrication based on top-down and process driven methods are often not adequate to produce complex structures due to the lack of control on scaffold architecture, porosity, and cellular interactions. The proposed projection stereolithography (PSL) platform can be used to design intricate 3D tissue scaffolds that can be engineered to mimic the microarchitecture of tissues, based on computer aided design (CAD). The PSL system was developed, programmed and optimized to fabricate 3D scaffolds using gelatin methacrylate (GelMA). Variation of the structure and prepolymer concentration enabled tailoring the mechanical properties of the scaffolds. A dynamic cell seeding method was utilized to improve the coverage of the scaffold throughout its thickness. The results demonstrated that the interconnectivity of pores allowed for uniform human umbilical vein endothelial cells (HUVECs) distribution and proliferation in the scaffolds, leading to high cell density and confluency at the end of the culture period. Moreover, immunohistochemistry results showed that cells seeded on the scaffold maintained their endothelial phenotype, demonstrating the biological functionality of the microfabricated GelMA scaffolds.

Responsive micromolds for sequential patterning of hydrogel microstructures.

Microscale hydrogels have been shown to be beneficial for various applications such as tissue engineering and drug delivery. A key aspect in these applications is the spatial organization of biological entities or chemical compounds within hydrogel microstructures. For this purpose, sequentially patterned microgels can be used to spatially organize either living materials to mimic biological complexity or multiple chemicals to design functional microparticles for drug delivery. Photolithographic methods are the most common way to pattern microscale hydrogels but are limited to photocrosslinkable polymers. So far, conventional micromolding approaches use static molds to fabricate structures, limiting the resulting shapes that can be generated. Herein, we describe a dynamic micromolding technique to fabricate sequentially patterned hydrogel microstructures by exploiting the thermoresponsiveness of poly(N-isopropylacrylamide)-based micromolds. These responsive micromolds exhibited shape changes under temperature variations, facilitating the sequential molding of microgels at two different temperatures. We fabricated multicompartmental striped, cylindrical, and cubic microgels that encapsulated fluorescent polymer microspheres or different cell types. These responsive micromolds can be used to immobilize living materials or chemicals into sequentially patterned hydrogel microstructures which may potentially be useful for a range of applications at the interface of chemistry, materials science and engineering, and biology.

Cell-laden microengineered pullulan methacrylate hydrogels promote cell proliferation and 3D cluster formation.

The ability to encapsulate cells in three-dimensional (3D) environments is potentially of benefit for tissue engineering and regenerative medicine. In this paper, we introduce pullulan methacrylate (PulMA) as a promising hydrogel platform for creating cell-laden microscale tissues. The hydration and mechanical properties of PulMA were demonstrated to be tunable through modulation of the degree of methacrylation and gel concentration. Cells encapsulated in PulMA exhibited excellent viability. Interestingly, while cells did not elongate in PulMA hydrogels, cells proliferated and organized into clusters, the size of which could be controlled by the hydrogel composition. By mixing with gelatin methacrylate (GelMA), the biological properties of PulMA could be enhanced as demonstrated by cells readily attaching to, proliferating, and elongating within the PulMA/GelMA composite hydrogels. These data suggest that PulMA hydrogels could be useful for creating complex, cell-responsive microtissues, especially for applications that require controlled cell clustering and proliferation.

Synthesis and characterization of tunable poly(ethylene glycol): gelatin methacrylate composite hydrogels.

Poly(ethylene glycol) (PEG) hydrogels are popular for cell culture and tissue-engineering applications because they are nontoxic and exhibit favorable hydration and nutrient transport properties. However, cells cannot adhere to, remodel, proliferate within, or degrade PEG hydrogels. Methacrylated gelatin (GelMA), derived from denatured collagen, yields an enzymatically degradable, photocrosslinkable hydrogel that cells can degrade, adhere to and spread within. To combine the desirable features of each of these materials we synthesized PEG-GelMA composite hydrogels, hypothesizing that copolymerization would enable adjustable cell binding, mechanical, and degradation properties. The addition of GelMA to PEG resulted in a composite hydrogel that exhibited tunable mechanical and biological profiles. Adding GelMA (5%-15% w/v) to PEG (5% and 10% w/v) proportionally increased fibroblast surface binding and spreading as compared to PEG hydrogels (p<0.05). Encapsulated fibroblasts were also able to form 3D cellular networks 7 days after photoencapsulation only within composite hydrogels as compared to PEG alone. Additionally, PEG-GelMA hydrogels displayed tunable enzymatic degradation and stiffness profiles. PEG-GelMA composite hydrogels show great promise as tunable, cell-responsive hydrogels for 3D cell culture and regenerative medicine applications.

Synthesis and characterization of photocrosslinkable gelatin and silk fibroin interpenetrating polymer network hydrogels.

To effectively repair or replace damaged tissues, it is necessary to design scaffolds with tunable structural and biomechanical properties that closely mimic the host tissue. In this paper, we describe a newly synthesized photocrosslinkable interpenetrating polymer network (IPN) hydrogel based on gelatin methacrylate (GelMA) and silk fibroin (SF) formed by sequential polymerization, which possesses tunable structural and biological properties. Experimental results revealed that IPNs, where both the GelMA and SF were independently crosslinked in interpenetrating networks, demonstrated a lower swelling ratio, higher compressive modulus and lower degradation rate as compared to the GelMA and semi-IPN hydrogels, where only GelMA was crosslinked. These differences were likely caused by a higher degree of overall crosslinking due to the presence of crystallized SF in the IPN hydrogels. NIH-3T3 fibroblasts readily attached to, spread and proliferated on the surface of IPN hydrogels, as demonstrated by F-actin staining and analysis of mitochondrial activity (MTT). In addition, photolithography combined with lyophilization techniques was used to fabricate three-dimensional micropatterned and porous microscaffolds from GelMA-SF IPN hydrogels, furthering their versatility for use in various microscale tissue engineering applications. Overall, this study introduces a class of photocrosslinkable, mechanically robust and tunable IPN hydrogels that could be useful for various tissue engineering and regenerative medicine applications.

Controlling the porosity of fibrous scaffolds by modulating the fiber diameter and packing density.

Porosity has been shown to be a key determinant of the success of tissue engineered scaffolds. A high degree of porosity and an appropriate pore size are necessary to provide adequate space for cell spreading and migration as well as to allow for proper exchange of nutrients and waste between the scaffold and the surrounding environment. Electrospun scaffolds offer an attractive approach for mimicking the natural extracellular matrix (ECM) for tissue engineering applications. The efficacy of electrospinning is likely to depend on the interaction between cells and the geometric features and physicochemical composition of the scaffold. A major problem in electrospinning is the tendency of fibers to accumulate densely, resulting in poor porosity and small pore size. The porosity and pore sizes in the electrospun scaffolds are mainly dependent on the fiber diameter and their packing density. Here we report a method of modulating porosity in three dimensional (3D) scaffolds by simultaneously tuning the fiber diameter and the fiber packing density. Nonwoven poly(ε-caprolactone) mats were formed by electrospinning under various conditions to generate sparse or highly dense micro- and nanofibrous scaffolds and characterized for their physicochemical and biological properties. We found that microfibers with low packing density resulted in improved cell viability, proliferation and infiltration compared to tightly packed scaffolds.

Cell-adhesive and mechanically tunable glucose-based biodegradable hydrogels.

The development of materials with biomimetic mechanical and biological properties is of great interest for regenerative medicine applications. In particular, hydrogels are a promising class of biomaterials due to their high water content, which mimics that of natural tissues. We have synthesized a hydrophilic biodegradable polymer, designated poly(glucose malate)methacrylate (PGMma), which is composed of glucose and malic acid, commonly found in the human metabolic system. This polymer is made photocrosslinkable by the incorporation of methacrylate groups. The resulting properties of the hydrogels can be tuned by altering the reacting ratio of the starting materials, the degree of methacrylation, and the polymer concentration of the resultant hydrogel. Hydrogels exhibited compressive moduli ranging from 1.8 ± 0.4 kPa to 172.7 ± 36 kPa with compressive strain at failure from 37.5 ± 0.9% to 61.2 ± 1.1%, and hydration by mass ranging from 18.7 ± 0.5% to 114.1 ± 1.3%. PGMma hydrogels also showed a broad range of degradation rates and were cell-adhesive, enabling the spreading of adherent cells. Overall, this work introduces a class of cell-adhesive, mechanically tunable and biodegradable glucose-based hydrogels that may be useful for various tissue engineering and cell culture applications.

Directed 3D cell alignment and elongation in microengineered hydrogels.

Organized cellular alignment is critical to controlling tissue microarchitecture and biological function. Although a multitude of techniques have been described to control cellular alignment in 2D, recapitulating the cellular alignment of highly organized native tissues in 3D engineered tissues remains a challenge. While cellular alignment in engineered tissues can be induced through the use of external physical stimuli, there are few simple techniques for microscale control of cell behavior that are largely cell-driven. In this study we present a simple and direct method to control the alignment and elongation of fibroblasts, myoblasts, endothelial cells and cardiac stem cells encapsulated in microengineered 3D gelatin methacrylate (GelMA) hydrogels, demonstrating that cells with the intrinsic potential to form aligned tissues in vivo will self-organize into functional tissues in vitro if confined in the appropriate 3D microarchitecture. The presented system may be used as an in vitro model for investigating cell and tissue morphogenesis in 3D, as well as for creating tissue constructs with microscale control of 3D cellular alignment and elongation, that could have great potential for the engineering of functional tissues with aligned cells and anisotropic function.

Interface-directed self-assembly of cell-laden microgels.

Cell-laden hydrogels show great promise for creating engineered tissues. However, a major shortcoming with these systems has been the inability to fabricate structures with controlled micrometer-scale features on a biologically relevant length scale. In this Full Paper, a rapid method is demonstrated for creating centimeter-scale, cell-laden hydrogels through the assembly of shape-controlled microgels or a liquid-air interface. Cell-laden microgels of specific shapes are randomly placed on the surface of a high-density, hydrophobic solution, induced to aggregate and then crosslinked into macroscale tissue-like structures. The resulting assemblies are cell-laden hydrogel sheets consisting of tightly packed, ordered microgel units. In addition, a hierarchical approach creates complex multigel building blocks, which are then assembled into tissues with precise spatial control over the cell distribution. The results demonstrate that forces at an air-liquid interface can be used to self-assemble spatially controllable, cocultured tissue-like structures.

Cell-laden microengineered gelatin methacrylate hydrogels.

The cellular microenvironment plays an integral role in improving the function of microengineered tissues. Control of the microarchitecture in engineered tissues can be achieved through photopatterning of cell-laden hydrogels. However, despite high pattern fidelity of photopolymerizable hydrogels, many such materials are not cell-responsive and have limited biodegradability. Here, we demonstrate gelatin methacrylate (GelMA) as an inexpensive, cell-responsive hydrogel platform for creating cell-laden microtissues and microfluidic devices. Cells readily bound to, proliferated, elongated, and migrated both when seeded on micropatterned GelMA substrates as well as when encapsulated in microfabricated GelMA hydrogels. The hydration and mechanical properties of GelMA were demonstrated to be tunable for various applications through modification of the methacrylation degree and gel concentration. The pattern fidelity and resolution of GelMA were high and it could be patterned to create perfusable microfluidic channels. Furthermore, GelMA micropatterns could be used to create cellular micropatterns for in vitro cell studies or 3D microtissue fabrication. These data suggest that GelMA hydrogels could be useful for creating complex, cell-responsive microtissues, such as endothelialized microvasculature, or for other applications that require cell-responsive microengineered hydrogels.

Controlling the porosity and microarchitecture of hydrogels for tissue engineering.

Tissue engineering holds great promise for regeneration and repair of diseased tissues, making the development of tissue engineering scaffolds a topic of great interest in biomedical research. Because of their biocompatibility and similarities to native extracellular matrix, hydrogels have emerged as leading candidates for engineered tissue scaffolds. However, precise control of hydrogel properties, such as porosity, remains a challenge. Traditional techniques for creating bulk porosity in polymers have demonstrated success in hydrogels for tissue engineering; however, often the conditions are incompatible with direct cell encapsulation. Emerging technologies have demonstrated the ability to control porosity and the microarchitectural features in hydrogels, creating engineered tissues with structure and function similar to native tissues. In this review, we explore the various technologies for controlling the porosity and microarchitecture within hydrogels, and demonstrate successful applications of combining these techniques.

Micro- and nanoscale control of the cardiac stem cell niche for tissue fabrication.

Advances in stem cell (SC) biology have greatly enhanced our understanding of SC self-renewal and differentiation. Both embryonic and adult SCs can be differentiated into a great variety of tissue cell types, including cardiac myocytes. In vivo studies and clinical trials, however, have demonstrated major limitations in reconstituting the myocardium in failing hearts. These limitations include precise control of SC proliferation, survival and phenotype both prior and subsequent to transplantation and avoidance of serious adverse effects such as tumorigenesis and arrhythmias. Micro- and nanoscale techniques to recreate SC niches, the natural environment for the maintenance and regulation of SCs, have enabled the elucidation of novel SC behaviors and offer great promise in the fabrication of cardiac tissue constructs. The ability to precisely manipulate the interface between biopolymeric scaffolds and SCs at in vivo scale resolutions is unique to micro- and nanoscale approaches and may help overcome limitations of conventional biological scaffolds and methods for cell delivery. We now know that micro- and nanoscale manipulation of scaffold composition, mechanical properties, and three-dimensional architecture have profound influences on SC fate and will likely prove important in developing the next generation of "transplantable SC niches" for regeneration of heart and other tissues. In this review, we examine two key aspects of micro- and nanofabricated SC-based cardiac tissue constructs: the role of scaffold composition and the role of scaffold architecture and detail how recent work in these areas brings us closer to clinical solutions for cardiovascular regeneration.

Modular Tissue Engineering: Engineering Biological Tissues from the Bottom Up.

Tissue engineering creates biological tissues that aim to improve the function of diseased or damaged tissues. To enhance the function of engineered tissues there is a need to generate structures that mimic the intricate architecture and complexity of native organs and tissues. With the desire to create more complex tissues with features such as developed and functional microvasculature, cell binding motifs and tissue specific morphology, tissue engineering techniques are beginning to focus on building modular microtissues with repeated functional units. The emerging field known as modular tissue engineering focuses on fabricating tissue building blocks with specific microarchitectural features and using these modular units to engineer biological tissues from the bottom up. In this review we will examine the promise and shortcomings of "bottom-up" approaches to creating engineered biological tissues. Specifically, we will survey the current techniques for controlling cell aggregation, proliferation and extracellular matrix deposition, as well as approaches to generating shape-controlled tissue modules. We will then highlight techniques utilized to create macroscale engineered biological tissues from modular microscale units.

Hemodynamics and axial strain additively increase matrix remodeling and MMP-9, but not MMP-2, expression in arteries engineered by directed remodeling.

We previously demonstrated the ability to create engineered arteries by carefully controlling the mechanical environment of intact arteries perfused ex vivo, yielding engineered arteries with native appearance and vasoactive response. Increased axial strain was sufficient to increase length up to 20% in 9 days through a growth and remodeling response. The amount of the achievable length increase, however, was highly dependent on the hemodynamic conditions acting through unknown mechanisms. Because matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) activity is increased, and often required, in mechanically induced remodeling in vivo, MMP-2 and MMP-9 expression was investigated to elucidate the hemodynamic mediation of artery length. Carotid arteries from 30 kg pigs were perfused for 9 days ex vivo at either in situ axial strain or with a gradual 50% increase in axial strain, under either arterial or reduced hemodynamics ( approximately 10% of arterial hemodynamics). MMP-2 protein expression increased roughly twofold, while MMP-9 expression increased threefold under either reduced hemodynamics or increased axial strain (p < 0.05). The combination of reduced hemodynamics with increased axial strain demonstrated an additive increase in MMP-9 protein (p < 0.05) with no further change in MMP-2 expression. To investigate the mechanism by which axial strain and hemodynamics could additively increase MMP-9 expression, the expression of nuclear factor kappa B (NF-kappaB) subunits p50 and p65 was evaluated. Axial strain stimulated p65 expression and localization, while hemodynamics increased p50 expression, with both molecules being expressed only when both mechanical stimuli were applied. These data suggest that MMP-9 expression can be simultaneously stimulated by separate mechanical stimuli mediated by p50 and p65 expression, and that by using conditions that maximize MMP-9 expression, we can create an optimal remodeling environment to better direct the growth of engineered arteries and other tissues.

Co-culture induces alignment in engineered cardiac constructs via MMP-2 expression.

Cardiac tissue engineering has been limited by the inability to recreate native myocardial structural features. We hypothesized that heart cell elongation and alignment in 3D engineered cardiac constructs would be enhanced by using physiologic ratios of cardiomyocytes (CM) and cardiac fibroblasts (CF) via matrix metalloprotease (MMP)-dependent mechanisms. Co-cultured CM and CF constructs were compared to CM-enriched constructs using either basal media or media with a general MMP inhibitor for 8 days. Co-cultured constructs exhibited significantly increased cell alignment (p<0.0002), which was eliminated by MMP inhibition. Co-cultured constructs expressed substantial active MMP-2 protein that was not present in CM-enriched constructs, increased pro-MMP-2 (p<0.001), and reduced pro-MMP-9 (p<0.001) expression. Apoptosis was decreased by co-culture (p<0.05), independent of MMP inhibition. These results demonstrated that co-culture of CF in physiologic ratios within engineered cardiac constructs improved cell elongation and alignment via increased MMP-2 expression and activation, and also improved viability independent of MMP activity.

A biodegradable and biocompatible gecko-inspired tissue adhesive.

There is a significant medical need for tough biodegradable polymer adhesives that can adapt to or recover from various mechanical deformations while remaining strongly attached to the underlying tissue. We approached this problem by using a polymer poly(glycerol-co-sebacate acrylate) and modifying the surface to mimic the nanotopography of gecko feet, which allows attachment to vertical surfaces. Translation of existing gecko-inspired adhesives for medical applications is complex, as multiple parameters must be optimized, including: biocompatibility, biodegradation, strong adhesive tissue bonding, as well as compliance and conformability to tissue surfaces. Ideally these adhesives would also have the ability to deliver drugs or growth factors to promote healing. As a first demonstration, we have created a gecko-inspired tissue adhesive from a biocompatible and biodegradable elastomer combined with a thin tissue-reactive biocompatible surface coating. Tissue adhesion was optimized by varying dimensions of the nanoscale pillars, including the ratio of tip diameter to pitch and the ratio of tip diameter to base diameter. Coating these nanomolded pillars of biodegradable elastomers with a thin layer of oxidized dextran significantly increased the interfacial adhesion strength on porcine intestine tissue in vitro and in the rat abdominal subfascial in vivo environment. This gecko-inspired medical adhesive may have potential applications for sealing wounds and for replacement or augmentation of sutures or staples.

A porous photocurable elastomer for cell encapsulation and culture.

Encapsulating cells within a polymer matrix creates a three-dimensional (3D) scaffold that may more accurately represent the native microenvironment and cell organization. Here we report a porous scaffold prepared from a photocurable elastomer, poly(glycerolco-sebacate)-acrylate (PGSA). The scaffold porosity, swelling, mass loss, toxicity and mechanical properties, suggest that porous PGSA could be used to support the growth and differentiation of encapsulated cells. Neuroblastoma (NB) and human embryonic stem cells (hESCs) were encapsulated into the matrix and found to adhere to the material and interact with each other within 24h. After 7 days, encapsulated NB cells were found to grow, and form matrix fibrils and tissue. Undifferentiated hESCs proliferated and differentiated in the PGSA scaffold. In vivo experiments showed that both porous scaffolds have similar biocompatibility profiles as non-porous PGSA, but porous PGSA promotes tissue ingrowth, as compared to non-porous PGSA. We therefore propose that porous PGSA scaffolds can provide a logistical template for 3D growth of cells and tissue engineering.

Micropatterned polymer surfaces improve retention of endothelial cells exposed to flow-induced shear stress.

The use of synthetic polymeric vascular grafts is limited by the thrombogenecity of most biomaterials. Efforts to reduce thrombogenicity by seeding grafts with endothelial cells, the natural non-thrombogenic lining of blood vessels, have been thwarted by flow-induced cell detachment. We hypothesized that by creating well-defined micro-textured patterns on a surface, fluid flow at the surface can be altered to create discrete regions of low shear stress. We further hypothesized that, due to reduced shear stress, these regions will serve as sanctuaries for endothelial cells and promote their retention. To test these hypotheses, well-defined micro-textured polyurethane (PU) surfaces consisting of arrays of parallel 95-micron wide and 32-micron deep channels were created using an etched silicon template and solvent casting techniques. Based on computational fluid dynamics, under identical bulk flow conditions, the average local shear stress in the channels (46 dyn/cm2) was 28% lower than unpatterned surfaces (60 dyn/cm2). When PU surfaces pre-seeded with endothelial cells (EC) were exposed to the same bulk flow rate, EC retention was significantly improved on the micropatterned surfaces relative to un-patterned surfaces (92% vs. 58% retention).