Pinar Zorlutuna

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.

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.

Direct-write bioprinting of cell-laden methacrylated gelatin hydrogels.

Fabrication of three dimensional (3D) organoids with controlled microarchitectures has been shown to enhance tissue functionality. Bioprinting can be used to precisely position cells and cell-laden materials to generate controlled tissue architecture. Therefore, it represents an exciting alternative for organ fabrication. Despite the rapid progress in the field, the development of printing processes that can be used to fabricate macroscale tissue constructs from ECM-derived hydrogels has remained a challenge. Here we report a strategy for bioprinting of photolabile cell-laden methacrylated gelatin (GelMA) hydrogels. We bioprinted cell-laden GelMA at concentrations ranging from 7 to 15% with varying cell densities and found a direct correlation between printability and the hydrogel mechanical properties. Furthermore, encapsulated HepG2 cells preserved cell viability for at least eight days following the bioprinting process. In summary, this work presents a strategy for direct-write bioprinting of a cell-laden photolabile ECM-derived hydrogel, which may find widespread application for tissue engineering, organ printing and the development of 3D drug discovery platforms.

Directed endothelial cell morphogenesis in micropatterned gelatin methacrylate hydrogels

Engineering of organized vasculature is a crucial step in the development of functional and clinically relevant tissue constructs. A number of previous techniques have been proposed to spatially regulate the distribution of angiogenic biomolecules and vascular cells within biomaterial matrices to promote vascularization. Most of these approaches have been limited to two-dimensional (2D) micropatterned features or have resulted in formation of random vasculature within three-dimensional (3D) microenvironments. In this study, we investigate 3D endothelial cord formation within micropatterned gelatin methacrylate (GelMA) hydrogels with varying geometrical features (50-150 μm height). We demonstrated the significant dependence of endothelial cells proliferation, alignment and cord formation on geometrical dimensions of the patterned features. The cells were able to align and organize within the micropatterned constructs and assemble to form cord structures with organized actin fibers and circular/elliptical cross-sections. The inner layer of the cord structure was filled with gel showing that the micropatterned hydrogel constructs guided the assembly of endothelial cells into cord structures. Notably, the endothelial cords were retained within the hydrogel microconstructs for all geometries after two weeks of culture; however, only the 100 μm-high constructs provided the optimal microenvironment for the formation of circular and stable cord structures. Our findings suggest that endothelial cord formation is a preceding step to tubulogenesis and the proposed system can be used to develop organized vasculature for engineered tissue constructs.

Patterning the differentiation of C2C12 skeletal myoblasts

Mammalian cells are sensitive to the physical properties of their micro-environment such as the stiffness and geometry of the substrate. It is known that the stiffness of the substrate plays a key role in the process of mammalian myogenesis. However, the effect of geometrical constraints on the process of myogenic differentiation needs to be explored further. Here, we show that the geometrical cues of substrates can significantly influence the differentiation process of C2C12 skeletal myoblasts. Three different geometries including lines of different widths, tori of different inner diameters, and hybrid structures (linear and circular features with different arc degrees) were created by micro-contact printing of fibronectin on the surface of Petri dishes. The differentiation of C2C12 cells was studied over a period of seven days and was quantified; we report the differentiation parameters of (1) fusion index, (2) degree of maturation, (3) alignment, and (4) response to electrical pulse stimulation (EPS). Hybrid structures with the smallest arc degree (hybrid 30°) showed the best results for all four differentiation parameters. The hybrid 30° pattern exhibits an ~2-fold increase in the fusion index when compared to the line patterns and an ~3-fold increase when compared to the toroid patterns. The hybrid 30° also showed a higher maturation index compared to the line or the toroid patterns. In response to electrical stimulation (20 V, 50 ms pulse, 1 Hz), mature myotubes on hybrid 30° patterns showed an ~2-fold increase in cellular displacement when compared to myotubes on the line and torus patterns. We tested the influence of C2C12 cell density on fusion and maturation indices, and the results suggest that density does not exert significant influence on cellular differentiation under these conditions. Our results can have important implications in engineering skeletal muscle tissues and designing muscle cell bio-actuators.

Nanobiomaterials: a review of the existing science and technology, and new approaches

Nanotechnology has made great strides forward in the creation of new surfaces, new materials and new forms which also find application in the biomedical field. Traditional biomedical applications started benefiting from the use nanotechnology in an array of areas, such as biosensors, tissue engineering, controlled release systems, intelligent systems and nanocomposites used in implant design. In this manuscript a review of developments in these areas will be provided along with some applications from our laboratories.

Nanopatterning of Collagen Scaffolds Improve the Mechanical Properties of Tissue Engineered Vascular Grafts

Tissue engineered constructs with cells growing in an organized manner have been shown to have improved mechanical properties. This can be especially important when constructing tissues that need to perform under load, such as cardiac and vascular tissue. Enhancement of mechanical properties of tissue engineered vascular grafts via orientation of smooth muscle cells by the help of topographical cues have not been reported yet. In the present study, collagen scaffolds with 650, 500, and 332.5 nm wide nanochannels and ridges were designed and seeded with smooth muscle cells isolated from the human saphenous vein. Cell alignment on the construct was shown by SEM and fluorescence microscopy. The ultimate tensile strength (UTS) and Youngs modulus of the scaffolds were determined after 45 and 75 days. Alamar Blue assay was used to determine the number of viable cells on surfaces with different dimensioned patterns. Presence of nanopatterns increased the UTS from 0.55 +/- 0.11 to as much as 1.63 +/- 0.46 MPa, a value within the range of natural arteries and veins. Similarly, Youngs modulus values were found to be around 4 MPa, again in the range of natural vessels. The study thus showed that nanopatterns as small as 332.5 nm could align the smooth muscle cells and that alignment significantly improved mechanical properties, indicating that nanopatterned collagen scaffolds have the potential for use in the tissue engineering of small diameter blood vessels.

Engineered cell-laden human protein-based elastomer.

Elastic tissue equivalence is a vital requirement of synthetic materials proposed for many resilient, soft tissue engineering applications. Here we present a bioelastomer made from tropoelastin, the human protein that naturally facilitates elasticity and cell interactions in all elastic tissues. We combined this proteins innate versatility with fast non-toxic fabrication techniques to make highly extensible, cell compatible hydrogels. These hydrogels can be produced in less than a minute through photocrosslinking of methacrylated tropoelastin (MeTro) in an aqueous solution. The fabricated MeTro gels exhibited high extensibility (up to 400%) and superior mechanical properties that outperformed other photocrosslinkable hydrogels. MeTro gels were used to encapsulate cells within a flexible 3D environment and to manufacture highly elastic 2D films for cell attachment, growth, and proliferation. In addition, the physical properties of this fabricated bioelastomer such as elasticity, stiffness, and pore characteristics were tuned through manipulation of the methacrylation degree and protein concentration. This photocrosslinkable, functional tissue mimetic gel benefits from the innate biological properties of a human elastic protein and opens new opportunities in tissue engineering.

Fiber-reinforced hydrogel scaffolds for heart valve tissue engineering

Heart valve-related disorders are among the major causes of death worldwide. Although prosthetic valves are widely used to treat this pathology, current prosthetic grafts cannot grow with the patient while maintaining normal valve mechanical and hemodynamic properties. Tissue engineering may provide a possible solution to this issue through using biodegradable scaffolds and patients’ own cells. Despite their similarity to heart valve tissue, most hydrogel scaffolds are not mechanically suitable for the dynamic stresses of the heart valve microenvironment. In this study, we integrated electrospun poly(glycerol sebacate) (PGS)–poly(ɛ-caprolactone) (PCL) microfiber scaffolds, which possess enhanced mechanical properties for heart valve engineering, within a hybrid hydrogel made from methacrylated hyaluronic acid and methacrylated gelatin. Sheep mitral valvular interstitial cells were encapsulated in the hydrogel and evaluated in hydrogel-only, PGS–PCL scaffold-only, and composite scaffold conditions. Although the cellular viability and metabolic activity were similar among all scaffold types, the presence of the hydrogel improved the three-dimensional distribution of mitral valvular interstitial cells. As seen by similar values in both the Young’s modulus and the ultimate tensile strength between the PGS–PCL scaffolds and the composites, microfibrous scaffolds preserved their mechanical properties in the presence of the hydrogels. Compared to electrospun or hydrogel scaffolds alone, this combined system may provide a more suitable three-dimensional structure for generating scaffolds for heart valve tissue engineering.

Directed differentiation of size-controlled embryoid bodies towards endothelial and cardiac lineages in RGD-modified poly(ethylene glycol) hydrogels.

Recent advances in stem cell research have demonstrated the importance of microenvironmental cues in directing stem cell fate towards specific cell lineages. For instance, the size of the embryoid body (EB) was shown to play a role in stem cell differentiation. Other studies have used cell adhesive RGD peptides to direct stem cell fate towards endothelial cells. In this study, materials and cell-based approaches are combined by using microwell arrays to produce size-controlled EBs and encapsulating the resulting aggregates in high molecular weight PEG-4 arm acrylate with and without conjugated RGD to study their effect on stem cell differentiation in a 3D microenvironment. Increasing EB size is observed along with a decrease in the total number of EBs in pristine PEG hydrogel, regardless of the initial EB size. In correlation with this aggregation, EBs in PEG show enhanced cardiogenic differentiation compared to RGD-PEG hydrogel. Both aggregation and cardiogenic differentiation are significantly reduced when RGD peptides are introduced to the microenvironment, while endothelial cell differentiation is accelerated by 3 to 5 days, depending on the EB size, and doubled over the course of cell culture for both EB sizes. Presented results indicate that RGD sequence has a dominant effect in driving endothelial cell differentiation in size-controlled EBs, while pristine multi-arm, high molecular weight PEG can induce cardiogenic differentiation, possibly through EB aggregation. The photopatternable nature of the hydrogel used in this study enabled patterning of such domains devoid or abundant of cell attachment sequences. Therefore, these hydrogels can potentially be used for spatially patterned embryonic stem cell differentiation, which may be beneficial for tissue engineering and regenerative medicine applications.