Sandra Hofmann

Structural and material approaches to bone tissue engineering in powder-based three-dimensional printing

This article reviews the current state of knowledge concerning the use of powder-based three-dimensional printing (3DP) for the synthesis of bone tissue engineering scaffolds. 3DP is a solid free-form fabrication (SFF) technique building up complex open porous 3D structures layer by layer (a bottom-up approach). In contrast to traditional fabrication techniques generally subtracting material step by step (a top-down approach), SFF approaches allow nearly unlimited designs and a large variety of materials to be used for scaffold engineering. Todays state of the art materials, as well as the mechanical and structural requirements for bone scaffolds, are summarized and discussed in relation to the technical feasibility of their use in 3DP. Advances in the field of 3DP are presented and compared with other SFF methods. Existing strategies on material and design control of scaffolds are reviewed. Finally, the possibilities and limiting factors are addressed and potential strategies to improve 3DP for scaffold engineering are proposed.

Silk fibroin as biomaterial for bone tissue engineering

UNLABELLED Silk fibroin (SF) is a fibrous protein which is produced mainly by silkworms and spiders. Its unique mechanical properties, tunable biodegradation rate and the ability to support the differentiation of mesenchymal stem cells along the osteogenic lineage, have made SF a favorable scaffold material for bone tissue engineering. SF can be processed into various scaffold forms, combined synergistically with other biomaterials to form composites and chemically modified, which provides an impressive toolbox and allows SF scaffolds to be tailored to specific applications. This review discusses and summarizes recent advancements in processing SF, focusing on different fabrication and functionalization methods and their application to grow bone tissue in vitro and in vivo. Potential areas for future research, current challenges, uncertainties and gaps in knowledge are highlighted. STATEMENT OF SIGNIFICANCE Silk fibroin is a natural biomaterial with remarkable biomedical and mechanical properties which make it favorable for a broad range of bone tissue engineering applications. It can be processed into different scaffold forms, combined synergistically with other biomaterials to form composites and chemically modified which provides a unique toolbox and allows silk fibroin scaffolds to be tailored to specific applications. This review discusses and summarizes recent advancements in processing silk fibroin, focusing on different fabrication and functionalization methods and their application to grow bone tissue in vitro and in vivo. Potential areas for future research, current challenges, uncertainties and gaps in knowledge are highlighted.

Controlled Positioning of Cells in Biomaterials—Approaches Towards 3D Tissue Printing

Current tissue engineering techniques have various drawbacks: they often incorporate uncontrolled and imprecise scaffold geometries, whereas the current conventional cell seeding techniques result mostly in random cell placement rather than uniform cell distribution. For the successful reconstruction of deficient tissue, new material engineering approaches have to be considered to overcome current limitations. An emerging method to produce complex biological products including cells or extracellular matrices in a controlled manner is a process called bioprinting or biofabrication, which effectively uses principles of rapid prototyping combined with cell-loaded biomaterials, typically hydrogels. 3D tissue printing is an approach to manufacture functional tissue layer-by-layer that could be transplanted in vivo after production. This method is especially advantageous for stem cells since a controlled environment can be created to influence cell growth and differentiation. Using printed tissue for biotechnological and pharmacological needs like in vitro drug-testing may lead to a revolution in the pharmaceutical industry since animal models could be partially replaced by biofabricated tissues mimicking human physiology and pathology. This would not only be a major advancement concerning rising ethical issues but would also have a measureable impact on economical aspects in this industry of today, where animal studies are very labor-intensive and therefore costly. In this review, current controlled material and cell positioning techniques are introduced highlighting approaches towards 3D tissue printing.

Tunable hydrogel composite with two-step processing in combination with innovative hardware upgrade for cell-based three-dimensional bioprinting.

Three-dimensional (3-D) bioprinting is the layer-by-layer deposition of biological material with the aim of achieving stable 3-D constructs for application in tissue engineering. It is a powerful tool for the spatially directed placement of multiple materials and/or cells within the 3-D sample. Encapsulated cells are protected by the bioink during the printing process. Very few materials are available that fulfill requirements for bioprinting as well as provide adequate properties for cell encapsulation during and after the printing process. A hydrogel composite including alginate and gelatin precursors was tuned with different concentrations of hydroxyapatite (HA) and characterized in terms of rheology, swelling behavior and mechanical properties to assess the versatility of the system. Instantaneous as well as long-term structural integrity of the printed hydrogel was achieved with a two-step mechanism combining the thermosensitive properties of gelatin with chemical crosslinking of alginate. Novel syringe tip heaters were developed for improved temperature control of the bioink to avoid clogging. Human mesenchymal stem cells mixed into the hydrogel precursor survived the printing process and showed high cell viability of 85% living cells after 3 days of subsequent in vitro culture. HA enabled the visualization of the printed structures with micro-computed tomography. The inclusion of HA also favors the use of the bioink for bone tissue engineering applications. By adding factors other than HA, the composite could be used as a bioink for applications in drug delivery, microsphere deposition or soft tissue engineering.

Chondrocyte redifferentiation in 3D: The effect of adhesion site density and substrate elasticity †

To obtain sufficient cell numbers for cartilage tissue engineering with autologous chondrocytes, cells are typically expanded in monolayer culture. As a result, they lose their chondrogenic phenotype in a process called dedifferentiation, which can be reversed upon transfer into a 3D environment. We hypothesize that the properties of this 3D environment, namely adhesion site density and substrate elasticity, would influence this redifferentiation process. To test this hypothesis, chondrocytes were expanded in monolayer and their phenotypical transition was monitored. Agarose hydrogels manipulated to give different RGD adhesion site densities and mechanical properties were produced, cells were incorporated into the gels to induce redifferentiation, and constructs were analyzed to determine cell number and extracellular matrix production after 2 weeks of 3D culture. The availability of adhesion sites within the gels inhibited cellular redifferentiation. Glycosaminoglycan production per cell was diminished by RGD in a dose-dependent manner and cells incorporated into gels with the highest RGD density, remained positive for collagen type I and produced the least collagen type II. Substrate stiffness, in contrast, did not influence cellular redifferentiation, but softer gels contained higher cell numbers and ECM amounts after 2 weeks of culture. Our results indicate that adhesion site density but not stiffness influences the redifferentiation process of chondrocytes in 3D. This knowledge might be used to optimize the redifferentiation process of chondrocytes and thus the formation of cartilage-like tissue.

Injectable and porous PLGA microspheres that form highly porous scaffolds at body temperature

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New depowdering-friendly designs for three-dimensional printing of calcium phosphate bone substitutes

Powder-based three-dimensional printing (3DP) is a versatile method that allows creating synthetic calcium phosphate (CaP) scaffolds of complex shapes and structures. However, one major drawback is the difficulty of removing all remnants of loose powder from the printed scaffolds, the so-called depowdering step. In this study, a new design approach was proposed to solve this problem. Specifically, the design of the printed scaffolds consisted of a cage with windows large enough to enable depowdering while still trapping loose fillers placed inside the cage. To demonstrate the potential of this new approach, two filler geometries were used: sandglass and cheese segment. The distance between the fillers was varied and they were either glued to the cage or free to move after successful depowdering. Depowdering efficiency was quantified by microstructural morphometry. The results showed that the use of mobile fillers significantly improved depowdering. Based on this study, large 3DP scaffolds can be realized, which might be a step towards a broader clinical use of 3D printed CaP scaffolds.

Effect of sterilization on structural and material properties of 3-D silk fibroin scaffolds

The development of porous scaffolds for tissue engineering applications requires the careful choice of properties, as these influence cell adhesion, proliferation and differentiation. Sterilization of scaffolds is a prerequisite for in vitro culture as well as for subsequent in vivo implantation. The variety of methods used to provide sterility is as diverse as the possible effects they can have on the structural and material properties of the three-dimensional (3-D) porous structure, especially in polymeric or proteinous scaffold materials. Silk fibroin (SF) has previously been demonstrated to offer exceptional benefits over conventional synthetic and natural biomaterials in generating scaffolds for tissue replacements. This study sought to determine the effect of sterilization methods, such as autoclaving, heat-, ethylene oxide-, ethanol- or antibiotic-antimycotic treatment, on porous 3-D SF scaffolds. In terms of scaffold morphology, topography, crystallinity and short-term cell viability, the different sterilization methods showed only few effects. Nevertheless, mechanical properties were significantly decreased by a factor of two by all methods except for dry autoclaving, which seemed not to affect mechanical properties compared to the native control group. These data suggest that SF scaffolds are in general highly resistant to various sterilization treatments. Nevertheless, care should be taken if initial mechanical properties are of interest.

3D Bioprinting of complex channels-Effects of material, orientation, geometry, and cell embedding.

Creating filled or hollow channels within 3D tissues has become increasingly important in tissue engineering. Channels can serve as vasculature enhancing medium perfusion or as conduits for nerve regeneration. The 3D biofabrication seems to be a promising method to generate these structures within 3D constructs layer-by-layer. In this study, geometry and interface of bioprinted channels were investigated with micro-computed tomography and fluorescent imaging. In filament printing, size and shape of printed channels are influenced by their orientation, which was analyzed by printing horizontally and vertically aligned channels, and by the ink, which was evaluated by comparing channels printed with an alginate-gelatin hydrogel or with an emulsion. The influence of geometry and cell-embedding in the hydrogel on feature size and shape was investigated by printing more complex channels. The generation of hollow channels, induced through leaching of a support phase, was monitored over time. Horizontally aligned channels provided 16× smaller cross-sectional areas than channels in vertical orientation. The smallest feature size of hydrogel filaments was twice as large compared to emulsion filaments. Feature size and shape depended on the geometry but did not alter when living cells were embedded. With that knowledge, channels can be consciously tailored to the particular needs.

Micro-Computed Tomography Based Computational Fluid Dynamics for the Determination of Shear Stresses in Scaffolds Within a Perfusion Bioreactor

Perfusion bioreactors are known to exert shear stresses on cultured cells, leading to cell differentiation and enhanced extracellular matrix deposition on scaffolds. The influence of the scaffold’s porous microstructure is investigated for a polycaprolactone (PCL) scaffold with a regular microarchitecture and a silk fibroin (SF) scaffold with an irregular network of interconnected pores. Their complex 3D geometries are imaged by micro-computed tomography and used in direct pore-level simulations of the entire scaffold–bioreactor system to numerically solve the governing mass and momentum conservation equations for fluid flow through porous media. The velocity field and wall shear stress distribution are determined for both scaffolds. The PCL scaffold exhibited an asymmetric distribution with peak and plateau, while the SF scaffold exhibited a homogenous distribution and conditioned the flow more efficiently than the PCL scaffold. The methodology guides the design and optimization of the scaffold geometry.