Symposium SM10 : Biofabrication-Based Biomaterials and Tissues

Hydrogels are attractive biomaterials for repair and potential regeneration of articular cartilage. Moreover, hydrogels are also particularly suitable as “bioinks” for biofabrication as they recapitulate several features of the natural extracellular matrix and allow cell encapsulation in a highly hydrated mechanically supportive 3D environment. Additionally, they allow for efficient and homogeneous cell seeding, can provide biologically-relevant chemical and physical signals and can be formed in various shapes and biomechanical characteristics. Optimization of – intrinsically weak – hydrogels to address the physico-chemical demands of the biofabrication process, whilst ensuring the right conditions for cell survival is currently regarded as an important research topic. Nevertheless, there exists an additional significant challenge in the biofabrication of articular cartilage tissue constructs: to address the harsh in vivo mechanical environment. We have developed novel hydrogel-based bioink formulations, as well as multimaterial biofabrication approaches that allow for the construction of intricate stable 3D structures, whilst providing the cells with a biologically suitable environment.

Introduction
Hydrogels are widely used as ECM mimetic three dimensional (3D) matrices for cell encapsulation. They can also be designed to comply with 3D printing technologies, opening the possibility to create hierarchical tissue-like structures. The number of available printable hydrogels is however still limited [1,2]. Here we introduce a novel 3D printable hydrogel based on polyglycidols and hyaluronic acid, cross-linked via thiol-ene click chemistry. We present optimization of rheological properties, dispense plotting, cell loading into the gels and in vitro evaluation up to 21 days, and in vivo evaluation for biocompatibility in small and large animal models.

Materials & Methods
Thiol-functionalized hyaluronic acid (HA-SH) and Poly(glycidyl-co-allylglycidylether) (P(G-co-AGE)) were synthesized based on literature [3,4] and characterized by NMR and GPC. For hydrogel formation both polymers were mixed with Irgacure 2959 in PBS buffer and cross-linked by UV irradiation at 365 nm. Gels were prepared with different polymer ratios and characterized regarding swelling, morphology (cryo-SEM) and mechanical properties. For cell loading, human mesenchymal stem cells (hMSCs) were added to the hydrogel solution directly before cross-linking. For 3D printing, the formulation was systematically optimized regarding rheological properties and printing conditions.

Results and Discussion
Thiol-ene cross-linking yielded uniform P(AGE-co-G) / HA-SH hydrogels that allowed for vital encapsulation of hMSCs with high cell survival for at least 21 d. Through optimization of rheological properties, hydrogel formulations could be obtained that exhibit very good printability with dispense plotting and homogeneous scaffolds with defined structures could be prepared. Printing is highly reproducible and pure hyrogel constructs with more than 20 layers could be prepared. The gels (non-printed and printed) underwent biocompatibility screening in vivo in mice, minipigs and in horses, demonstrating good compatibility of the gels in all species.

Conclusion
We present a new hybrid hydrogel system based on thiol-functional hyaluronic acid and allyl-functional poly(glycidol)s, cross-linked through thiol-ene chemistry, allowing for vital encapsulation of hMSCs over at least 21 days, 3D printing via dispense plotting and exhibiting biocompatibility in different animal models. It thus presents a promising new bio-ink that is at the moment evaluated for more complex morphologies.

Acknowledgement
We thank the European Union (FP7) for funding under grant agreement no. 309962 (project HydroZONES).

References
[1] J. Malda et al., Adv. Mater. 2013, 25, 5011-5028.
[2] T. Jüngst et al., Chem. Rev. 2016, DOI: 10.1021/acs.chemrev.5b00303.
[3] X. Z. Shu et al., Biomacromolecules 2002, 3, 1304-1311.
[4] J. Groll et al., Journal of Polymer Science Part A: Polymer Chemistry 2009, 47, 5543.

A key factor in scaffold-based tissue and organ regeneration relies on enhancing (stem) cell-material interactions to obtain the same original functionality. Different approaches include delivery of biological factors and surface topography modifications. Although both strategies have proved to augment cell activity on biomaterials, they are still characterized by limited control in space and time, which hampers the proper regeneration of complex tissues. Here, we present a few examples where the integration of biofabrication technology platforms allowed enhanced spatial organization via the generation of a new library of 3D scaffolds with tailored biological, physical, and chemical cues at the macro, micro, and nano scale.

3D scaffolds with tailored mechanical, physical, and biological properties at different scales have been successfully designed and fabricated by additive manufacturing and electrospinning technologies. By engineering their topological properties, these porous biomaterials influenced the activity of seeded cells, thereby initiating the regeneration of skeletal, vascular, and neural tissues.

Future efforts should aim at further improving our understanding of scaffold topological properties to achieve a fine control on cell fate at multiple scales. Furthermore, current efforts towards the use of supramolecular based systems to provide enhanced temporal control over the fabrication process and cellular growth will be presented. These future efforts will enable the regeneration of complex tissues including vasculature and innervation, which will result in enhanced in vivo integration with surrounding tissues. By doing so, the gap from tissue to organ regeneration will be reduced, bringing regenerative medicine technologies closer to the clinics.

Ca-carbonate is the inorganic scaffold that forms the spicules of the calcareous marine sponges. Recent results disclosed that the Ca-carbonate/biocalcite-based spicular skeleton of these animals is formed via an enzymatic mechanism. The underlying enzyme that mediates this Ca-carbonate deposition has been identified as a carbonic anhydrase (CA), which has been cloned from the calcareous sponge species Sycon raphanus [1]. Ca-carbonate deposits are also found as constituents in vertebrate bones, besides of the main component, Ca-phosphate/hydroxyapatite (HA). Evidence has been presented that during the initial phase of HA synthesis poorly crystalline carbonated apatite is deposited. We succeeded to show that during early bone formation, Ca-carbonate deposits act as (potential) bioseeds for the precipitation of Ca-phosphate mineral during bone-formation by osteoblasts [2-5].
By using the human osteogenic SaOS-2 cells, or mesenchymal stem cells it could be shown that after exposure of those cells to Ca-carbonate (amorphous) in vitro, a significant increase of Ca-deposit formation results onto the bone cells. In parallel, the gene expressions of the carbonic anhydrase-II and IX (CA-II and CA-IX) are upregulated [6,7].
These new data support the view that the Caphosphate/hydroxyapatite deposition reactions in bone tissue are preceded by a Ca-carbonate precipitation, a process that is enzymatically driven by the CA. The proposed hypothesis, the enzymatic synthesis of Ca-carbonate via CA, leaves room for future detailed localization of the deposits formed by poorly crystalline Ca-carbonate or by carbonated apatite in the vicinity of the plasma membrane. Our discovery that Ca-carbonate deposits act as bioseeds in human bone formation may allow the development of novel biomimetic scaffolds for bone tissue engineering. Na-alginate hydrogels, enriched with bicarbonate, polyphosphate and biosilica, have recently been demonstrated as a suitable matrix to embed bone forming cells for rapid prototyping bioprinting/3D cell printing applications [8,9].

References
1. Mueller WEG, Wang XH, Grebenjuk VA, Korzhev M, Wiens M, Schlossmacher U and Schroeder HC. PLoS ONE. 2012, 7(4): e34617.
2. Mueller WEG, Schroeder HC, Schlossmacher U and Wang XH. Biomaterials. 2013, 34: 8671-8680.
3. Wang XH, Schroeder HC and Mueller WEG. Trends Biotechnol. 2014, 32: 441-447.
4. Wang XH, Schroeder HC and Mueller WEG. Int Rev Cell Mol Biol. 2014, 313: 27-77.
5. Mueller WEG, Schlossmacher U, Schroeder HC, Lieberwirth I, Glasser G, Korzhev M, Neufurth M and Wang XH. Acta Biomaterialia. 2014, 10: 450-462.
6. Mueller WEG, Schroeder HC, Feng QL and Wang XH. J Tissue Engin Regener Med. 2013, doi: 10.1002/term.1745.
7. Mueller WEG, Schroeder HC, Tolba E, Neufurth M, Wang XH. FEBS J. 2015, doi: 10.1111/febs.13552.
8. Link T, Wang XH, Feng QL, Schroeder HC and Mueller WEG. RSC Advances. 2013, 3, 11140-11147.
9. Wang XH, Schroeder HC and Mueller WEG. Beilstein J Nanotechnol. 2014, 5: 610-621.

The initial mineralization centers during human bone formation onto osteoblasts are composed of CaCO₃ [1,2,3]. Those bioseeds are enzymatically formed via carbonic anhydrase(s) in close association with the cell surface of the osteoblasts. Subsequently, the bicarbonate/carbonate anions are exchanged non-enzymatically by inorganic phosphate [P_i]. One source for the supply of P_i is polyphosphate [polyP] which is a physiological polymer, formed in the osteoblasts as well as in the platelets.
This physiological inorganic polymer, polyP, is built of multiple phosphate units and linked by high-energy phosphoanhydride bonds, so it can provide an extracellular system for energy transport and delivery required for bone mineral deposition in a dual way: (i) as a metabolic fuel transferring metabolic energy through the extracellular space; and (ii) as a signaling molecule that amplifies energy/ATP production in mitochondria. Several metabolic pathways are triggered by polyP, among them biomineralization/hydroxyapatite formation onto bone cells. The accumulation of polyP in the platelets allows long-distance transport of the polymer in the extracellular space. The discovery of polyP as metabolic fuel and signaling molecule initiated the development of novel techniques for encapsulation of polyP into nanoparticles. They facilitate cellular uptake of the polymer by receptor-mediated endocytosis and allow the development of novel strategies for therapy of metabolic diseases associated with deviations in energy metabolism or mitochondrial dysfunctions [4,5].
After having successfully established the morphogenetic activity of polyP in in vitro and in vivo (rat calvarial defect model) assays [1], we showed that polyP can be used as a biomimetic material to fabricate bone scaffolds/implants for biomedical applications in orthopedics to provide regenerative alternatives of autologous tissues transplantation. The data gathered indicated that polyP is one component of functionalized three-dimensional template/scaffold, together with N,O-carboxymethyl chitosan (N,O-CMC) and alginate. The two polymers, N,O-CMC and polyP, are linked together via Ca²⁺ bridges. This N,O-CMC and polyP material was proven to be printable and durable for layers and tissue units which retain their properties to induce SaOS-2 bone-like cells to biomineralization. In turn, N,O-CMC/polyP represents a promising hybrid material for potential custom-designed scaffold/implant fabrication [4,5].

References
[1] Müller WEG, Tolba E, Schröder HC, Neufurth M, Wang S, Link T, Al-Nawas B and Wang XH (2015) J Mat Chem B 3:1722-1730.
[2] Wang XH, Schröder HC and Müller WEG (2014) Trends Biotechnol 32:441-447.
[3] Müller WEG, Schröder HC, Schlossmacher U, Grebenjuk VA, Ushijima H and Wang XH (2013) Biomaterials 34:8671-8680.
[4] Wang XH, Schröder HC, Müller WEG (2015) Biotechnol J. doi: 10.1002/biot.201500168.
[5] Müller WEG, Schröder HC, Tolba E, Diehl-Seifert B, Wang XH (2015) FEBS J; in press.

Amyloid formation causes many human diseases but E. coli produces functional amyloid fibers which are called as curli. These fibers are secreted via special machinery and play roles in community behaviors. CsgA and CsgB proteins are forming bacterial biofilms and these proteins are responsible for the formation of bacterial nanofibers. CsgA is the major subunit of curli and CsgB is the minor subunit. CsgB is responsible for nucleation while CsgA is responsible for seeding. We have designed a cellular machine that can secrete the CsgA and CsgB with designed functionalities. Controlling the CsgA and CsgB, we are able to control the shape and size of bacterial nanofibers for material growth and biocatalysis applications. We aim to develop a cellular system where we can easily control chemical, mechanical properties of bacterial nanofibers enabled by synthetic gene circuits. Currently we aim to explore the fiber formation characteristics of functional amyloids. In this sense we are probing the fiber formation with purified CsgA and CsgB proteins. Protein nanomaterial shape and size as a function of CsgA and CsgB presence and their concentration were analyzed using molecular characterization tools and SEM, TEM and AFM. Additionally, using the QCM-D we assayed the fiber formation characteristics of CsgA and CsgB proteins through real monitoring of amyloid formation. Curli protein promise a wide range of possibilities to build up protein based nanomaterials for bio-nanotechnology applications to be used in biomedicine and biotechnology. This study is kindly supported by TUBITAK 114M163.

Tissue engineering scaffolding is the external media or structure in which cell growth, migration and reproduction is enabled in order to stimulate tissue regeneration. In order to promote tissue regeneration, scaffolding materials are required to have certain properties such as biocompatibility, adequate mechanical properties and surface topographical features in order to provide specific biological signals to promote cell attachment and proliferation [1].

Bacterial cellulose is the most abundant, inexpensive and readily available carbohydrate polymer in the world and it is traditionally extracted from plants or their wastes [2]. Although the plant itself is the major contributor of cellulose, various types of bacteria are able to produce cellulose and it is termed bacterial cellulose [3]. Bacterial cellulose is a well suited scaffold for tissue regeneration due to its biocompatibility, mechanical properties and its ability to be combined with other structures such calcium phosphates [4], which can create composites with intrinsic properties that meet the requirements of the different tissues of the human body [5].

Through additive manufacturing, highly complex structures can be created which are similar to those found in nature. This work will explore the different ways to produce biomimetic structures for tissue engineering applications through the combination of bacterial cellulose and additive manufacturing producing complex structures of a highly a biocompatible material for a range of different biomedical applications [6]. In addition to the manufacturing and processing techniques, the use of mango (juice/peel) as a complex carbon source for the production of bacterial cellulose was investigated.

1. Helenius, G. et al., 2006. In vivo biocompatibility of bacterial cellulose. Journal of Biomedical Materials Research - Part A, 76(2), pp.431–438.
2. Esa, F., Tasirin, S.M. & Rahman, N.A., 2014. Overview of Bacterial Cellulose Production and Application. Agriculture and Agricultural Science Procedia, 2, pp.113–119.
3. Jozala, A.F. et al., 2014. Bacterial cellulose production by Gluconacetobacter xylinus by employing alternative culture media. Applied Microbiology and Biotechnology, 99(3), pp.1181–1190.
4. Luo, H. et al., 2015. Surface controlled calcium phosphate formation on three-dimensional bacterial cellulose-based nanofibers. Materials science & engineering. C, Materials for biological applications, 49, pp.526–33.
5. Petersen, N. & Gatenholm, P., 2011. Bacterial cellulose-based materials and medical devices: Current state and perspectives. Applied Microbiology and Biotechnology, 91(5), pp.1277–1286.
6. Markstedt, K. et al., 2015. 3D Bioprinting Human Chondrocytes with Nanocellulose-Alginate Bioink for Cartilage Tissue Engineering Applications. Biomacromolecules.

The significant role of biofilms in pathogenicity has spurred research into preventing their formation and promoting their disruption, resulting in overlooked opportunities to develop biofilms as a synthetic biological platform for self-assembling functional materials. Here we present Biofilm-Integrated Nanofiber Display (BIND) as a strategy for the molecular programming of the bacterial extracellular matrix material by genetically appending peptide domains to the amyloid protein CsgA, the dominant proteinaceous component in Escherichia coli biofilms. These engineered CsgA fusion proteins are successfully secreted and extra- cellularly self-assemble into amyloid nanofibre networks that retain the functions of the displayed peptide domains. We show the use of BIND to confer diverse artificial functions to the biofilm matrix, such as nanoparticle biotemplating, substrate adhesion, covalent immobilization of proteins or a combination thereof. BIND is a versatile nanobiotechnological platform for developing robust materials with programmable functions, demonstrating the potential of utilizing biofilms as large-scale designable biomaterials.

3D printing techniques possess great potential to fabricate complex and multiscale structures as in vitro models or for tissue engineering applications. Such strategies may use open-source technologies that are economical and permit diverse printing approaches, such as extrusion-based printing. In this case, the properties of the biomaterial ink are important and involve the transition of a flowing ink into a solid material, such as with shear-thinning hydrogels or with a crosslinking step (e.g., photopolymerization). We are pursuing two approaches for scaffold fabrication.
The first approach involves the printing of a shear-thinning and self-healing hydrogel ink into another support hydrogel in 3D space, where both are formed through guest-host chemistry (e.g., cyclodestrin/adamantane) with modified hyaluronic acid macromers. Shear forces disrupt the hydrogel structure for extrusion and also to receive the extruded material, with resolutions dependent on needle diameter, printing speed, and extrusion rate. This approach allowed for the printing of cells, multiple inks into the support gel, and pockets of materials. Additionally, open and perfusable structures (e.g., channels) could be fabricated with secondary photocrosslinking of the support hydrogel and washing of the ink.
The second approach involves the extrusion of a viscous photopolymerizable precursor that is subsequently exposed to UV light for crosslinking in the presence of a photoinitiator. We synthesized both acrylate-modified poly(glycerol sebacate) (PGS) and norborne-modified PGS that polymerize via radical photoinitiated polymerization or thiol-ene reactions in the presence of a di-thiol and light, respectively. These elastomers have tunable mechanical properties, are biodegradable, and form macroporous printed structures. Both of these printing procedures are useful for the fabrication of biomedical scaffolds for diverse applications.

Introduction: Hair loss is a common disorder that affects men, women, and children due to aging, diseases, and medical treatments. Over the last few years, as a new approach for hair loss regenerative medicine of hair has been increasingly expected. In in vivo development, generation of hair follicle germs is governed by interactions between epithelial and mesenchymal layers. Recent studies revealed that hair follicle germs can be fabricated in vitro by merging two pellets of embryotic epithelial and mesenchymal cells under a microscope. Indeed, fabricated and intracutaneously transplanted germs formed hair follicles which enabled repeated hair cycles. This would be an excellent approach, but one drawback is to require labor steps for the preparation of the germs particularly considering that hundreds of thousands of hair follicles are necessary for a patient. In this study, microfabricated culture chips were employed for one-step preparation of a large number of hair follicle germs with a uniform diameter and spacing.
Methods: Mouse epithelial and mesenchymal cells were prepared from embryonic skins. The isolated epithelial and mesenchymal cells were suspended in a culture medium and seeded in a poly(dimethylsiloxane) microarray chip which had cylindrical wells of 1 mm diameter at a density of 100 wells/cm². To distinguish two cell types, dermal cells were stained by alkaline phosphatase dye. Hair follicle germs formed in the wells were encapsulated in a hydrogel and intracutaneously transplanted to the back skin of nude mice. Immunostaining was performed to characterize hair follicle stem cells at 18 days after transplantation.
Results: What we found in this study is that epithelial and mesenchymal cells formed a single aggregate in each microwell but these cell types were spatially separated each other in the aggregate spontaneously. This indicates that, unlike the previous approach, a large number of hair follicle germs can be prepared by simply mixing two cell types and seeding them on the microfabricated chip. Alkaline phosphatase activity, which is an indicator of the hair follicle inductivity of dermal papilla cells, was expressed in the fabricated germs at 3 days of culture. Further, the germs transplanted intracutaneously formed hair follicles and spatially aligned hairs generated at the transplanted site after 18 days of transplantation. The regenerated hair follicles also showed the hair cycle through the rearrangement of follicular stem cells.
Conclusions: A large number of epithelial and mesenchymal cell aggregates with a uniform diameter and spacing could be prepared by simply seeding a suspension solution of two cell types on a poly(dimethylsiloxane) microarray chip. Fabricated aggregates possessed the ability to regenerate hair follicles in vivo. This technology can be a fundamental technique for hair regenerative medicine.

The development of organ-on-chip models has attracted great attentions in recent years based on its revolutionary significance in pharmaceutical research. Potentially, organ-on-chip opens up an alternative route for drug development besides conventional animal models which are expensive and often fail to predict drug toxicity to human model. One of the major challenges in organ-on-chip development is to incorporate appropriate extracellular matrix (ECM). ECM is widely known as a key component in regulating cell morphology and cell fate. Hence, to mimic the exact physiological system and obtain proper cell functionality, it is crucial to integrate specific ECM into organ-on-chip model. Here we explore the possibility of utilizing the continuous electrojet writing (cEJW) technique to construct the defined biochemical and physical inputs of an ECM. In particular, cEJW allows direct printing of designed biomimetic fibril structures with tuneable fibril resolution ranging between 100nm to hundreds of microns. Two main advantages of cEJW include wide material library (both native proteins and biodegradable polymers can be fabricated) and substrate flexibility (deposition substrates include conductive and insulating material and pre-fabricated structures such as microfluidic devices made of PDMS and hydrogels). More specifically, cEJW can be used to fabricate single-layered bio-interface with controllable thickness and porosity features which can be used to develop tissue-to-tissue interface in vitro. In this project, we hope to demonstrate the ability of using cJEW to create highly specialised microenvironment for specific organ-on-chip systems, such as a kidney-on-chip model.

Reconstruction of functional tissues by tissue engineering and regenerative medicine methods is typically based upon one of three fundamental approaches: cell-based strategies, scaffold-based approaches, and/or bioactive molecule-based approaches. Regardless of the starting point, all three components obviously must eventually combine to form functional tissues and organs. A variety of tools are utilized in each of the above mentioned approaches including: incubators, bioprinters, electrospinning devices, lyophilizers, and tools for terminal sterilization, among others. Functional outcomes are critically dependent upon the methods used.
Numerous studies have shown the effect of biofabrication methods upon structure/function relationships within engineered tissue. This presentation will describe the central role of biofabrication methods in the practice of tissue engineering and regenerative medicine.

Recent years have seen significant progress in two distinct tissue engineering fields: 3D biomaterial printing and processing and utilization of decellularized extra cellular matrix (dECM). We present a new, efficient, and highly versatile method for creating a broad range of tissue-specific simple and complex constructs through integration of novel material casting and 3D-printing technologies recently developed by the authors. 2D-castable and 3D-printable, liquid-based inks (inks) are synthesized through combination of several organic solvents, biocompatible elastomer (30 vol.%), and particulate material (70 vol.%). In this instance, the particulate material is tissue specific dECM, created by decellularizing porcine and bovine tissues and organs followed by lyophilization and mechanical milling. Once incorporated within the ink formulation via simple mixing, the ink may be immediately cast into a container. After several minutes of drying, “tissue paper” results. Similar to standard paper, the dECM can be cut, folded, crumpled, rolled, and even sutured. Although the fabrication process and, to a lesser extent, resulting mechanical properties are independent of the tissue or organ being utilized, structural evaluation via scanning electron microscopy (SEM) reveal that papers derived from distinct tissues are themselves distinct. To create thicker or more complex tissue constructs comprised of the same or several distinct dECM types, the paper can be stacked. Small solvent volumes, similar to those used in creating the initial ink, can be applied to individual layers and subsequently fused together. Through this process, thick, multi-layered single or multi-tissue-type constructs can be created by fusing together dECM paper of the same or differing type, respectively. These tissue papers can be integrated with 3D-printed biomaterials derived from the exact same particle-based ink system including the highly osteogenic Hyperelastic Bone (HB) and highly neurogenic 3D-Graphene (3DG) to create three-dimensional, porous multi-functional constructs. Due to the similarity in composition, these extruded 3D-inks instantly fuse with tissue papers, acting as printing substrates, upon contact. Utilizing this process, we illustrate that complex, 3D-printed constructs can be directly printed onto the tissue paper, ultimately creating biomaterial structures mimetic of multi-tissue structures. Additionally, we present methods for fabricating functional tissue papers from porcine lung, liver, heart, kidney, dermal, muscle, and adipose tissues, present their mechanical and microstructural properties and in vitro biological properties.

3D-printing (3DP) technologies have emerged as exceptionally promising fabrication tools with applications across medical fields, including tissue engineering and regenerative medicine. However, the current palette of 3D-printable biomaterials does not come close to collectively emulating the biological, chemical, and physical properties of the incredibly diverse and expansive spectrum of tissues and organs that comprise the human body. In order for 3D-printing to be a technically, commercially, and clinically practical approach to tissue and organ engineering, new approaches for 3D biomaterial ink design need to be developed and implemented. In this talk, new strategies for expanding the biomaterial toolbox for 3D printing developed in the Shah Tissue Engineering and Additive Manufacturing (TEAM) lab will be presented. The first is a single bioink method for synthesis of hydrogel inks for cell printing. Utilizing PEG cross-linkers (PEGX), soft and extrudable yet self-supporting hydrogels are produced for direct extrusion that maintain well-defined as-printed architectures. Hydrogel polymer concentration, hydrogel polymer type (i.e. gelatin, fibrinogen, PEG), degree of cross-linking, cross-linker properties (linear, multi-arm, various molecular weights) and cross-linking chemistry were manipulated to generate over 100 printable formulations. This method allows tuning of hydrogel bioink properties without compromising printability. Furthermore, it is independent of a specific cross-linking chemistry and can be used to develop printable materials of various functionalities for expanding the number of 3D printable materials. The second comprehensive material approach is a new class of particle-laden 3D-printable liquid inks comprised of the particles of interest (60-80 vol.% solids content), an elastomeric, biocompatible and biodegradable polymer (20-40 vol.% solids content), and a series of organic solvents. These inks can be prepared under ambient conditions and rapidly 3D-printed via syringe extrusion at linear deposition rates upwards of 15 cm/s into highly bioactive and ready to use structures comprised of as many as hundreds or thousands of layers, with no drying time required prior to handling. Two promising new 3D-printable particle-based biomaterials will be highlighted: Osteogenic Hyperelastic “Bone”, so named because of its highly elastic mechanical properties despite being 90wt% bioceramic, and the neurogenic 3D-Printed Graphene. The mechanisms governing the ability to directly 3D-print these new ink systems into self-supporting constructs will be presented, as well as the microstructural, physical, and biological properties of the resulting 3D-printed constructs. We hope that these new versatile biomaterial ink technologies will enable biofabrication of compositionally and structurally complex structures and functional tissues for drug testing and studying tissue morphogenesis, as well as for transplantable tissue and organ substitutes.

The 1980s both gave start to both Tissue Engineering through the groundbreaking paper by Robert Langer and Joseph P. Vacanti, as well as development of the first 3D printing technologies, namely stereolithography developed by Charles W. Hull and fused deposition modeling developed by S. Scott Crump. Initial work in Tissue Engineering was accomplished by modifying existing technology for the printing of biomaterials into 3 dimensional scaffolds, or even of cell suspensions by adapting ink jet technology as published by Thomas Boland in 2003. Observing a widespread use of self-made printers in Tissue Engineering, Rüdiger Landers and Rolf Mülhaupt published in 2000 the development of an extrusion based 3D printed for the unique purpose of printing biomaterials and cell suspensions into 3D scaffolds. Based on their work, Envisiontec GmbH announced the first commercial 3D bioprinter in 2001. This presentation will discuss the evolution of the 3D-Bioplotter in the 15 years since its inception, as well as how it helped further the research of Tissue Engineering as the most widespread commercial 3D bioprinter.

Textile technologies have opened a new area in tissue engineering. Precise control over the distribution of different cell types and microstructure of fabricated constructs are considered as key advantages of textile techniques. Insufficient mechanical properties of cell-carrying fibers made of hydrogels have limited their usage in textile processes. Thus, the concept of composite fibers (CFs) that can withstand textile processing has been recently introduced to address this challenge. Here, we discuss the processes for addition of functionality to CFs and their utilization in various biomedical applications including muscle and tendon tissue engineering and smart wound dressing platforms. The engineered CFs could support cellular attachment and provide a suitable environment for cell proliferation and can accurately control cellular alignment. Similarly, by incorporation of stimuli-responsive hydrogels, CFs can be used for engineering smart platforms for on demand release of drugs and growth factors. CFs containing fibroblasts, C2C12 myoblasts were created and the thickness of the gel layer could be changed from 20µm to 600µm by adjusting the drawing speed and the prepolymer concentrations. The encapsulated cells showed high cellular viability. In addition, the metabolic activity of the encapsulated cells was assessed and showed a continuous increase over 7 days of culture. Immunostaining results confirmed normal cellular morphology and alignment of encapsulated cells along fiber’s axis and formation of highly organized myotubes. Similarly, NIPAM-based drug microcarriers were embedded within the hydrogel layer coated on electrically conductive threads serving as heaters for controlling the release profile of drugs. Antibiotics were loaded into the drug microcarriers and their effectiveness for eradication of bacterial infection was demonstrated.

Tremendous strides have been achieved over the past several decades in the field of tissue engineering to construct implantable thin tissue constructs such as skin, cornea, and bladder. However, obtaining functional, physiologically relevant tissues is still a major challenge due to the necessity of a vascular system to supply nutrients and remove waste in thick constructs. Additionally, tissue engineers have historically focused on fabricating tissue constructs containing a single vascular network. However, a key feature of complex biological systems is the presence of interpenetrating networks, such as the respiratory tree with its pervading blood vessel network, which are central to mammalian physiology yet has remained difficult to fabricate in vitro. To this end, we sought to develop a stereolithography (SLA)-based 3D printing system for fabrication of tissue constructs on the order of several centimeters containing complex interpenetrating networks. Photorheological characterization of prepolymer formulations containing poly(ethylene glycol) diacrylate (PEGDA) and a photoinitiator lithium acylphosphinate (LAP) was performed to understand the gelation kinetics and modulus of a single layer of hydrogel. Next, architectures akin to physiologic vascular networks were used to explore parametric designs which contain multiple interpenetrating networks. Print fidelity was investigated by comparing designed models to volumetric renderings of micro-computed tomography (µCT) scans of printed gels. Bioluminescent and fluorescent images of printed hydrogels containing encapsulated cells demonstrated cell survival that was dependent on the printed network architecture. We expect the performed studies will provide insight to the field by demonstrating architectural features required to build living tissues the size of human organs.

Tissue engineering and regenerative medicine seek to augment the healing process through th fabrication of autologous tissue grafts eliminating the immune response in the host. It has been suggested that we will be able to build complete organs from scratch, and the term “organ printing” has been coined and used by the popular press. Although such a goal is unlikely to be attainable in the near future, the fabrication technologies used may be able to build model organ-on-a-chip structures in the nearer term.

Tissue engineering regards the scaffold, a three-dimensional (3D), highly porous substrate, as a key enabling component. Cells donated by the patient are expanded in culture and then transferred to the scaffold. The scaffold provides a surface on which cells adhere, thrive, multiply, and generate the extracellular matrix (ECM) that make up living tissue. Within a tissue scaffold, all cells must be supplied with the means to maintain life, and this is achieved initially by providing a highly porous open structure to allow the uninterrupted flow and access of culture media in a bioreactor. Traditionally, porous scaffolds have been made by a number of routes with a random architecture and a limited control of scale. However, the development of additive manufacturing techniques has enabled fabrication of fine-scale porous structures allowing a true engineering of the scaffold. There are a number of materials challenges that must be overcome to allow us to use the full capabilities of these advanced manufacturing tools in a biofabrication context.

Inkjet printing is a highly versatile method for the precise positioning and patterning of cells and biomaterials. It is a non-contact method for patterning surfaces with very small droplets of ink (typically picolitres). In addition, because it is a liquid dispensing method, it is possible to dispense biological materials in an aqueous environment at physiological temperature. It is now well established that a range of cell types, including human embryonic stem cells, can be printed successfully using this method. Thus it is possible to build 3D structures containing living cells as a precursor to true tissue analogue structures.

growing topic of interest due to their applications for drug-screening, and physiological study. However, the complexity of living organs, including complicated cell-cell and cell-extracellular matrix interconnection, hampers the advance of in vitro cell culture models. Especially, the basement membrane, which is extremely thin and nanofibrous structure, still remains as an obstacle in developing in vitro cell culture models mimicking living organs. Electrospinning, which is simple and versatile technique to fabricate nanofibers, has been intensively studied to fabricate nanofibrous structure. A metal collector has been commonly utilized to accumulate nanofibers during electrospinning, but however, the collected nanofibers were adhered to the metal surface, which required additional steps including chemical or mechanical peel-off and transfer to be free-standing and to be integrated with other devices. To overcome the problem, we introduced electrolyte solution in electrospinning process instead of the metal collector. In this research, we fabricated a random or aligned, free-standing nanofiber membrane integrated with a transwell by electrolyte-assisted electrospinning. The electrolyte solution was utilized as a collector for nanofibers during electrospinning instead of the metal collector. By removing electrolyte after electrospinning process, we could achieve free-standing, random or aligned nanofiber membrane integrated with a transwell. On the nanofiber membrane, brain endothelial cells were cultured, and tight junctions of the cells were evaluated.

In the field of tissue engineering and regenerative medicine, it is still a big challenge to produce complex tissues by engineering approach, despite the tremendous efforts by many researchers. Biofabrication were started to overcome such challenging issues.
Recently, the definition of Biofabrication for tissue engineering and regenerative medicine was officially defined by the International Society for Biofabrication (ISBF) as “the automated generation of biologically functional products with structural organization from living cells, bioactive molecules, biomaterials, cell aggregates such as micro-tissues, or hybrid cell-material constructs, through Bioprinting or Bio-assembly and subsequent tissue maturation processes.” [1]
To date, we have cultured cells on the micro-patterned culture discs or in the micro-fabricated molds and found that the micro-patterned cultured cells or micro-fabricated cells did not only proliferate or aggregate but also generate tissue-like structures, such as capillary-like tubular structures and the bundle-like tissues with linearly aligned muscular cells. Based on those findings, we tried to assemble such pre-cultured pre-tissue-forming cell constructs by transfer printing and by assembling procedure. 3D tissues with aligned fiber-like muscular tissues were successfully fabricated through those assembling procedures. Our results suggest that the bioprinting and bio-assembly with pre-cultured tissue components can provide a promising biofabrication method for hierarchical tissues.

Ref:[1] Groll J, Boland T, et.al., Biofabrication: reappraising the definition of an evolving field. Biofabrication. 2016 8(1):013001. doi: 10.1088/1758-5090/8/1/013001.

Bioprinting of cell-laden constructs within the body yields great promise for in-situ tissue regeneration applications such as arthroscopic cartilage repair procedures. Ideally, in-situ bioprinting requires a small printing device that could be applied for the deposition of cell-laden hydrogels at the defect site. However, several limitations still hamper the current generation of these devices, for example clogging of the required nozzles, the size of the device, or the requirement of compressed air for operation.
To resolve these issues, we present a small-scale (~1cm3), hand-held hydrogel printer that can print hydrogel constructs without a need for compressed air. We demonstrate rapid (~2ml/min) printing of hydrogel constructs with a size of order 1 cm³, which we print onto walls with arbitrary directions with respect to gravity. This size easily exceeds the capillary length scale of ~3mm (with a corresponding volume of 27 mm³), which is the physical size limit for 3D printing of non-solidifying (or slowly solidifying) materials onto inclined or overhanging walls. We adjust the liquid flow rates of the hydrogel and crosslinker to optimize the resolution, deposition rate, and the mechanical properties of the solid hydrogel. Furthermore, we demonstrate printing of cell-laden constructs with a cell viability exceeding 80% and various biocompatible hydrogel types. The proposed omnidirectional bioprinting device is cheap and highly versatile, and is therefore expected to enable rapid translation into the clinic.

Fundamental understanding of interactions between different cells and their environment is essential for cell-based therapies in regenerative medicine. Common ex vivo cell studies in two-dimensional cell cultures have significant limitations and are not appropriate to simulate complex interactions in three-dimensional (3D) tissues and cell–microenvironments in vivo, since cell behavior differs dramatically in 3D. To bridge the gap between common cell culture conditions in vitro and animal models, 3D cell systems are necessary.
In this presentation, we discuss laser-based techniques applied for precise generation of 3D scaffolds, with sub-micron resolution, and for printing biological cells into 3D patterns.
For the scaffold generation, two-photon polymerization (2PP) technique is applied, which allows writing CAD structures directly into the volume of photosensitive polymer solutions. The polymerization occurs in the laser focus only. Thereby, resolutions below the diffraction limit down to the sub-100-nanometer range have been achieved. Scaffolds from different biomaterials like organic-Inorganic Sol-Gel-Composites (e.g., zirconium-hybrids), biodegradable polymers (e.g., polylactic acid (PLA), polycaprolactone (PCL), polyethylene glycol (PEG)), and hydrogels (e.g., gelatin, hyaluronic acid, chitosan, alginate, gellan gum) or hydrogel blends, have been generated with this technique. The effect of the micro-structure on cell behavior will be discussed.
For arranging cells in 3D patterns, laser-assisted bioprinting (LAB) based on the laser-induced forward transfer process is used. Different cell types, including primary cells and stem cells embedded in hydrogels as extra-cellular matrix, have been printed. Thereby, 3D stem cell grafts, skin tissue, and cell patterns for studying cell-cell interactions have been generated.
Both 2PP and LAB techniques are capable of advancing 3D cell culture towards CAD defined and precisely arranged 3D cell models and “organ-on-chip” systems. Such innovative 3D cell models could provide new insights in understanding of cell behavior, tissue functions and their regeneration. Printed tissue, for example skin, can be used for analyzing the effect of agents like pharmaceuticals or cosmetics ex vivo and, by applying human primary cells, it might be applied instead of animal tests.

Aim:
Fibre based strategy is attractive for creating 3D complex hierarchical human tissues in vitro because they mimic fibrous human tissues such as tendon, ligament and muscle. These fibres need to offer an extra cellular matrix-like environment for the interaction with cells and also to be mechanically strong so it is robust during handling and can bear in vivo loading after implantation. Here we report a study on 3D printing of fibres which are mechanically robust as well as have chemistry suitable for cell survival and function.

Method:
In this study, a modified commercial 3D printer was used to print the fibres which are mechanical strong and encapsulate cells. Fibres were made from a composite material that exhibited superior biological and mechanical properties compared to its individual components. These fibres were characterised by mechanical testing; cell viability and function within these fibres were also characterised. These fibres were printed into 3D structures.

Results:
The composite material showed a 3 fold increase in ultimate tensile strength compared to its individual components. The failure strain was also significantly increased. The cells that are encapsulated in the material showed good viability and proliferation, which suggested that the material allowed sufficient nutrient and oxygen transport into the encapsulated cells. The material could be extruded during 3D printing to form different 3D structures such as tubes and meshes.

Conclusion:
Fibres with good biological and mechanical properties have been made and laid into different structures using 3D printing. These fibres can potentially be used in load bearing applications.

Chlorella is a green, photosynthetic single-celled genus of algae. It can be 3D printed as a suspension in sodium alginate and gelled with calcium solutions. We have made “log pile” structures with channels between the gel lines to allow easy transport of nutrients and products. Under white light and immersed in solutions of bicarbonate and phosphate and urea “plant food” the algae multiply with the gel and produce oxygen at a rate comparable to that reported for suspensions of Chlorella. The system is stable for one or two weeks at least. In principle this can be extended to other plant tissues but there are concerns relating to bacterial and fungal infection and toxicity of the gel components. In addition a tougher gel is needed if this was to be converted to a practical bioreactor system.

Liver tissue engineering aims to provide transplantable tissues to replace or regenerate liver tissue as a supplement to the nearly 16,000 patients currently awaiting transplants. Hepatocytes are notoriously difficult to culture in vitro and require novel 3D scaffolds that can optimally support growth and function. Hepatocyte aggregation is known to have a positive influence on viability, however the influence scaffold pore size and shape has on aggregation and gene expression has yet to be fully investigated. Threee dimensional (3D) printing has been implemented in tissue engineering, and is particularly amenable to tissues of a repetitive nature such as the liver. 3D printing allows for precise control over scaffold size, shape, porosity, and uniformity in a manner unrivaled in scaffold fabrication. Here we utilize 3D printed gelatin to demonstrate the effect varying scaffold pore size, shape, and tortuosity has on hepatocyte proliferation, viability, and gene expression. Porcine gelatin type A (10% w/v, PBS) was 3D printed using a 3D BioPlotter (EnvisionTEC GMBH, Germany) by heating the solution and extruding through a 200 μm diameter nozzle. Scaffold struts were spaced 650 μm apart, into 6 layer scaffolds with strut orientations of either 0° and 90° (0-90), or 0°, 60°, and 120° (60° advancing angle, 60AA). Scaffolds were then cross-linked with EDC/NHS and 4 mm diameter scaffold biopsy punches were seeded with HUH7 human hepatocytes and cultured in DMEM with 10% FBS. Six well culture plates were coated in gelatin and cultured as a control. Viability was imaged using live/dead (calcein/ethidium) and confocal microscopy. Proliferation was quantified using PicoGreen. Gene expression was assayed using qRT PCR. Relative expression levels were normalized to human cyclophilin 1. The torturous pore geometry provided by 60AA scaffold architectures led to enhanced seeding efficiency, evident by cell numbers at day 1 and imaging. Cell numbers were not significantly different between 60AA and 0-90 scaffolds on subsequent time points. However, viability imaging indicates significant differences in cell aggregation. By day 14, dense cell aggregates prevented useful image acquisition. Gene expression data indicates little significant change from 2D culture at days 1 and 3, aside from albumin expression. By day 7, drastic changes in gene expression are observed, chiefly between the scaffolds of differing geometry. The primary differences are in expression levels of CYP3A4, CYP3A7, CYP2C9, NDUFA2, and Albumin. Proliferation on the scaffold surface is hypothesized to dampen gene detection of those hepatocytes subject to the effects of differing pore geometry. Despite no significant difference in cell number, gene expression levels of hepatocytes vary substantially depending or scaffold pore shape. These studies provide useful insight into optimal 3D printing schemes that may incorporate primary hepatocytes and co-cultures of non-parenchymal cells.

Micro- and nanoscale technologies are emerging as powerful techniques for the development of highly organized and functional three-dimensional (3D) complex constructs. In addition, hydrogel biomaterials have been increasingly used in various tissue engineering applications since they provide cells with a hydrated 3D microenvironment that mimics the native extracellular matrix. In our lab we have developed various approaches to merge microfabrication techniques such as 3D bioprinting, photolithography, and microfuidic with hydrogel biomaterials for directing cell organization and generating complex 3D tissue. In this talk, I will introduce a number of bioprinting advances and highlight their potential for tissue engineering and biosensing applications.

During the last decades direct-write fabrication on the micro- and nano-scale has attracted enormous attention in science and technology due to simpler, faster and more flexible process capabilities. Within this class, focused electron beam induced processing (FEBIP) is getting increasingly important as it combines real nanoscale resolution with high flexibility concerning the pattern geometries which is indispensable for rapid prototyping applications. Although FEBIP is mostly used as bottom-up (deposition) and top-down (etching) method, it also allows highly localized, chemical conversion of functional precursor into the desired material with theoretical nanoscale resolution. In this contribution we combine this focused electron beam induced conversion (FEBIC) approach with biological materials by transferring a cellulose precursor into cellulose. For that thin tri-methyl-silyl-cellulose (TMSC) spin cast films are exposed to a nanosized electron beam which induce a local dissociation into cellulose and volatile products which can leave the solid for sufficiently thin precursor films. Depending on the total exposure dose, three regimes can be identified: 1) incomplete dissociation; 2) ideal conversion; and 3) decomposition which irreversibly damage the material. If processed in the ideal range, complex cellulose patterns can be written into initial TMSC film followed by a final wet chemical step to remove unexposed TMSC areas. By that, cellulose structures down to 100 nm can be fabricated which are barely possible via other techniques. The maintained cellulose functionality is demonstrated by classical enzyme degradation studies which confirm the suitability of FEBIC for the fabrication of highly complex, on-demand cellulose structures in science and technology.

3D bio-printing shows great promise as a technique to deliver future generations of customised products with great clinical utility. Manufacturing of these products will be challenging. Some of these challenges are a consequence of the regulatory environment for these new products. This presentation will examine the overall manufacturing process for these products to identify where the important regulatory challenges are and where new science is required to address them on behalf of the community.

Bioprinting tissues and organs has been big news recently. Scientists have already printed blood vessels, organ tissues, and even an ear. Some people question whether the scientists who manufacture vessels and organs – objects that already exist in nature – should be able to obtain patents on these new technologies. But the patenting of bioprinting techniques has quietly been going on for years. This presentation will discuss patents covering the three process phases of bioprinting, the exceptions to patent infringement for experimental uses, and the prospects for further patenting and patent infringement lawsuits.

With growing demands for tailored cellular microenvironments in bioengineering, there is tremendous potential in combining nanotechnology and new biomaterial fabrication techniques to construct the defined biochemical and physical inputs of an extracellular matrix (ECM). Major challenges in fabricating biointerface geometries in a nano-scale are associated with the precise deposition of one dimensional (1D) nano-structures and the capacity to construct ordered three dimensional (3D) configurations. Here we introduce a new direct writing technique, low voltage continuous electrospinning writing (LV-cEW), to directly print out 1D nano fibres into 2D and 3D scaffolds with designed features. Using very low applied voltage (as low as 50 V), LV-cEW offers several advantages over traditional electrospinning protocols in the controllability of printing. In particular, LV-cEW can define the attachment of single fibre on the substrate, or fabricate nano-fibres as bridges across a 2 cm long channel. The spatial arrangement of fibres can also be tuned easily. This technique can be applied to a wide range of materials, including proteins (gelatin and collagen type-I) and biodegradable polymers. There is also no specific requirement for processing solution selections. Beyond the ability to precisely print composites in 2D and 3D with a wide range of material selections, LV-cEW also extends the application to print encapsulated bio-element with minimized external influence (such as by avoiding a strong electric field and shearing force in the polymeric jet). The LV-cEW technique reported here illuminates new avenues for flexible rapid prototyping of biointerfaces and well-defined ECM-like 3D scaffolds. It also opens up the potential of direct printing of electric-sensitive elements.

A physical microenvironment of cells, such as topography and stiffness of extracellular matrix (ECM) is one of important factors to regulate cell fate and functions. Many previous works have focused on the topographical effects on the cell behaviors and tried to control cell functions including orientation, migration, proliferation, and differentiation. Here, we suggest two different types of nanoengineered cell culture platforms, which are for mimicking the geometrical topography of the ECM.
Nanoengineered polystyrene (PS) cell culture platforms, named nano Petri dishes, were introduced. The PS was selected as the substrate material of the culture platforms because it is a well-known thermoplastic material that is commonly used for plastic labware in the cell culture history. The nanoengineered cell culture platforms with relatively large area were realized through the mass-production technologies based on precision nano injection molding and thermal nano imprinting with a use of metal mold inserts. The nickel mold inserts with nanopore/pillar arrays were manufactured through a two-step electrochemical oxidation process followed by a nickel electroforming process. On the nano Petri dishes, we have extensively investigated promoted cell functions and fate of migration, elongation, proliferation, and differentiation of many different types of cells including adult stem cells and embryonic stem cells.
And recently, a free-standing, spatially controllable nanofiber membranes fabricated by electrolyte-assisted electrospinning (ELES) were introduced to produce a nanotopography. Conventional electrospinning process required a metal electrode to collect as-spun nanofibers, and generally produced a nanofiber membrane strongly adhered to the metal surface, which requires complicate post-processing to realize a free-standing membrane. In this work, we developed ELES to facilitate the fabrication of free-standing, spatially controllable nanofiber membranes on from a two-dimensional flat surface to three-dimensional curved geometry by utilizing the fluidic nature of an electrolyte solution. On free-standing, aligned nanofiber membrane integrated with a microfluidics device, we have developed in vitro blood-vessel model with aligned endothelial cells.

Advances in regenerative medicine have been recently dominated by the increasingly popular implementation of 3D printing in the field of tissue engineering. The utilization of 3D printing allows for a repaired model of damaged tissue to be constructed from a patient's CT scan or MRI and printed within 24 hours. Thus, the patient can receive an individualized, biocompatible tissue that is of the correct size and geometry which can support incorporation of the patient's own cells to better promote healing and acceptance in vivo. The challenge with 3D printing of biomaterials remains the correct selection of a material with optimal biocompatibility and "printability," or rheological properties that allow it to be extruded through a thin nozzle and maintain a stable printed 3D structure once deposited. Synthetic polymers have been frequently used in 3D printing due to their exceptional printability but often possess limited biocompatibility and biodegradability. Thus, more biocompatible alternatives, such as natural biopolymers, that also exhibit excellent printability have gained recent interest as 3D printing materials.

In this study, a novel alginate-hydroxyapatite bioink with optimal rheological properties for 3D bioprinting of MC3T3 cells has been developed. The combination of alginate, a natural biopolymer derived from algae, and hydroxyapatite, the main mineral component of natural bone, results in a bioink with both printability and biocompatibility. The optimal concentrations of alginate, phosphate and calcium have been determined to result in a polymer composition with ideal gelation time and viscosity which allows the hydrogel to be extruded and recover as it's deposited. The bioink was printed into 1.5 cm diameter cylinders using a HyRel System 30 3D printer. Crosslinking of alginate prints was completed in a 100mM CaCl₂ bath for 1 hour to allow high cell viability throughout the process and prevent cell death due to calcium exposure. Degradation studies in α-MEM cell culture media showed that the 3D printed alginate-apatite scaffolds remained in-tact with low mechanical properties for 14 days. These results proved the potential of the scaffolds to support cell life for this time period, which has been reported as sufficient time in which the first signs of bone deposition occurs. The incorporation of MC3T3 cells in the bioink maintained good viability through the completion of the printing process and crosslinking in the calcium bath, which was measured via MTT assay. Thus, 3D printing of this alginate-hydroxyapatite bioink has great potential for bone regeneration in vivo of non-load bearing applications.

Among biodegradable polymers, thermoplastic polybutylene succinate (PBS) has desirable melt processability and splendid mechanical properties, closely comparable to those of widely-used polyethylene (PE) and polypropylene (PP). Additionally, PBS, whose ester bonds can be chemically degraded by water, has a remarkable reprocessability. Because of these excellent properties, it could be found as packaging film, tableware, flushable hygiene products and biomedical materials.
Scaffold is a temporary structure used to support the cell growth. Tissue engineering scaffolds should ideally provide suitable three – dimensional(3D) microenvironment with desirable mechanical support for cell proliferation and extracellular matrix deposition. Of course the scaffold must be biocompatible and biodegradable. Scaffold can be fabricated by various techniques such as electrospinning, freeze drying, gas foaming, salt/sugar particulate leaching and thermally induced phase separation. There are some advantages as well as disadvantages associated with each technique. Among the available techniques, electrospinning method is one of the simplest and economic technique available for scaffold fabrication.
Electrospinning is a simple and effective method used to fabricate ultra-fine fibers with nanometer scale range diameters. The ultra-fine fibers are collected on the metal collector in a form of flat nonwoven mats. Due to its ultra-fine diameter, the nanofibrous mat has high surface-to-volume ratios and can serve as adequate carriers for active ingredients. There have been a lot of studies on the application of active ingredients containing electrospun nanofibers in scaffold.
In this study, we suggest a novel electrospinning method to make a 3-D nanofiber scaffold with a large pore size as well as high porosity using PBS, known as an excellent biodegradable polymer. We dissolved PBS with chloroform or dichloromethane. PBS solution was electrospun using typical electrospinning process except collecting part of the process. The PBS nanofibers were dropped directly into coagulation bath containing water.
We successfully fabricated biodegradable PBS 3D scaffold. Electrospun nanofibrous PBS s caffold can provide similar architecture to the ECM leading to enhancement of cell adhesion, proliferation, migration. The morphological appearance of the electrospun PBS scaffold was observed with SEM. FTIR were useful to find out the secondary structure of PBS scaffold. MTT assay will be conduct to cytotoxicity of PBS scaffold.

Tissue engineering has potential to address this need through the combination of biomaterials, growth factors, and cells. Highly porous scaffolds are generally used as the substrate for anchorage dependent cells and to facilitate nutrient and metabolite distribution to guide cell growth leading to new bone tissue formation. For bone tissue engineering, biodegradable synthetic polymers such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and copolymers of poly(DL-lactic-glycolic acid) (PLGA), and biodegradable naturally derived polymers such collagen and fibrin.
Regenerated Bombyx mori silk fibroin (SF) has excellent biological and mechanical properties, including biocompatibility, programmable biodegradability, and remarkable strength and toughness. Diverse and adaptable properties of Sf are possible by varying the structural form using different processing conditions. One of the important physical forms for biomaterials is the formation of hydrogels, which has been extensively studied for a variety of polymers. The sol-gel transition depended on the concentration of the protein, temperature, and pH. In the SF hydrogel, random coil to β-sheet (physical cross-linking) structural transitions were noted during the process of hydrogelation. Due to the β-sheet formation, SF exhibits relatively slow degradation in vitro and in vivo, compared to collagen and many other biopolymers.
Hydroxyapatite (HAP) has been investigated for bone replacement since this material mimics natural bone mineral features. HAP has been studied extensively in cell culture and possesses osteoconductivity.
In this study, the synthesis and characterization of bone-like mineral HAP into highly porous biodegradable silk fibroin scaffold with via chemical cross-linking reaction of SF by gamma-ray (g-ray) were investigated. These 3-D SF scaffolds had different secondary structures, elasticity and nanostructure compared with β-sheet induced hydrogels. The effect of degradation rate, elasticity and mineralization on osteogenic responses of osteoblast was assessed with respect to bone tissue engineering.

In order to study specific adhesion of nanoparticles modified by monoclonal IgG M75 antibody to HT-29 cancer cells, expressing carbonic anhydrase IX antigen, we developed a 3D cell culture model with characterized fluid flow conditions. Through the combination of engineering, biology, and advanced analytics, this model represents a link between simplistic 2D in vitro assays and complex in vivo animal models. The model was printed by a 3D printer and consists of a flow cell with a slide-in rack where 3D scaffolds in the form of lattices overgrown by cells can be put. Two cell lines of colorectal carcinoma were used for cultivation on the 3D scaffolds: HT-29 cells and DLD1 cells used as a negative control. Laminarity of the flow within the model was verified by MRI (Magnetic Resonance Imaging), and flow velocities were calculated using CFD (Computational Fluid Dynamics). Nanoparticles used for adhesion experiments are fluorescently labeled and contain magnetite; therefore, evaluation of adhesion efficiency can be observed qualitatively by MRI and confocal microscopy and also quantitatively by fluorescent spectrometry and flow cytometry. Adhesion experiments in stationery medium and preliminary experiments in the perfusion model showed specific adhesion of IgG M75 nanoparticles to HT-29 cells. Further experiments will be performed using the 3D cell culture model under flow velocities similar to those of blood and other body fluids.

In this study, we checked about the mechanical properties of the composite scaffold possessing negative Poisson's ratio(NPR). The composite scaffolds were prepared with poly(lactide-co-glycolide)(PLGA) and hydroxyapatite(HA). For materializing negative Poisson’s ratio, we fabricated scaffold with salt-solvent casting method first, and then, applying 3-directional permanent volumetric compression under appropriate temperature. NaCl particle was used for making porous scaffold and its size was 500~600um range. The ratio of volumetric compression was 2.37:1. Control group were made only PLGA and experimental group were prepared as a composite form. The matrix was PLGA and HA(5, 10 and 15wt%) particles were incorporated.
The measurement of the Poisson’s ratio was analyzed by recording the specified displacement using a digital camera at the point under the compressive strain at 0, 5, 10, 20 and 25 % by using MTS(material testing system). As a result, the lowest NPR was shown at the 10wt% HA/PLGA scaffold at 10% strain.
The compression test of the scaffold was measured at 5 and 10% strain using MTS. Mechanical properties were increased with increment of HA contents. Contact angle measurement was done by using contact angle analyzer according to HA contents (0, 5, 10 and 15 wt%). The 10wt% HA/PLGA scaffold was shown appropriate hydrophilicity.
Mechanical properties of the scaffold in the wet state of the scaffold were investigated in order to analyze the variation of the physical properties of the scaffold in cell culture conditions. Recovery rate after applying compression was 90% on dry-state and 60% on wet-state in 10wt% HA/PLGA scaffold group. It was shown that 10wt% HA/PLGA scaffold has higher recovery rate and mechanical property than only PLGA scaffold.
Cell proliferation about MG-63 on scaffold was checked by CCK-8. It was shown that 10wt% HA/PLGA scaffolds have about 20% higher proliferation rate than that of PLGA scaffolds.
NPR composite scaffolds of this study will be useful to bone tissue regeneration by applying an effective isotropic compression to stimulate proliferation of bone cells.

At $3.07 billion in 2013, the 3D printing industry was projected to reach $12.8 billion in 2018 and exceed $21 billion by 2020 (Wohlers and Caffrey, 2013). A lucrative part of this expanding industry includes printing biocompatible medical implants, devices and tissue scaffolds. A common problem encountered with traditional devices and implants, is that they are not unique to the patient, making the surgeries more difficult and less effective. Tissue scaffolds could also benefit from increased strength and biocompatibility. To answer these demands, customizable devices are being produced from patient medical scans and CAD designs using 3D printers. Traditionally, plastics such as polylactic acid (PLA) or poly-(lactic co-glycolic) acid (PLGA) are used in 3D printers because of their thermoplastic properties, which make them easy to print. These plastics are typically regarded as biocompatible but can degrade to less biocompatible forms in the body and leave the implant site, causing inflammatory and foreign body responses. Because of these problems, there has been a focus on developing new biomaterials for making customizable and highly biocompatible, resorbable implants. Spider silk is a natural protein polymer that is stronger than steel or Kevlar and more elastic than nylon. It has also been shown to be more biocompatible than many materials currently used in 3D printers. In previous animal studies, spider silk has proven to not cause an inflammatory response upon degradation which makes it a desired resorbable implant material (Lewis, 2006). A 3D printer system comprised of a synthetic spider silk resin and a modified 3D printer was developed. A fused filament 3D printer, purchased for under $600, was modified with a custom syringe pump design. This syringe pump allowed for the extrusion of spider silk proteins through a needle, producing defined structures. Cell studies were performed on these structures which showed favorable cell attachment and growth. Capable of entering various emerging industries, spider silk offers an alternative in 3D printed biomaterials.

Lewis, R., 2006, Spider Silk: Ancient Ideas for New Biomaterials: Chemical Reviews, v. 106, p. 3762-3774.

Wohlers, T., and T. Caffrey, 2013, Additive manufacturing and 3D printing state of the industry annual worldwide progress report. 2014: Wohlers Associates.

Hydrogels were investigated in various biomedical fields, especially to study the effects of three-dimensional matrix properties on encapsulated cells or tissues. Regards to fabrication of hydrogels, the chemical crosslinking reactions have received considerable attention recently for biomedical application because these reactions make gelation rapid and efficient in biological conditions. Also, after primary chemical crosslinking reaction, the effect of changes in properties of hydrogel on cell behaviors have been studied through the additional crosslinking reaction. However, to realize the dual mode gelation system to fabricate hydrogels with adjustable properties, secondary crosslinking reactions with additional macromers, photoinitiators, or UV irradiation can produce more radicals, resulting in more cytotoxicity. To minimize these problems and to fabricate hydrogels with tunable properties, we used thiol-ene photo click reaction as primary chemical crosslinking reaction and silk crystallization as secondary physical crosslinking reaction.
Using thiol-ene photo click reaction, fabricated hydrogels have homogeneous network and good mechanical properties. Also, UV-mediated thiol-ene reactions occur rapidly right after the UV irradiation and minimize the exposure of UV light and radicals to encapsulated cells. For these reasons, the thiol-ene reaction can be used as primary chemical crosslinking reaction. And physical crosslinking of silk fibroin can be utilized as further crosslinking reaction after primary gelation, due to low cytotoxicity of silk fibroin and its physical crosslinking.
In this study, we used biocompatible silk fibroin (SF) and poly (ethylene glycol) (PEG). For fabricating the hydrogels, introduction of norbornene (NB) groups to SF and 4-arm PEG was proceeded and modified products, SF-NB and PEG4NB, were used with DTT as a thiol group-containing crosslinker and LAP as a photoinitiator using thiol-ene photo click reaction under UV light irradiation. Chemically crosslinked SF-NB/PEG4NB hydrogels were characterized according to manufacture conditions and in situ gelation behavior and physical properties of hydrogels were observed using rheometer. Then, the secondary physical crosslinking reaction was carried out sequentially and changes in physical properties were also observed using rheometer. We successfully fabricated SF-NB/PEG4NB hydrogels using dual mode gelation system, chemical and physical crosslinking reaction in sequence. It can give an overview of their tunable properties and feasible use of these hydrogels in biomedical applications.

PBAT(Poly (butylene adipate co-terephthalate) is a random copolymer, specially a copolyester of adipic acid, 1,4-butanediol and dimethyl terephthalate. It is a biodegradable alternative to low density polyethylene, having many similar properties including flexibility and resilience, and it is usually used for plastic bags and wraps. Due to its random structure, it has wide melting point, low modulus and stiffness, but high flexibility and toughness. The most fascinating advantage is that PBAT has been proven to be fully biodegradable and harmlessly disappeared due to the containment of butylene adipate groups.
Electrospinning is a technique for producing fibers with diameters ranging from several tens of nanaometers to several micrometers. This process includes solvent evaporation and deposition of electrospun fiber in the form of nonwoven fibrous webs which have highly porosity. Due to these characteristics of electrospinning, the fiber mat has large specific surface area ratio to volume or mass and high porosity, so it can be a good candidate for various biomaterials such as scaffolds for cell/tissue culture, carriers for drug delivery, and wound-dressing.
In this study, we found the optimum conditions for electrospinning of PBAT. The parameters broadly classified into 3 categories. The first is solution properties such as concentration of dope solution, solution viscosity, elasticity, conductivity, surface tension. The second is processing conditions. Electric field voltage, flow rate, the needle to collector distance, and take-up rate are the examples. The last category is the ambient conditions such as temperature and RH of the environment. We mainly focused on the first and second parameters in this experiment. The third condition was fixed as constant. The fiber diameters, morphology, and pore density were characterized through scanning electron microscope (SEM). Both PBAT dope solution and electrospun PBAT mat were analyzed by Fourier transform infrared spectroscopy (FT-IR) to determine the chemical and structural changes of the PBAT polymer.
Nanofiber mat of PBAT were successfully produced by electrospinning techniques. And the properties of nanofibers can be controlled by various parameters. Among many other materials of electrospinning, usage of PBAT is eco-friendly due to its biodegradable feature. So it can produce high molecular weight but flexible biomaterials, and we can expect various application as biomaterials.