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In microfluidic tissue chips, angiogenesis processes are spatially guided by combinatorial signals such as interstitial flows and growth factor gradients. It is not always clear how endothelial cells respond to single signaling cues or a combination of signals during their organization into network patterns within hydrogels. We are developing hybrid cellular Potts models combined with a finite element approach to investigate the effect of fluid flows and growth factor gradients on microvascular network formation and organization. Here, we show the simulation results that indicate the emergence of a variety of networks, due to the stimulation with diverse growth factor gradients. With this approach, our long-term aim is to include multifactorial cues such as matrix stiffness, fluid flows, multiple growth factors and cell types within a single model, to create virtual multiscale vascular networks. This provides us with valuable information that can be translated to in vitro settings, in order to create fully functional engineered tissues that can be controlled and perfused.

Introduction Spatiotemporally controlled growth factors delivering systems are crucial for tissue engineering. However, most of the current strategies for growth factors delivery often focuses on the immobilization or coupling of growth factors within the engineered matrices (hydrogel) via various linker proteins or peptides. These systems provide passive release rates and growth factor delivery on demand, but fail to adapt their release rates in accordance with the tissue development. To overcome this limitation, the present study employed nucleic acid based aptamers for achieving spatiotemporally controlled growth factor delivery. Aptamers are affinity ligands selected from DNA/RNA libraries to recognize proteins with high affinity and specificity.1 Aptamer based growth factor delivery systems are able to load/release multiple growth factors on demand with high specificity. In the present study, the authors have 3D-bioprinted aptamer-functionalized bio-inks to evaluate their potential for growth factor sequestering, programmable release and for studying their effect on vascular network formation. Methods The aptamer-functionalized hydrogels were prepared via photo-polymerization of gelatin methacryloyl (GelMA) and acrydite functionalized aptamers having sequence specific for binding to vascular endothelial growth factor (VEGF165). Visible light photoinitiator, tris(2,2′-bipyridyl)dichloro-ruthenium(II) hexahydrate with sodium persulfate was used. The 3D-bioprinting experiments were carried out using Rokit Invivo 3D printer. The viscoelastic properties of the bio-inks were evaluated and compared with control GelMA bio-ink. To study the programmable growth factor release efficiency, VEGF antibody immunostaining was used. For studying the effect of triggered growth factor release on vascular network formation, human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs) were encapsulated within the bio-inks. Results & Discussion The results obtained from VEGF antibody immunostainings confirmed the sequestration and triggered release of VEGF in response to complementary sequence addition from the 3D bioprinted construct after 5 days of culture. The bioprinted construct showed high cellular viability. The F-Actin/DAPI staining showed cellular sprouting and vascular network formation within the 3D printing aptamer functionalized bio-ink regions. In addition, the endothelial cells showed variations in cellular organization based on the VEGF bound aptamer availability within the bioprinted construct. These observations altogether confirms the bioactivity of VEGF bound aptamers within the printed constructs. Conclusions The present study shows the vasculogenic potential of 3D bioprinted aptamer-functionalized bio-inks via spatiotemporally controlling VEGF availability within the hydrogel system. Acknowledgements: This work is supported by an ERC Consolidator Grant under grant agreement no 724469. References 1. M.R. Battig, et. al., J. Am. Chem. Soc. 134 (2012) 12410-12413.

INTRODUCTION: To be clinically effective, engineered tissues should be mechanically stable with surgical compatibility and have a multiscale hierarchical organization resembling vascular tree. Apart from organization, hemodynamic parameters such as blood flow velocity, patency and vascular permeability are the important parameters which control the vascular structures to pass over remodeling and maturation phase [1]. However, how developing vascular network acquire hierarchical organization, what signals drive the organization, and what parameters are involved behind the scenes, remains poorly understood. Thisstudy reports the application of optical techniques, namely laser speckle contrast imaging (LSCI), laser Doppler perfusion imaging (LDPI) and monitoring (LDPM) and side-stream dark field imaging (SDF) [2] to explore and quantify theabovementioned hemodynamic parameters within developing chicken embryo. METHODS: To follow how the vascular networks form around the yolk, fertilized chicken eggs were cracked on day 3 into a PDMS container. The imaging procedure of different techniques is listed below;1. Non-invasive (LSCI and LDPI): The embryo surface is illuminated by a uniform coherent light distribution and imaged viacameras.2. Invasive: (a) LDPM: Optical probe containing a pair of fibers to illuminate coherent light and detect Doppler shifted photonsis mounted on a vessel. (b) SDF: Optical probe including visible light emitting diodes (LEDs) and photodetector is mounted on the capillaries to imagethe dark field. RESULTS: Both qualitative and quantitative assessment of spatiotemporal blood flow perfusion maps and RBCs velocity measurement were performed within macro- and microvascular structures. Results show that relative perfusion levels of individual blood vessels can be imaged with LSCI and LDPI in full-field with millimeter resolution. It can also be locally monitored in time domain using LDPM. Moreover, SDF helps to probe the formation of microcapillaries, and quantitatively measure the blood flow within the capillaries with micrometer resolution. DISCUSSION & CONCLUSIONS: By developing an ex ovo chicken embryo system that is compatible with multiple imaging technologies, we have developed a powerful tool to better understand the processes of vascular organization and maturation. Next steps will focus on the perturbation of the vascular network using mechanical and chemical signals to study how these signals regulate the organization. This will provide crucial information to design mechanically stable organized vascular networks within engineered tissues.Acknowledgements:This work is supported by an ERC Consolidator Grant (724469) and NWO Dutch funding (14538). References:[1] Le Noble F, et al. Development 2004;131(2):361-375[2] Nadort A, et al. Sci Rep 2016;6:25258

Introduction The human vasculature is a highly structured and organized system to supply each cell in the body with oxygen and nutrients and remove metabolic waste. Blood vessels mainly consist of three different cell types. The inner layer (Tunica Intima) of endothelial cells which form a monolayer which are responsible for new blood vessel formation, allow blood flow and also act as a semi-selective barrier to the surrounding tissue followed by the middle layers (Tunica media) of mural cells (smooth muscle cells and pericytes) which are responsible for the vessel maturation and stabilisation. The outer layer (Tunica adventitia) consists mostly of fibrous connective tissue and myofibroblast which can act as progenitor cells. One promising tool to recreate a vascular network in artificial tissue constructs is the use of 3D printable spheroids. 3D printing in general offers the opportunity to have spatial control about release of different materials depending on its properties. In this project we are aiming for using cell spheroids as a 3D printable material. Spheroids display a scaffold free multicellular tissue construct which is characterized by three dimensional cellular self-assembly and by strong cell-cell and cell-ECM interactions1. Furthermore spheroids represent more physiological and functional characteristics compared to 2D cell culture 3,4. By using spheroids consisting of cells which reflect the physiological cell composition and having the ability to fuse with each other to larger scale multicellular constructs, they show a promising potential for usage as printable building blocks Aim Cultivation and characterisation of cell spheroids consisting of different cell types regarding their time related compaction rate and fusion ability as prediction for use as 3D printable building blocks. Additional analysis of cell survival and cell proliferation at different states of the spheroid characterization. Materials and Methods We use manufactured SU8/Si wafer as a positive mold to create patterned elastomeric stamps of poly (dimethylsiloxane) (PDMS) for microwell array platforms. A 4 % agarose solution was cast onto the PDMS stamp for non-adherent cell spheroid culture to cultivate spheroids made of 100 % smooth muscle cells (SMC), 100 % mesenchymal stem cells (MSC) and a co-culture of 90 % SMC and 10 % human umbilical vein endothelial cells (HUVEC). Cell spheroids, consisting each of approximately 267 cells, were cultivated for 9 days to analyse the spheroid formation and spheroid compaction over time using a custom developed MATLAB based program for automate data analysis. Spheroids at day 9 were stained for Live/Dead and Ki67. Afterwards ,the spheroids were transferred to another agarose made microwell array platform to analyse the fusion between different spheroids of the same cell types (MSC-Spheroid : MSC-Spheroid, SMC-Spheroid : SMC-Spheroid, SMC-HUVEC-Spheroid : SMC-HUVEC-Spheroid) and spheroids of different cell types (SMC-Spheroid : MSC-Spheroid, SMC-HUVEC-Spheroid : MSC-Spheroid) for 48 hours by using different cell trackers with subsequent staining for Live/Dead and Ki67. Figure 1. Schematic overview of the current workflow to analyse spheroid cultivation, compaction and fusion as a final use as 3D printable building blocks Results and Conclusions Depending on the cellular composition of the spheroids, different compaction times could be observed, resulting in a final size range of 70-120µm despite the same cell number in each spheroid type. After the final compaction it could be shown, that the spheroids didn’t developed a necrotic core or specific proliferation zones. Due to the differences in final compaction size, heterologous spheroid fusion showed a different dynamic compared to homolog spheroid fusion. References 1. Whitesides GM, Grzybowski B. Self-assembly at all scales. Science 2002; 295: 2418–213 2. Fukuda J, Nakazawa K. Orderly Arrangement of Hepatocyte Spheroids on a Microfabricated Chip. Tissue Eng 2005; 11: 1254–1262. 3. Bhang SH, Cho S-W, La W-G, Lee T-J, Yang HS, Sun A-Y et al. Angiogenesis in ischemic tissue produced by spheroid grafting of human adipose-derived stromal cells. Biomaterials 2011; 32: 2734–2747. Acknowledgements This work is supported by an ERC Consolidator Grant under grant agreement no 724469.

Background Methods to visualize vasculature exist, but additional tools to perturbate the developing vascular networks are still needed to study the angiogenesis and vascular organization with response to external mechano-biochemical stimuli like fluid flow and growth factor gradients. One difficulty in studying vascular organization over development arise from the complexity of interface between the local microenvironment and the vascular system. Here, we report the development of an investigational platform, (similar to W Huang et.al., 2017) used to probe the vasculature within developing chicken embryo. On this platform, using laser doppler perfusion imaging (LDPI) setup, we acquire both structural and perfusion speckle patterns, in order to extract blood flow velocity and structural information. In near future, this system will be used to control the developing vascular network towards the direction of fluid flow and gradient of growth factor pattern. The significance of this system attracts the scientific community and clinicians as it could offers the tool to study natural organization and controlled local microenvironment in a single system. Objective To develop 3D system for probing vascular networks with response to fluid flow and growth factor gradients in a developing chicken embryo Methods The artificial eggshell (core) system consists of an poly-lactic acid (PLA) frame (printed from Shapeways, Netherlands) for mechanical support and six PDMS sheets of ~0.3mm thickness; all the elements are sticked together using PDMS glue. External to the core system the artificial egg shell systems were placed in the incubator and syringe pump would be connected to flow channels in patterned PDMS sheet which would be placed on the developing embryo (see figure). Fertilized chicken eggs were bought from the local poultry farm (Elshuis, Albergen) and transferred to the incubator. Temperature 38°C and 65% humidity were maintained throughout the entire culturing process. Initially for first 3 days before cracking, the eggs were turned 12 times a days with the interval of every 2 hours for 15 seconds to make sure that the embryo does not stick to the wall of egg shell. After 3-day incubation period, chicken embryo was transferred to the PDMS based eggshell under sterile laminar flow hood by removing 3-5mL of albumen from the egg to maintain the osmotic balance and so there won’t be damage to the vasculature during transfer. Results and conclusion In the present study we have optimized the culture conditions of chicken embryo within PDMS based artificial egg shell. Also, we have compared different ex-ovo methods for survivability of chicken embryo. Using LDPI setup, we quantified the blood flow maps of the developing vasculature. Figure. 3D platform for quantifying vascular networks of developing embryo Future plans In future, this optimized system will be used to study the response of vascular networks to fluid flow and patterned growth factor gradients in a developing chicken embryo. References 1. W Huang et.al., Egg-in-Cube: Design and Fabrication of a Novel Artificial Eggshell with Functionalized Surface, PLoS One, 2015 2. W Huang et.al., An angiogenesis platform using a cubic artificial eggshell with patterned blood vessels on chicken chorioallantoic membrane, PLoS One, 2017 Acknowledgements This work is supported by an ERC Consolidator Grant under grant agreement no 724469 and NWO Dutch funding with project number 14538.

Adipose tissues hold great promise in bone tissue engineering since they are available in large quantities as a waste material. The buccal fat pad (BFP) is a specialized mass of adipose tissue that can be easily obtained via the oral cavity without injury to the external body surface. Another advantage of BFP over subcutaneous fat is that its size appears to be similar among different people, independent of body weight and fat distribution. However, limited studies have been conducted on the osteogenic capability of stem cells derived from BFP (BFPSCs). In this study, the BFPSCs were characterized for their osteogenic differentiation potential especially in contact with a synthetic scaffold in a perfusion bioreactor. The features of BFPSCs were compared with bone marrow-derived stem cells (BMSCs) as a well known cell source for bone tissue engineering. Comparing BFPSCs with BMSCs indicated similar morphology, but faster proliferation rate of BMSCs. Moreover, when properly induced for two weeks, BFPSCs resembled BMSCs in the production of bone-specific markers, such as alkaline phosphatase, collagen, bone morphogenic protein (BMP), Runx2, and osteocalcin. Both cell types attached nicely to the pores of a gelatin-coated β-Tricalcium phosphate scaffolds. More osteogenic differentiation potential was observed for both cells under dynamic culture in a perfusion bioreactor compared with static culture. The highest collagen content and BMP production were observed in BFPSCs cultured in the bioreactor for two weeks. These results define BFP as a new, rich, and accessible source of stem cells for tissue engineering purposes.

Adipose tissues hold great promise in bone tissue engineering since they are available in large quantities as a waste material. The buccal fat pad (BFP) is a specialized mass of adipose tissue that can be easily obtained via the oral cavity without injury to the external body surface. Another advantage of BFP over subcutaneous fat is that its size appears to be similar among different people, independent of body weight and fat distribution. However, limited studies have been conducted on the osteogenic capability of stem cells derived from BFP (BFPSCs). In this study, the BFPSCs were characterized for their osteogenic differentiation potential especially in contact with a synthetic scaffold in a perfusion bioreactor. The features of BFPSCs were compared with bone marrow-derived stem cells (BMSCs) as a well known cell source for bone tissue engineering. Comparing BFPSCs with BMSCs indicated similar morphology, but faster proliferation rate of BMSCs. Moreover, when properly induced for two weeks, BFPSCs resembled BMSCs in the production of bone-specific markers, such as alkaline phosphatase, collagen, bone morphogenic protein (BMP), Runx2, and osteocalcin. Both cell types attached nicely to the pores of a gelatin-coated β-Tricalcium phosphate scaffolds. More osteogenic differentiation potential was observed for both cells under dynamic culture in a perfusion bioreactor compared with static culture. The highest collagen content and BMP production were observed in BFPSCs cultured in the bioreactor for two weeks. These results define BFP as a new, rich, and accessible source of stem cells for tissue engineering purposes.

In order for engineered tissue grafts and eventually organs to successfully integrate in a clinical setting, a functional vascular network is imperative. Without vasculature, the tissue constructs cannot receive nutrients essential for their survival, but also lack the stimuli that determine the tissue’s biophysical properties i.e. cell fate determination, cell to cell junctions, and cell orientation. In order for the vascular network to functionally connect to the patient, a hierarchical organization, resembling the vascular tree, is important. From previous studies it is known that fluid flow is a crucial component in controlling the formation of the vascular tree, and that the organization of the vascular network can be further controlled using gradients of angiogenic growth factors such as VEGF. By utilizing spheroid bioprinting within a microgel suspension, an artificial vessel structure was assembled. The deposited spheroids maintained viability and fused over time into perfusable vessels.The subsequent formation of small-diameter vascular structures and capillaries was regulated by an on-demand flow through the bioprinted vessel, resulting in controllable fluid flow shear stresses. Furthermore, VEGF was spatially patterned in the tissue block by locally doping the suspension with growth factor releasing microparticles. By varying both these stimuli, the location of vascular sprout formation and subsequent growth of the new vascular structures could be influenced. This spheroid 3D bioprinting platform offers a dynamic, customizable and accurate method to trigger and control the process of angiogenesis in vitro. By stimulating an artificial blood vessel with controlled fluid flow and growth factor gradients, a vascular complex vascular network can be produced and modulated. The combination of this approach with a gradual replacement of the microgel suspension with cells, can pave the way for the production of vascularized tissue blocks.

Introduction: While tissue engineering offers great potential, its successful application in clinics is currently hampered. The integration of engineered tissues after implantation is limited due to the lack of a vascular network. Therefore, vascularization has emerged as one of the major problems that prevent large-scale translation of the engineered tissues to the clinic. To this end, addition of pre-vascularized network into the engineered tissues during in vitro culture has been proved to enhance tissue survival after implantation. For pre-vascularized tissue engineering, the two basic strategies relies either on natural organization of endothelial cells or on the patterning of endothelial cells into desired vascular geometry using different micro-fabrication technologies. However, in the natural system there are various strong biochemical cues, e.g. growth factors, present to guide the endothelial cells organization towards a vascular tree. Due to the prolonged presence of these strong cues on the endothelial cells, the vascular structures tend to resist remodeling towards a random organization over time which provides long-term functionality to the network. Therefore, there is a need to strategically study the spatial and temporal effect of growth factors on the engineered tissues. This approach would result in a designable vascular network with long-term stability and anastomosis compatibility which could be further used in the formation of tissue building blocks for multi-structural tissue engineering. Aim: To develop vasculogenic hydrogels and evaluate their cellular compatibility by using bone marrow derived human mesenchymal stem cells in 3D culture conditions. Materials and Methods:The vasculogenic hydrogel was prepared by photopolymerization of gelatin methacrylate (GelMA) using Irgacure 2959 as photoinitiator. For cell viability (live/dead analysis) and cell adhesion (DAPI/Alexa fluor 546 Phalloidin) studies, bone marrow derived human mesenchymal stem cells (hMSCs) were used. Results and Conclusions: The physicochemical properties analysis of the synthesized GelMA and vasculogenic hydrogel confirmed the successful crosslinking of gelatin backbone with methacrylate groups by FTIR and NMR spectroscopy. The scanning electron microscope results of the vasculogenic hydrogel revealed interconnected microporous structure with average pore size of 40-80 μm. The developed vasculogenic hydrogels supported the growth of hMSCs in terms of cell viability and adhesion over a span of 7 days within the hydrogels. The cells cultured in the hydrogel showed round morphology and cellular behavior quite similar to native tissue-like growth. Future Plans: In the present study we have optimized the vasculogenic hydrogel conditions in terms of its physicochemical properties and biocompatibility. In future, the optimized results from this study will be used to synthesize vasculogenic hydrogels for patterning growth factors (growth factor mimicking peptides) within the hydrogel and study their effect on the endothelial cells and with co-cultures of endothelial cells and mesenchymal stem cells (see Scheme 1).

Laser-induced periodic surface structures (LIPSS) are highly regular, but at the same time contain a certain level of disorder. The application of LIPSS is a promising method to functionalize biomaterials. However, the absorption of laser energy of most polymer biomaterials is insufficient for the direct application of LIPSS. Here, we report the application of LIPSS to relevant biomaterials using a two-step approach. First, LIPSS are fabricated on a stainless steel surface. Then, the structures are replicated onto biomaterials using the steel as a mold. Results show that LIPSS can be transferred successfully using this approach, and that human mesenchymal stromal cells respond to the transferred structures. With this approach, the range of biomaterials that can be supplied with LIPSS increases dramatically.

In regenerative medicine studies, cell seeding efficiency is not only optimized by changing the chemistry of the biomaterials used as cell culture substrates, but also by altering scaffold geometry, culture and seeding conditions. In this study, the importance of seeding parameters, such as initial cell number, seeding volume, seeding concentration and seeding condition is shown. Human mesenchymal stem cells (hMSCs) were seeded into cylindrically shaped 4 × 3 mm polymeric scaffolds, fabricated by fused deposition modelling. The initial cell number ranged from 5 × 104 to 8 × 105 cells, in volumes varying from 50 µl to 400 µl. To study the effect of seeding conditions, a dynamic system, by means of an agitation plate, was compared with static culture for both scaffolds placed in a well plate or in a confined agarose moulded well. Cell seeding efficiency decreased when seeded with high initial cell numbers, whereas 2 × 105 cells seemed to be an optimal initial cell number in the scaffolds used here. The influence of seeding volume was shown to be dependent on the initial cell number used. By optimizing seeding parameters for each specific culture system, a more efficient use of donor cells can be achieved. Copyright © 2013 John Wiley & Sons, Ltd.