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Bone tissue is highly vascularized. The crosstalk of vascular and osteogenic cells is not only responsible for the formation of the strongly divergent tissue types but also for their physiological maintenance and repair. Extrusion-based bioprinting presents a promising fabrication method for bone replacement. It allows for the production of large-volume constructs, which can be tailored to individual tissue defect geometries. In this study, we used the all-gelatin-based toolbox of methacryl-modified gelatin (GM), non-modified gelatin (G) and acetylated GM (GMA) to tailor both the properties of the bioink towards improved printability, and the properties of the crosslinked hydrogel towards enhanced support of vascular network formation by simple blending. The vasculogenic behavior of human dermal microvascular endothelial cells (HDMECs) and human adipose-derived stem cells (ASCs) was evaluated in the different hydrogel formulations for 14 days. Co-culture constructs including a vascular component and an osteogenic component (i.e. a bone bioink based on GM, hydroxyapatite and ASCs) were fabricated via extrusion-based bioprinting. Bioprinted co-culture constructs exhibited functional tissue-specific cells whose interplay positively affected the formation and maintenance of vascular-like structures. The setup further enabled the deposition of bone matrix associated proteins like collagen type I, fibronectin and alkaline phosphatase within the 30-day culture.
The coculture of osteogenic and angiogenic cells and the resulting paracrine signaling via soluble factors are supposed to be crucial for successfully engineering vascularized bone tissue equivalents. In this study, a coculture system combining primary human adiposederived stem cells (hASCs) and primary human dermal microvascular endothelial cells (HDMECs) within two types of hydrogels based on methacryloyl‐modified gelatin (GM) as three‐dimensional scaffolds was examined for its support of tissue specific cell functions. HDMECs, together with hASCs as supporting cells, were encapsulated in soft GM gels and were indirectly cocultured with hASCs encapsulated in stiffer GM hydrogels additionally containing methacrylate‐modified hyaluronic acid and hydroxyapatite particles. After 14 days, the hASC in the stiffer gels (constituting the “bone gels”) expressed matrix proteins like collagen type I and fibronectin, as well as bone‐specific proteins osteopontin and alkaline phosphatase. After 14 days of coculture with HDMEC‐laden hydrogels, the viscoelastic properties of the bone gels were significantly higher compared with the gels in monoculture. Within the soft vascularization gels, the formed capillary‐like networks were significantly longer after 14 days of coculture than the structures in the control gels. In addition, the stability as well as the complexity of the vascular networks was significantly increased by coculture. We discussed and concluded that osteogenic and angiogenic signals from the culture media as well as from cocultured cell types, and tissue‐specific hydrogel composition all contribute to stimulate the interplay between osteogenesis and angiogenesis in vitro and are a basis for engineering vascularized bone.
New approaches to respiratory assist: bioengineering an ambulatory, miniaturized bioartificial lung
(2019)
Although state-of-the-art treatments of respiratory failure clearly have made some progress in terms of survival in patients suffering from severe respiratory system disorders, such as acute respiratory distress syndrome (ARDS), they failed to significantly improve the quality of life in patients with acute or chronic lung failure, including severe acute exacerbations of chronic obstructive pulmonary disease or ARDS as well. Limitations of standard treatment modalities, which largely rely on conventional mechanical ventilation, emphasize the urgent, unmet clinical need for developing novel(bio)artificial respiratory assist devices that provide extracorporeal gas exchange with a focus on direct extracorporeal CO2 removal from the blood. In this review, we discuss some of the novel concepts and critical prerequisites for such respiratory lung assist devices that can be used with an adequate safety profile, in the intensive care setting, as well as for long-term domiciliary therapy in patients with chronic ventilatory failure. Specifically, we describe some of the pivotal steps, such as device miniaturization, passivation of the blood-contacting surfaces by chemical surface modifications, or endothelial cell seeding, all of which are required for converting current lung assist devices into ambulatory lung assist device for long-term use in critically ill patients. Finally, we also discuss some of the risks and challenges for the long-term use of ambulatory miniaturized bioartificial lungs.
Size and function of bioartificial tissue models are still limited due to the lack of blood vessels and dynamic perfusion for nutrient supply. In this study, we evaluated the use of cytocompatible methacryl-modified gelatin for the fabrication of a hydrogel-based tube by dip-coating and subsequent photo-initiated cross-linking. The wall thickness of the tubes and the diameter were tuned by the degree of gelatin methacryl-modification and the number of dipping cycles. The dipping temperature of the gelatin solution was adjusted to achieve low viscous fluids of approximately 0.1 Pa s and was different for gelatin derivatives with different modification degrees. A versatile perfusion bioreactor for the supply of surrounding tissue models was developed, which can be adaped to several geometries and sizes of blood-vessel mimicking tubes. The manufactured bendable gelatin tubes were permeable for water and dissolved substances, like Nile Blue and serum albumin. As a proof of concept, human fibroblasts in a three-dimensional collagen tissue model were sucessfully supplied with nutrients via the central gelatin tube under dynamic conditions for 2 days. Moreover, the tubes could be used as scaffolds to build-up a functional and viable endothelial layer. Hence, the presented tools can contribute to solving current challenges in tissue engineering.
Methacrylated gelatin and mature adipocytes are promising components for adipose tissue engineering
(2016)
In vitro engineering of autologous fatty tissue constructs is still a major challenge for the treatment of congenital deformities, tumor resections or high-graded burns. In this study, we evaluated the suitability of photo-crosslinkable methacrylated gelatin (GM) and mature adipocytes as components for the composition of three-dimensional fatty tissue constructs. Cytocompatibility evaluations of the GM and the photoinitiator Lithium phenyl-2,4,6 trimethylbenzoylphosphinate (LAP) showed no cytotoxicity in the relevant range of concentrations. Matrix stiffness of cell-laden hydrogels was adjusted to native fatty tissue by tuning the degree of crosslinking and was shown to be comparable to that of native fatty tissue. Mature adipocytes were then cultured for 14 days within the GM resulting in a fatty tissue construct loaded with viable cells expressing cell markers perilipin A and laminin. This work demonstrates that mature adipocytes are a highly valuable cell source for the composition of fatty tissue equivalents in vitro. Photo-crosslinkable methacrylated gelatin is an excellent tissue scaffold and a promising bioink for new printing techniques due to its biocompatibility and tunable properties.
Though bioprinting is a forward-looking approach in bone tissue engineering, the development of bioinks which are on the one hand processable with the chosen printing technique, and on the other hand possess the relevant mechanical as well as osteoconductive features remains a challenge. In the present study, polymer solutions based on methacrylated gelatin and methacrylated hyaluronic acid modified with hydroxyapatite (HAp) particles (5 wt%) were prepared. Encapsulation of primary human adipose derived stem cells in the HAp-containing gels and culture for 28 d resulted in a storage moduli significantly increased to 126% ± 9.6% compared to the value on day 1 by the sole influence of the HAp. Additional use of osteogenic media components resulted in an increase of storage module up to 199% ± 27.8%. Similarly, the loss moduli was increased to 370% ± 122.1% under the influence of osteogenic media components and HAp. Those changes in rheological material characteristics indicate a distinct change in elastic and viscous hydrogel properties, and are attributed to extensive matrix production in the hydrogels by the encapsulated cells, what could also be proven by staining of bone matrix components like collagen I, fibronectin, alkaline phosphatase and osteopontin. When using the cell-laden polymer solutions as bioinks to build up relevant geometries, the ink showed excellent printability and the printed grid structure's integrity remained intact over a culture time of 28 d. Again, an intense matrix formation as well as upregulation of osteogenic markers by the encapsulated cells could be shown. In conclusion, we demonstrated that our HAp-containing bioinks and hydrogels on basis of methacrylated gelatin and hyaluronic acid are on the one hand highly suitable for the build up of relevant three-dimensional geometries with microextrusion bioprinting, and on the other hand exhibit a significant positive effect on bone matrix development and remodeling in the hydrogels, as indicated by rheological measurements and staining of bone components. This makes the developed composite hydrogels an excellent material for bone bioprinting approaches.
In bioprinting approaches, the choice of bioink plays an important role since it must be processable with the selected printing method, but also cytocompatible and biofunctional. Therefore, a crosslinkable gelatin-based ink was modified with hydroxyapatite (HAp) particles, representing the composite buildup of natural bone. The inks’ viscosity was significantly increased by the addition of HAp, making the material processable with extrusion-based methods. The storage moduli of the formed hydrogels rose significantly, depicting improved mechanical properties. A cytocompatibility assay revealed suitable ranges for photoinitiator and HAp concentrations. As a proof of concept, the modified ink was printed together with cells, yielding stable three-dimensional constructs containing a homogeneously distributed mineralization and viable cells.
Blood vessel reconstruction is still an elusive goal for the development of in vitro models as well as artificial vascular grafts. In this study, we used a novel photo curable cytocompatible polyacrylate material (PA) for freeform generation of synthetic vessels. We applied stereolithography for the fabrication of arbitrary 3D tubular structures with total dimensions in the centimeter range, 300 µm wall thickness, inner diameters of 1 to 2 mm and defined pores with a constant diameter of approximately 100 µm or 200 µm. We established a rinsing protocol to remove remaining cytotoxic substances from the photo-cured PA and applied thio-modified heparin and RGDC-peptides to functionalize the PA surface for enhanced endothelial cell adhesion. A rotating seeding procedure was introduced to ensure homogenous endothelial monolayer formation at the inner luminal tube wall. We showed that endothelial cells stayed viable and adherent and aligned along the medium flow under fluid-flow conditions comparable to native capillaries. The combined technology approach comprising of freeform additive manufacturing (AM), biomimetic design, cytocompatible materials which are applicable to AM, and biofunctionalization of AM constructs has been introduced as BioRap® technology by the authors.