Tilman Ahlfeld

Bio: Tilman Ahlfeld is an academic researcher from Dresden University of Technology. The author has contributed to research in topics: Medicine & 3D bioprinting. The author has an hindex of 16, co-authored 31 publications receiving 737 citations.

Development of a clay based bioink for 3D cell printing for skeletal application.

Abstract: Three-dimensional printing of cell-laden hydrogels has evolved as a promising approach on the route to patient-specific or complex tissue-engineered constructs. However, it is still challenging to print structures with both, high shape fidelity and cell vitality. Herein, we used a synthetic nanosilicate clay, called Laponite, to build up scaffolds utilising the extrusion-based method 3D plotting. By blending with alginate and methylcellulose, a bioink was developed which allowed easy extrusion, achieving scaffolds with high printing fidelity. Following extrusion, approximately 70%-75% of printed immortalised human mesenchymal stem cells survived and cell viability was maintained over 21 days within the plotted constructs. Mechanical properties of scaffolds comprised of the composite bioink decreased over time when stored under cell culture conditions. Nevertheless, shape of the plotted constructs was preserved even over longer cultivation periods. Laponite is known for its favourable drug delivery properties. Two model proteins, bovine serum albumin and vascular endothelial growth factor were loaded into the bioink. We demonstrate that the release of both growth factors significantly changed to a more sustained profile by inclusion of Laponite in comparison to an alginate-methylcellulose blend in the absence of Laponite. In summary, addition of a synthetic clay, Laponite, improved printability, increased shape fidelity and was beneficial for controlled release of biologically active agents such as growth factors.

Bioprinting of mineralized constructs utilizing multichannel plotting of a self-setting calcium phosphate cement and a cell-laden bioink.

Abstract: Due to their characteristic resemblance of the mineral component of bone, calcium phosphates are widely accepted as optimal bone substitute materials. Recent research focused on the development of pasty calcium phosphate cement (CPC) formulations, which can be fabricated into various shapes by low-temperature extrusion-based additive manufacturing, namely 3D plotting. While it could be demonstrated that sensitive substances like growth factors can be integrated in such printed CPC scaffolds without impairment of their biological activity live cells cannot be suspended in CPC as they may not be functional when enclosed in a solid and stiff matrix. In contrast, 3D bioprinting of soft cell-laden hydrogels (bioinks) enables the fabrication of constructs with spatially defined cell distribution, which has the potential to overcome problems of conventional cell seeding techniques-but such objects lack mechanical stability. Herein, we combine 3D plotting of CPC and bioprinting of a cell-laden bioink for the first time. As model bioink, an alginate-methylcellulose blend (alg/mc) was used, previously developed by us. Firstly, a fabrication regime was established, enabling optimal setting of CPC and cell survival inside the bioink. As the cells are exposed to the chemical changes of CPC precursors during setting, we studied the compatibility of the complex system of CPC and cell-laden alg/mc for a combined extrusion process and characterized the cellular behavior of encapsulated human mesenchymal stroma cells within the bioink at the interface and in direct vicinity to the CPC. Furthermore, biphasic scaffolds were mechanically characterized and a model for osteochondral tissue grafts is proposed. The manuscript discusses possible impacts of the CPC setting reaction on cells within the bioink and illustrates the advantages of CPC in bioprinting as alternative to the commonly used thermoplasts for bone tissue engineering.

Design and Fabrication of Complex Scaffolds for Bone Defect Healing: Combined 3D Plotting of a Calcium Phosphate Cement and a Growth Factor-Loaded Hydrogel

Abstract: Additive manufacturing enables the fabrication of scaffolds with defined architecture. Versatile printing technologies such as extrusion-based 3D plotting allow in addition the incorporation of biological components increasing the capability to restore functional tissues. We have recently described the fabrication of calcium phosphate cement (CPC) scaffolds by 3D plotting of an oil-based CPC paste under mild conditions. In the present study, we have developed a strategy for growth factor loading based on multichannel plotting: a biphasic scaffold design was realised combining CPC with VEGF-laden, highly concentrated hydrogel strands. As hydrogel component, alginate and an alginate-gellan gum blend were evaluated; the blend exhibited a more favourable VEGF release profile and was chosen for biphasic scaffold fabrication. After plotting, two-step post-processing was performed for both, hydrogel crosslinking and CPC setting, which was shown to be compatible with both materials. Finally, a scaffold was designed and fabricated which can be applied for testing in a rat critical size femur defect. Optimization of CPC plotting enabled the fabrication of highly resolved structures with strand diameters of only 200 µm. Micro-computed tomography revealed a precise strand arrangement and an interconnected pore space within the biphasic scaffold even in swollen state of the hydrogel strands.

3D Bioprinting of osteochondral tissue substitutes - in vitro-chondrogenesis in multi-layered mineralized constructs

Abstract: For the generation of multi-layered full thickness osteochondral tissue substitutes with an individual geometry based on clinical imaging data, combined extrusion-based 3D printing (3D plotting) of a bioink laden with primary chondrocytes and a mineralized biomaterial phase was introduced. A pasty calcium phosphate cement (CPC) and a bioink based on alginate-methylcellulose (algMC) - both are biocompatible and allow 3D plotting with high shape fidelity - were applied in monophasic and combinatory design to recreate osteochondral tissue layers. The capability of cells reacting to chondrogenic biochemical stimuli inside the algMC-based 3D hydrogel matrix was assessed. Towards combined osteochondral constructs, the chondrogenic fate in the presence of CPC in co-fabricated and biphasic mineralized pattern was evaluated. Majority of expanded and algMC-encapsulated cells survived the plotting process and the cultivation period, and were able to undergo redifferentiation in the provided environment to produce their respective extracellular matrix (ECM) components (i.e. sulphated glycosaminoglycans, collagen type II), examined after 3 weeks. The presence of a mineralized zone as located in the physiological calcified cartilage region suspected to interfere with chondrogenesis, was found to support chondrogenic ECM production by altering the ionic concentrations of calcium and phosphorus in in vitro culture conditions.

A versatile method for combining different biopolymers in a core/shell fashion by 3D plotting to achieve mechanically robust constructs.

Abstract: Three-dimensional extrusion of two different biomaterials in a core/shell (c/s) fashion has gained much interest in the last couple of years as it allows for fabricating constructs with novel and interesting properties. We now demonstrate that combining high concentrated (16.7 wt%) alginate hydrogels as shell material with low concentrated, soft biopolymer hydrogels as core leads to mechanically stable and robust 3D scaffolds. Alginate, chitosan, gellan gum, gelatin and collagen hydrogels were utilized successfully as core materials-hydrogels which are too soft for 3D plotting of open-porous structures without an additional mechanical support. The respective c/s scaffolds were characterized concerning their morphology, mechanical properties and swelling behavior. It could be shown that core as well as shell part can be loaded with growth factors and that the release depends on core composition and shell thickness. Neither the plotting process nor the crosslinking with 1M CaCl2 denatured the proteins. When core and shell were loaded with different growth factors (VEGF and BMP-2, respectively) a dual release was achieved. Finally, live human endothelial cells were integrated in the core material, demonstrating that this new strategy can be used for bioprinting purposes as well.

Recent trends in bioinks for 3D printing.

Abstract: The worldwide demand for the organ replacement or tissue regeneration is increasing steadily. The advancements in tissue engineering and regenerative medicine have made it possible to regenerate such damaged organs or tissues into functional organ or tissue with the help of 3D bioprinting. The main component of the 3D bioprinting is the bioink, which is crucial for the development of functional organs or tissue structures. The bioinks used in 3D printing technology require so many properties which are vital and need to be considered during the selection. Combination of different methods and enhancements in properties are required to develop more successful bioinks for the 3D printing of organs or tissue structures. This review consists of the recent state-of-art of polymer-based bioinks used in 3D printing for applications in tissue engineering and regenerative medicine. The subsection projects the basic requirements for the selection of successful bioinks for 3D printing and developing 3D tissues or organ structures using combinations of bioinks such as cells, biomedical polymers and biosignals. Different bioink materials and their properties related to the biocompatibility, printability, mechanical properties, which are recently reported for 3D printing are discussed in detail. Many bioinks formulations have been reported from cell-biomaterials based bioinks to cell-based bioinks such as cell aggregates and tissue spheroids for tissue engineering and regenerative medicine applications. Interestingly, more tunable bioinks, which are biocompatible for live cells, printable and mechanically stable after printing are emerging with the help of functional polymeric biomaterials, their modifications and blending of cells and hydrogels. These approaches show the immense potential of these bioinks to produce more complex tissue/organ structures using 3D bioprinting in the future.

The return of a forgotten polymer : Polycaprolactone in the 21st century

Abstract: During the resorbable-polymer-boom of the 1970s and 1980s, polycaprolactone (PCL) was used in the biomaterials field and a number of drug-delivery devices. Its popularity was soon superseded by faster resorbable polymers which had fewer perceived disadvantages associated with long term degradation (up to 3-4 years) and intracellular resorption pathways; consequently, PCL was almost forgotten for most of two decades. Recently, a resurgence of interest has propelled PCL back into the biomaterials-arena. The superior rheological and viscoelastic properties over many of its aliphatic polyester counterparts renders PCL easy to manufacture and manipulate into a large range of implants and devices. Coupled with relatively inexpensive production routes and FDA approval, this provides a promising platform for the production of longer-term degradable implants which may be manipulated physically, chemically and biologically to possess tailorable degradation kinetics to suit a specific anatomical site. This review will discuss the application of PCL as a biomaterial over the last two decades focusing on the advantages which have propagated its return into the spotlight with a particular focus on medical devices, drug delivery and tissue engineering.

A definition of bioinks and their distinction from biomaterial inks.

Abstract: Biofabrication aims to fabricate biologically functional products through bioprinting or bioassembly (Groll et al 2016 Biofabrication 8 013001). In biofabrication processes, cells are positioned at defined coordinates in three-dimensional space using automated and computer controlled techniques (Moroni et al 2018 Trends Biotechnol. 36 384-402), usually with the aid of biomaterials that are either (i) directly processed with the cells as suspensions/dispersions, (ii) deposited simultaneously in a separate printing process, or (iii) used as a transient support material. Materials that are suited for biofabrication are often referred to as bioinks and have become an important area of research within the field. In view of this special issue on bioinks, we aim herein to briefly summarize the historic evolution of this term within the field of biofabrication. Furthermore, we propose a simple but general definition of bioinks, and clarify its distinction from biomaterial inks.

3D printing of hydrogels: Rational design strategies and emerging biomedical applications

Abstract: 3D printing alias additive manufacturing can transform 3D virtual models created by computer-aided design (CAD) into physical 3D objects in a layer-by-layer manner dispensing with conventional molding or machining. Since the incipiency, significant advancements have been achieved in understanding the process of 3D printing and the relationship of component, structure, property and application of the created objects. Because hydrogels are one of the most feasible classes of ink materials for 3D printing and this field has been rapidly advancing, this Review focuses on hydrogel designs and development of advanced hydrogel-based biomaterial inks and bioinks for 3D printing. It covers 3D printing techniques including laser printing (stereolithography, two-photon polymerization), extrusion printing (3D plotting, direct ink writing), inkjet printing, 3D bioprinting, 4D printing and 4D bioprinting. It provides a comprehensive overview and discussion of the tailorability of material, mechanical, physical, chemical and biological properties of hydrogels to enable advanced hydrogel designs for 3D printing. The range of hydrogel-forming polymers covered encompasses biopolymers, synthetic polymers, polymer blends, nanocomposites, functional polymers, and cell-laden systems. The representative biomedical applications selected demonstrate how hydrogel-based 3D printing is being exploited in tissue engineering, regenerative medicine, cancer research, in vitro disease modeling, high-throughput drug screening, surgical preparation, soft robotics and flexible wearable electronics. Incomparable by thermoplastics, thermosets, ceramics and metals, hydrogel-based 3D printing is playing a pivotal role in the design and creation of advanced functional (bio)systems in a customizable way. An outlook on future directions of hydrogel-based 3D printing is presented.