Jasper Van Hoorick

Hybrid Tissue Engineering Scaffolds by Combination of Three-Dimensional Printing and Cell Photoencapsulation

Three-dimensional (3D) printing offers versatile possibilities for adapting the structural parameters of tissue engineering scaffolds. However, it is also essential to develop procedures allowing efficient cell seeding independent of scaffold geometry and pore size. The aim of this study was to establish a method for seeding the scaffolds using photopolymerizable cell-laden hydrogels. The latter facilitates convenient preparation, and handling of cell suspension, while distributing the hydrogel precursor throughout the pores, before it is cross-linked with light. In addition, encapsulation of living cells within hydrogels can produce constructs with high initial cell loading and intimate cell-matrix contact, similar to that of the natural extra-cellular matrix (ECM). Three dimensional scaffolds were produced from poly(lactic) acid (PLA) by means of fused deposition modeling. A solution of methacrylamide-modified gelatin (Gel-MOD) in cell culture medium containing photoinitiator Li-TPO-L was used as a hydrogel precursor. Being an enzymatically degradable derivative of natural collagen, gelatin-based matrices are biomimetic and potentially support the process of cell-induced remodeling. Preosteoblast cells MC3T3-E1 at a density of 10 × 106 cells per 1 mL were used for testing the seeding procedure and cell proliferation studies. Obtained results indicate that produced constructs support cell survival and proliferation over extended duration of our experiment. The established two-step approach for scaffold seeding with the cells is simple, rapid, and is shown to be highly reproducible. Furthermore, it enables precise control of the initial cell density, while yielding their uniform distribution throughout the scaffold. Such hybrid tissue engineering constructs merge the advantages of rigid 3D printed constructs with the soft hydrogel matrix, potentially mimicking the process of ECM remodeling.

Cryogel-PCL combination scaffolds for bone tissue repair.

The present work describes the development and the evaluation of cryogel-poly-ε-caprolactone combinatory scaffolds for bone tissue engineering. Gelatin was selected as cell-interactive biopolymer to enable the adhesion and the proliferation of mouse calvaria pre-osteoblasts while poly-ε-caprolactone was applied for its mechanical strength required for the envisaged application. In order to realize suitable osteoblast carriers, methacrylamide-functionalized gelatin was introduced into 3D printed poly-ε-caprolactone scaffolds created using the Bioplotter technology, followed by performing a cryogenic treatment which was concomitant with the redox-initiated, covalent crosslinking of the gelatin derivative (i.e. cryogelation). In a first part, the efficiency of the cryogelation process was determined using gel fraction experiments and by correlating the results with conventional hydrogel formation at room temperature. Next, the optimal cryogelation parameters were fed into the combinatory approach and the scaffolds developed were characterized for their structural and mechanical properties using scanning electron microscopy, micro-computed tomography and compression tests respectively. In a final part, in vitro biocompatibility assays indicated a good colonization of the pre-osteoblasts and the attachment of viable cells onto the cryogenic network. However, the results also show that the cellular infiltration throughout the entire scaffold is suboptimal, which implies that the scaffold design should be optimized by reducing the cryogel density.

Cross-Linkable Gelatins with Superior Mechanical Properties Through Carboxylic Acid Modification: Increasing the Two-Photon Polymerization Potential

The present work reports on the development of photo-cross-linkable gelatins sufficiently versatile to overcome current biopolymer two-photon polymerization (2PP) processing limitations. To this end, both the primary amines as well as the carboxylic acids of gelatin type B were functionalized with photo-cross-linkable moieties (up to 1 mmol/g) resulting in superior and tunable mechanical properties (G′ from 5000 to 147000 Pa) enabling efficient 2PP processing. The materials were characterized in depth prior to and after photoinduced cross-linking using fully functionalized gelatin-methacrylamide (gel-MOD) as a benchmark to assess the effect of functionalization on the protein properties, cross-linking efficiency, and mechanical properties. In addition, preliminary experiments on hydrogel films indicated excellent in vitro biocompatibility (close to 100% viability) both in the presence of MC3T3 preosteoblasts and L929 fibroblasts. Moreover, 2PP processing of the novel derivative was superior in terms of applied laser power (≥40 vs ≥60 mW for gel-MOD at 100 mm/s) as well as post-production swelling (0–20% vs 75–100% for gel-MOD) compared to those of gel-MOD. The reported novel gelatin derivative (gel-MOD-AEMA) proves to be extremely suitable for direct laser writing as both superior mimicry of the applied computer-aided design (CAD) was obtained while maintaining the desired cellular interactivity of the biopolymer. It can be anticipated that the present work will also be applicable to alternative biopolymers mimicking the extracellular environment such as collagen, elastin, and glycosaminoglycans, thereby expanding current material-related processing limitations in the tissue engineering field.

Indirect Rapid Prototyping: Opening Up Unprecedented Opportunities in Scaffold Design and Applications

Over the past decades, solid freeform fabrication (SFF) has emerged as the main technology for the production of scaffolds for tissue engineering applications as a result of the architectural versatility. However, certain limitations have also arisen, primarily associated with the available, rather limited range of materials suitable for processing. To overcome these limitations, several research groups have been exploring novel methodologies through which a construct, generated via SFF, is applied as a sacrificial mould for production of the final construct. The technique combines the benefits of SFF techniques in terms of controlled, patient-specific design with a large freedom in material selection associated with conventional scaffold production techniques. Consequently, well-defined 3D scaffolds can be generated in a straightforward manner from previously difficult to print and even “unprintable” materials due to thermomechanical properties that do not match the often strict temperature and pressure requirements for direct rapid prototyping. These include several biomaterials, thermally degradable materials, ceramics and composites. Since it can be combined with conventional pore forming techniques, indirect rapid prototyping (iRP) enables the creation of a hierarchical porosity in the final scaffold with micropores inside the struts. Consequently, scaffolds and implants for applications in both soft and hard tissue regeneration have been reported. In this review, an overview of different iRP strategies and materials are presented from the first reports of the approach at the turn of the century until now.

Cell Regeneration: Current Knowledge and Evolutions

Low back pain is one of the most common complaints throughout the modern western society. It can lead to a chronic disability for 10 % of the patients resulting in a huge economic burden for society and often an incapacitating life for the patient. As low back pain often goes concomitant with intervertebral disc (IVD) degeneration, tissue-engineering solutions for IVD gained increasing attention during the last decade.

Indirect Solid Freeform Fabrication of an Initiator-Free Photocrosslinkable Hydrogel Precursor for the Creation of Porous Scaffolds

In the present work, a photopolymerized urethane-based poly(ethylene glycol) hydrogel is applied as a porous scaffold material using indirect solid freeform fabrication (SFF). This approach combines the benefits of SFF with a large freedom in material selection and applicable concentration ranges. A sacrificial 3D poly(ε-caprolactone) structure is generated using fused deposition modeling and used as template to produce hydrogel scaffolds. By changing the template plotting parameters, the scaffold channel sizes vary from 280 to 360 μm, and the strut diameters from 340 to 400 μm. This enables the production of scaffolds with tunable mechanical properties, characterized by an average hardness ranging from 9 to 43 N and from 1 to 6 N for dry and hydrated scaffolds, respectively. Experiments using mouse calvaria preosteoblasts indicate that a gelatin methacrylamide coating of the scaffolds results in an increased cell adhesion and proliferation with improved cell morphology.