Methacrylamide-modified Gelatin Research Articles

Development of photo-crosslinkable collagen hydrogel building blocks for vascular tissue engineering applications: A superior alternative to methacrylated gelatin?

The present work targets the development of collagen-based hydrogel precursors, functionalized with photo-crosslinkable methacrylamide moieties (COL-MA), for vascular tissue engineering (vTE) applications. The developed materials were physico-chemically characterized in terms of crosslinking kinetics, degree of modification/conversion, swelling behavior, mechanical properties and in vitro cytocompatibility. The collagen derivatives were benchmarked to methacrylamide-modified gelatin (GEL-MA), due to its proven track record in the field of tissue engineering. To the best of our knowledge, this is the first paper in its kind comparing these two methacrylated biopolymers for vTE applications. For both gelatin and collagen, two derivatives with varying degrees of substitutions (DS) were developed by altering the added amount of methacrylic anhydride (MeAnH). This led to photo-crosslinkable derivatives with a DS of 74 and 96% for collagen, and a DS of 73 and 99% for gelatin. The developed derivatives showed high gel fractions (i.e. 74% and 84%, for the gelatin derivatives; 87 and 83%, for the collagen derivatives) and an excellent crosslinking efficiency. Furthermore, the results indicated that the functionalization of collagen led to hydrogels with tunable mechanical properties (i.e. storage moduli of [4.8–9.4 kPa] for the developed COL-MAs versus [3.9–8.4 kPa] for the developed GEL-MAs) along with superior cell-biomaterial interactions when compared to GEL-MA. Moreover, the developed photo-crosslinkable collagens showed superior mechanical properties compared to extracted native collagen. Therefore, the developed photo-crosslinkable collagens demonstrate great potential as biomaterials for vTE applications.

Engineering microvasculature by 3D bioprinting of prevascularized spheroids in photo-crosslinkable gelatin

To engineer tissues with clinically relevant dimensions by three-dimensional bioprinting, an extended vascular network with diameters ranging from the macro- to micro-scale needs to be integrated. Extrusion-based bioprinting is the most commonly applied bioprinting technique but due to the limited resolution of conventional bioprinters, the establishment of a microvascular network for the transfer of oxygen, nutrients and metabolic waste products remains challenging. To answer this need, this study assessed the potential and processability of spheroids, containing a capillary-like network, to be used as micron-sized prevascularized units for incorporation throughout the bioprinted construct. Prevascularized spheroids were generated by combining endothelial cells with fibroblasts and adipose tissue-derived mesenchymal stem cells as supporting cells. To serve as a viscous medium for the bioink-based deposition by extrusion printing, spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) and Irgacure 2959. The influence of gelMA encapsulation, the printing process and photo-crosslinking conditions on spheroid viability, proliferation and vascularization were analyzed by live/dead staining, immunohistochemistry, gene expression analysis and sprouting analysis. Stable spheroid-laden constructs, allowing spheroid outgrowth, were achieved by applying 10 min UV-A photo-curing (365 nm, 4 mW cm−2), while the construct was incubated in an additional Irgacure 2959 immersion solution. Following implantation in ovo onto a chick chorioallantoic membrane, the prevascular engineered constructs showed anastomosis with the host vasculature. This study demonstrated (a) the potential of triculture prevascularized spheroids for application as multicellular building blocks, (b) the processability of the spheroid-laden gelMA bioink by extrusion bioprinting and (c) the importance of photo-crosslinking parameters post printing, as prolonged photo-curing intervals showed to be detrimental for the angiogenic potential and complete vascularization of the construct post printing.

Tuning the Phenotype of Cartilage Tissue Mimics by Varying Spheroid Maturation and Methacrylamide-Modified Gelatin Hydrogel Characteristics.

In hybrid bioprinting of cartilage tissue constructs, spheroids are used as cellular building blocks and combined with biomaterials for dispensing. However, biomaterial intrinsic cues can deeply affect cell fate and to date, the influence of hydrogel encapsulation on spheroid viability and phenotype has received limited attention. This study assesses this need and unravels 1) how the phenotype of spheroid-laden constructs can be tuned through adjusting the hydrogel physico-chemical properties and 2) if the spheroid maturation stage prior to encapsulation is a determining factor for the construct phenotype. Articular chondrocyte spheroids with a cartilage specific extracellular matrix (ECM) are generated and different maturation stages, early-, mid-, and late-stage (3, 7, and 14 days, respectively), are harvested and encapsulated in 10, 15, or 20w/v% methacrylamide-modified gelatin (gelMA) for 14 days. The encapsulation of immature spheroids do not lead to a cartilage-like ECM production but when more mature mid- or late-stage spheroids are combined with a certain concentration of gelMA, a fibrocartilage-like as well as a hyaline cartilage-like phenotype can be induced. As a proof of concept, late-stage spheroids are bioprinted using a 10w/v% gelMA-Irgacure 2959 solution with the aim to test the processing potential of the spheroid-laden bioink.

Precise control of synthetic hydrogel network structure via linear, independent synthesis-swelling relationships.

Hydrogel physical properties are tuned by altering synthesis conditions such as initial polymer concentration and polymer-cross-linker stoichiometric ratios. Traditionally, differences in hydrogel synthesis schemes, such as end-linked poly(ethylene glycol) diacrylate hydrogels and cross-linked poly(vinyl alcohol) hydrogels, limit structural comparison between hydrogels. In this study, we use generalized synthesis variables for hydrogels that emphasize how changes in formulation affect the resulting network structure. We identify two independent linear correlations between these synthesis variables and swelling behavior. Analysis through recently updated swollen polymer network models suggests that synthesis-swelling correlations can be used to make a priori predictions of the stiffness and solute diffusivity characteristics of synthetic hydrogels. The same experiments and analyses performed on methacrylamide-modified gelatin hydrogels demonstrate that complex biopolymer structures disrupt the linear synthesis-swelling correlations. These studies provide insight into the control of hydrogel physical properties through structural design and can be used to implement and optimize biomedically relevant hydrogels.

Hybrid Bioprinting of Chondrogenically Induced Human Mesenchymal Stem Cell Spheroids.

To date, the treatment of articular cartilage lesions remains challenging. A promising strategy for the development of new regenerative therapies is hybrid bioprinting, combining the principles of developmental biology, biomaterial science, and 3D bioprinting. In this approach, scaffold-free cartilage microtissues with small diameters are used as building blocks, combined with a photo-crosslinkable hydrogel and subsequently bioprinted. Spheroids of human bone marrow-derived mesenchymal stem cells (hBM-MSC) are created using a high-throughput microwell system and chondrogenic differentiation is induced during 42 days by applying chondrogenic culture medium and low oxygen tension (5%). Stable and homogeneous cartilage spheroids with a mean diameter of 116 ± 2.80 μm, which is compatible with bioprinting, were created after 14 days of culture and a glycosaminoglycans (GAG)- and collagen II-positive extracellular matrix (ECM) was observed. Spheroids were able to assemble at random into a macrotissue, driven by developmental biology tissue fusion processes, and after 72 h of culture, a compact macrotissue was formed. In a directed assembly approach, spheroids were assembled with high spatial control using the bio-ink based extrusion bioprinting approach. Therefore, 14-day spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) as viscous printing medium to ensure shape fidelity of the printed construct. The photo-initiators Irgacure 2959 and Li-TPO-L were evaluated by assessing their effect on bio-ink properties and the chondrogenic phenotype. The encapsulation in gelMA resulted in further chondrogenic maturation observed by an increased production of GAG and a reduction of collagen I. Moreover, the use of Li-TPO-L lead to constructs with lower stiffness which induced a decrease of collagen I and an increase in GAG and collagen II production. After 3D bioprinting, spheroids remained viable and the cartilage phenotype was maintained. Our findings demonstrate that hBM-MSC spheroids are able to differentiate into cartilage microtissues and display a geometry compatible with 3D bioprinting. Furthermore, for hybrid bioprinting of these spheroids, gelMA is a promising material as it exhibits favorable properties in terms of printability and it supports the viability and chondrogenic phenotype of hBM-MSC microtissues. Moreover, it was shown that a lower hydrogel stiffness enhances further chondrogenic maturation after bioprinting.

Extrusion-based 3D printing of photo-crosslinkable gelatin and κ-carrageenan hydrogel blends for adipose tissue regeneration

Current soft tissue repair techniques for women with breast cancer remain associated with several drawbacks including surgical complications and a high resorption rate for lipofilling techniques. Hence, the need to develop improved adipose tissue reconstruction strategies. Additive manufacturing can be a promising tool towards the development of patient-specific scaffolds which are able to support adipose tissue engineering. In the present work, scaffolds composed of both methacrylamide-modified gelatin (Gel-MA) and methacrylated κ-carrageenan (Car-MA), i.e. hydrogel blends, were developed using extrusion-based 3D printing in order to establish a close resemblance to the native extracellular matrix. The hydrogel blends were benchmarked to scaffolds constituting of only Gel-MA. Our results indicate that both types of scaffolds remain stable over time (21 days), are able to absorb large amounts of water and exhibit mechanical properties comparable to those of native breast tissue (2 kPa). Furthermore, a similar cell viability (> 90%) and proliferation rate after 14 days was obtained for adipose tissue-derived stem cells (ASCs) upon seeding onto both types of scaffolds. Additionally, the ASCs were able to differentiate into the adipogenic lineage on the hydrogel blend scaffolds, although their differentiation potential was lower compared to that of ASCs seeded onto the Gel-MA scaffolds.

Additive manufacturing of photo-crosslinked gelatin scaffolds for adipose tissue engineering

There exists a clear clinical need for adipose tissue reconstruction strategies to repair soft tissue defects which outperform the currently available approaches. In this respect, additive manufacturing has shown to be a promising alternative for the development of larger constructs able to support adipose tissue engineering. In the present work, a thiol-ene photo-click crosslinkable gelatin hydrogel was developed which allowed extrusion-based additive manufacturing into porous scaffolds. To this end, norbornene-functionalized gelatin (Gel-NB) was combined with thiolated gelatin (Gel-SH). The application of a macromolecular gelatin-based thiolated crosslinker holds several advantages over conventional crosslinkers including cell-interactivity, less chance at phase separation between scaffold material and crosslinker and the formation of a more homogeneous network. Throughout the paper, these photo-click scaffolds were benchmarked to the conventional methacrylamide-modified gelatin (Gel-MA). The results indicated that stable scaffolds could be realized which were further characterized physico-chemically by performing swelling, mechanical and in vitro biodegradability assays. Furthermore, the seeded adipose tissue-derived stem cells (ASCs) remained viable (>90%) up to 14 days and were able to proliferate. In addition, the cells could be differentiated into the adipogenic lineage on the photo-click crosslinked scaffolds, thereby performing better than the cells supported by the frequently reported Gel-MA scaffolds. As a result, the developed photo-click crosslinked scaffolds can be considered a promising candidate towards adipose tissue engineering and a valuable alternative for the omnipresent Gel-MA. Statement of SignificanceThe field of adipose tissue engineering has emerged as a promising strategy to repair soft tissue defects. Herein, Gel-NB/Gel-SH gelatin-based hydrogel scaffolds were produced using extrusion-based additive manufacturing. Using a cell-interactive, thiolated gelatin crosslinker, a homogeneous network was formed and the risk of phase separation between norbornene-modified gelatin and macromolecular crosslinkers was reduced. UV-induced crosslinking of these materials is based on step growth polymerization which requires less free radicals to enable polymerization. Our results demonstrated the potential of the developed scaffolds, due to their favourable physico-chemical characteristics as well as their adipogenic differentiation potential when benchmarked to Gel-MA scaffolds. Hence, the hydrogels could be of great interest towards future development of adipose tissue constructs and tissue engineering in general.

Gelatin- and starch-based hydrogels. Part A: Hydrogel development, characterization and coating

The present work aims at constructing the ideal scaffold matrix of which the physico-chemical properties can be altered according to the targeted tissue regeneration application. Ideally, this scaffold should resemble the natural extracellular matrix (ECM) as close as possible both in terms of chemical composition and mechanical properties. Therefore, hydrogel films were developed consisting of methacrylamide-modified gelatin and starch-pentenoate building blocks because the ECM can be considered as a crosslinked hydrogel network consisting of both polysaccharides and structural, signaling and cell-adhesive proteins. For the gelatin hydrogels, three different substitution degrees were evaluated including 31%, 72% and 95%. A substitution degree of 32% was applied for the starch-pentenoate building block. Pure gelatin hydrogels films as well as interpenetrating networks with gelatin and starch were developed. Subsequently, these films were characterized using gel fraction and swelling experiments, high resolution-magic angle spinning 1H NMR spectroscopy, rheology, infrared mapping and atomic force microscopy. The results indicate that both the mechanical properties and the swelling extent of the developed hydrogel films can be controlled by varying the chemical composition and the degree of substitution of the methacrylamide-modified gelatin applied. The storage moduli of the developed materials ranged between 14 and 63kPa. Phase separation was observed for the IPNs for which separated starch domains could be distinguished located in the surrounding gelatin matrix. Furthermore, we evaluated the affinity of aggrecan for gelatin by atomic force microscopy and radiolabeling experiments. We found that aggrecan can be applied as a bioactive coating for gelatin hydrogels by a straightforward physisorption procedure. Thus, we achieved distinct fine-tuning of the physico-chemical properties of these hydrogels which render them promising candidates for tissue engineering approaches.

Crosslinking strategies for porous gelatin scaffolds

The present work reports on the application and the evaluation of a multitude of crosslinking approaches including high-energy irradiation, redox-initiating systems and conventional carbodiimide-coupling chemistry for frozen and/or freeze-dried porous gelatin scaffolds. The latter is particularly relevant for a plethora of biomedical applications such as tissue engineering supports, wound dressings, adhesive and absorbent pads for surgery, etc. Moreover, the results obtained for gelatin can be considered a proof-of-concept to be extrapolated to other polymer systems containing double bonds and/or amines and carboxylic acids to also realize scaffold crosslinking in dry or frozen state. The results showed that high-energy irradiation at −5 °C enabled sufficient segmental mobility to induce chemical crosslinking after performing a cryogenic treatment of methacrylamide-modified gelatin scaffolds. Alternatively, although several redox-initiating systems were unable to chemically crosslink functionalized gelatin, the combination of ammonium persulphate and TEMED resulted in the formation of scaffolds with a reasonable gel fraction. Interestingly, carbodiimide-coupling was found suitable to crosslink freeze-dried gelatin matrices.

Indirect additive manufacturing as an elegant tool for the production of self-supporting low density gelatin scaffolds.

The present work describes for the first time the production of self-supporting low gelatin density (<10w/v%) porous scaffolds using methacrylamide-modified gelatin as an extracellular matrix mimicking component. As porous scaffolds starting from low gelatin concentrations cannot be realized with the conventional additive manufacturing techniquesin the abscence of additives, we applied an indirect fused deposition modelling approach. To realize this, we have printed a sacrificial polyester scaffold which supported the hydrogel material during UV crosslinking, thereby preventing hydrogel structure collapse. After complete curing, the polyester scaffold was selectively dissolved leaving behind a porous, interconnective low density gelatin scaffold. Scaffold structural analysis indicated the success of the selected indirect additive manufacturing approach. Physico-chemical testing revealed scaffold properties (mechanical, degradation, swelling) to depend on the applied gelatin concentration and methacrylamide content. Preliminary biocompatibility studies revealed the cell-interactive and biocompatible properties of the materials developed.

Cross-linkable alginate-graft-gelatin copolymers for tissue engineering applications

When it comes to failing or injured tissues and organs, patients often end up on waiting lists for tissue or even organ transplantation negatively affecting the patient’s quality of life. The multidisciplinary research field of tissue engineering may offer more innovative ways to replace or ideally regenerate failing tissues and organs. A widely used material in this research field is gelatin because of its biocompatibility and interesting hydrogel forming properties. However, at body temperature gelatin’s mechanical properties are greatly reduced due to the dissolution of collagen-like triple helices. With the aim to obtain materials that retain their mechanical properties at body temperature, we propose to combine sodium alginate and methacrylamide-modified gelatin (Gel-MOD) in the form of a graft copolymer to obtain a material that closely resembles the extracellular matrix. The obtained materials can be cross-linked via three distinct pathways including cation mediated, temperature mediated or via covalent bond formation after UV irradiation in the presence of a photo-initiator. The current contribution covers the synthesis of the above mentioned alginate-graft-gelatin copolymers and the characterization of the resulting hydrogels. The materials developed are highly hydrophilic, showing high gel fractions and satisfactory mechanical properties. Moreover the attained storage moduli were tunable by divalent cation addition (72–275% increase at 21°C, 42–405% increase at 40°C). One formulation was found to outperform Gel-MOD in terms of mechanical properties at 40°C, thus indicating the proposed strategy can be used to improve the mechanical properties of gelatin-based hydrogels. Moreover, in vitro biocompatibility assays indicated that cell adhesion and proliferation improves with increasing gelatin content. The present paper illustrates that the developed triple cross-linkable materials are suitable cell carriers, promising to be applied for biomedical purposes.

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.

Hybrid Hydrogels Intended as Scaffolds for Soft Tissue Repair

This work describes the investigation of the properties of hydrogels based on methacrylamide-modified gelatin (GelMA) and polyvinylpyrrolidone (PVP) with potential applications in wound treatment. While the semi-natural polymer insures the biocompatibility and biodegradability of the synthesized materials, the synthetic polymer was selected due to its water affinity and interesting mechanical properties. The efficiency of the polymerization process and the stability of the PVP within the semi interpenetrated polymer network (semiIPN) were verified through gel fraction study. The water affinity, tensile strength and rheological properties of the hydrogels were also investigated.

High throughput micro-well generation of hepatocyte micro-aggregates for tissue engineering.

The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

Immunocompatibility evaluation of hydrogel‐coated polyimide implants for applications in regenerative medicine

Immunocompatibility of gelatin-based hydrogels to be applied as implant coatings for local regenerative treatment has been studied. First, the bio- and immuno-acceptability of the methacrylamide-modified gelatin hydrogels per se was screened. The results indicated that the hydrogels support cell growth. Metabolic activity of normal cells and permanent cell lines representing various cell types (endothelial, epithelial, fibroblast, and monocyte/macrophage) cultivated on the gelatin hydrogels was moderately lower compared to cells cultivated on tissue culture plastic. The cells cultivated on the hydrogels produced identical cytokines as the control cells although at lower levels. Importantly, no inflammatory activity, measured by nitric oxide and pro-inflammatory cytokine (IL-1α, IL-6, and TNFα) production, was observed in peritoneal cells and monocyte/macrophage RAW 264.7 cell line cultivated on the hydrogels. Finally, polyimide (PI) implantable membranes were surface-modified with gelatin hydrogels and screened for their in vivo immunocompatibility. Their histological examination performed after subcutaneous implantation in mice produced a sound proof of immunoacceptability. Normal tissue repair, mild cellular infiltration and edema mainly induced by the surgery were observed after 2 and 6 days. No adverse tissue responses were induced by the implants. Analysis performed after 4 and 9 weeks indicated areas of foreign body granuloma without formation of a fibrous capsule.

Impact of Endothelial Cells on 3D Cultured Smooth Muscle Cells in a Biomimetic Hydrogel

For the development of vascular tissue engineering, the impact of endothelial cells (ECs) on smooth muscle cell (SMC) spreading, proliferation, and differentiation is explored in the current study using a coculture model. In this coculture model, SMCs were encapsulated in a biomimetic hydrogel based on methacrylated dextran-graft-lysine (Dex-MA-LA) and methacrylamide-modified gelatin (Gel-MA), and exposed to a monolayer of ECs. With EC coculture, SMC proliferation in 3D hydrogel was promoted at initial period, and the formation of denser cellular networks was enhanced. ECs dynamically modulated SMC phenotype by promoting a more contractile SMC phenotype initially (on day 2), indicated by the upregulated expression of contractile genes α-actin, calponin, smooth muscle-myosin heavy chain (SM-MHC), and smoothelin; however, the onset of maximum expressions was delayed by ECs. Full differentiation of SMCs was not obtained even with EC coculture. Higher level of platelet-derived growth factor (PDGF)-BB and latent transforming growth factor (TGF)-β1 were detected in medium of coculture. These biochemical cues together with the physical cue of tensional force within cellular networks may be responsible for the dynamic modulation of SMC phenotype in coculture. Synthesis of elastin was promoted by ECs at transcriptional level. The formation of denser cellular networks and synthesis of elastin suggest that coculture with ECs is a potential method to construct functional vessel media layer in vitro.

Novel gelatin–PHEMA porous scaffolds for tissue engineering applications

In the present work, novel bicomponent polymeric hydrogels based on methacrylamide-modified gelatin (MAG) and 2-hydroxyethyl methacrylate (HEMA) have been prepared by cross-linking polymerization using photoinitiation. Five types of novel hydrogels have been prepared using different MAG/HEMA ratios between 1/0.5 and 1/10 w/w. Subsequently, porous scaffolds were obtained via a cryogenic treatment followed by freeze-drying. Physico-chemical measurements as well as in vitro degradation tests have been performed in order to correlate the material composition with the corresponding properties. Among the properties studied we have to mention the water uptake capacity, the rheological properties and the enzyme-mediated degradation behaviour. The results indicate that the HEMA content in the initial polymerization mixtures modulates the architecture of the porous scaffolds from straightforward, top-to-bottom oriented channels for hydrogels possessing the lowest HEMA content to a complex and dense internal porosity of the channels the case of higher HEMA loaded materials. While aiming at tissue engineering applications, it is important to notice that the covalently bound gelatin sequences significantly improve the biocompatibility of PHEMA based hydrogels.

Laser Fabrication of Three-Dimensional CAD Scaffolds from Photosensitive Gelatin for Applications in Tissue Engineering

In the present work, 3D CAD scaffolds for tissue engineering applications were developed starting from methacrylamide-modified gelatin (GelMOD) using two-photon polymerization (2PP). The scaffolds were cross-linked employing the biocompatible photoinitiator Irgacure 2959. Because gelatin is derived from collagen (i.e., the main constituent of the ECM), the developed materials mimic the cellular microenvironment from a chemical point of view. In addition, by applying the 2PP technique, structural properties of the cellular microenvironment can also be mimicked. Furthermore, in vitro degradation assays indicated that the enzymatic degradation capability of gelatin is preserved for the methacrylamide-modified derivative. An in depth morphological analysis of the 2PP-fabricated scaffolds demonstrated that the parameters of the CAD model are reproduced with great precision, including the ridge-like surface topography on the order of 1.5 μm. The developed scaffolds showed an excellent stability in culture medium. In a final part of the present work, the suitability of the developed scaffolds for tissue engineering applications was verified. The results indicated that the applied materials are suitable to support porcine mesenchymal stem cell adhesion and subsequent proliferation. Upon applying osteogenic stimulation, the seeded cells differentiated into the anticipated lineage. Energy dispersive X-ray (EDX) analysis showed the induced calcification of the scaffolds. The results clearly indicate that 2PP is capable of manufacturing precisely constructed 3D tissue engineering scaffolds using photosensitive polymers as starting material.

Effect of Cryogenic Treatment on the Rheological Properties of Gelatin Hydrogels

Gelatin has the ability to form a gel when cooled below the sol—gel temperature. In this study, the effects of various cryo-parameters, including the number of freeze—thaw cycles, cooling rate, thawing rate, and gelatin concentration, on the material properties were examined. The rheological properties of the cryogels improved with increasing cryo-cycles and decreasing cooling and thawing rates as well as were superior to those of the corresponding hydrogels formed at room temperature. In addition, the critical gelation concentration decreased after repeated cryo-treatments. Methacrylamide-modified gelatin was also treated cryogenically, followed by in situ UV irradiation to enable radical cross-linking. The cross-linking efficiency of specific gelatin concentrations improved with freeze—thawing. Cryogelation can be used to fine-tune the mechanical properties of hydrogels. This is of relevance to tissue engineering where porous gelatin hydrogels are used as biomaterials.

Comparative study of silanisation reactions for the biofunctionalisation of Ti-surfaces

In the present work, we report on the biofunctionalisation of silanised Ti-surfaces with gelatin. In recent years, a large number of papers have been published about the silanisation of different substrates including Ti-surfaces for biomedical applications. However, a comparative study evaluating the effects of different pre-treatment methods (cleaning and/or oxidation) and the efficiency of different silanisation reactions has to our information not yet been published. Since a clean and reproducible surface is required for studying structure–property relations, all Ti-surfaces were subjected to a three step procedure including a cleaning step, an oxidation step and a silanisation step. XPS analysis and contact angle measurements revealed that the pre-treatment of the Ti samples (cleaning + oxidation) had a drastic effect both on the surface composition and its wettability. A detailed study on the effect of different silanisation parameters indicated that the siloxane concentration, the siloxane type, the solvent and a catalyst affect the coupling efficiency of siloxanes to Ti-surfaces. Stability studies revealed a dependency between the siloxane type and the stability of the siloxane coating against hydrolytic cleavage from a Ti-surface. Since Ti-surfaces modified with a methacrylate containing silane possessed the highest hydrolytic stability, these surfaces were selected for the subsequent immobilisation of methacrylamide-modified gelatin via high energy irradiation induced cross-linking. The present work clearly demonstrates the need of a proper reaction strategy for immobilising ligands on Ti-surfaces.