Next to the classic tissue engineering (TE) approach of seeding cells onto a preexisting scaffold, bioprinting is getting more and more attention these days as a method to combine tissue specific cells and suitable biomaterials. Benefits over the classic TE approaches include the controlled positioning of cells and matrix components and the possibility to create space-resolved geometries to obtain functional units representing the characteristics of the natural tissue . Additionally, tissue equivalents with patient specific dimensions can be generated, turning bioprinting into a potential future method for tissue regeneration. A crucial point in the bioprinting process is the selection of a suitable base material for the bioink which is on the one hand processable with the respective printing system and on the other hand generates tissue equivalents which support encapsulated cells in their functionality and differentiation process.
A material that was already proven to be very suitable as a basis for bioprinting approaches is methacrylated gelatin (GM) , , , , . Functionalized with photo-curable methacrylic groups, the gelatin molecules can be crosslinked to form a hydrogel that is highly cytocompatible due to its collagenous origin and stays stable under culture conditions . Additionally, the properties of the inks concerning their crosslinking potential and viscosity can be influenced easily by varying the polymer content as well as the degree of methacrylation and masking of side groups , . For specific applications, the gelatin-based inks can be modified by the addition of further components which support a specific cell type or generate the desired matrix properties.
Since hydroxyapatite (HAp) forms the principal component of the anorganic bone phase, representing 50%–70% of the bone’s dry weight , extensive research has been done by several authors on the subject of introducing a mineral phase into the scaffold structures used for bone TE. A beneficial effect of a mineral phase onto the osteogenic differentiation of stem cells was found, especially when being available as nanostructures, resembling the appearance of the mineral phase in the natural bone , . With the composite approach, two main strategies are used, the biomimetic mineralization of a natural or synthetic scaffold material by incubation in a simulated body fluid containing a mixture of calcium and phosphate ions , , , and the introduction of mineral particles into the hydrogel solution, most often in the form of HAp , . Investigated approaches range from the use of porous scaffolds made of HAp  or tricalciumphosphates (TCP) to the fabrication of composite scaffolds consisting of hydrogels and anorganic particles [reviewed in ).
In the present study, we systematically analysed GM-based bioinks containing varying amounts of HAp particles in respect of their cytocompatibility and processability with a pneumatic bioprinting system, and the mechanical properties of the respective hydrogels. In a proof of concept, human adipose-derived stem cells (hASCs) were printed using different inks and encapsulated in 3D hydrogels by UV induced crosslinking.
For the modification of GM-based bioinks for the printing of bone tissue equivalents, the use of HAp particles on the one hand provides the opportunity to improve the ink properties for the processing with extrusion-based printing systems, and on the other hand introduces cues which can support the process of osteogenic differentiation. For the processabilty of the inks with extrusion-based systems, their viscosity plays an important role and needs to be considerably higher than for the use with inkjet-based systems , . The viscosities of GM-inks spiked with various amounts of HAp particles were systematically analysed. The mean viscosities determined at a constant shear rate of 500 s−1 were significantly increased by the addition of HAp up to 67.3±2.22 mPa s compared to the pure GM with 28.5±0.04 mPa s (Figure 1A). Additionally, over the range of shear rates from 0 to 1000 s−1, a tendency towards a shear-thinning behavior was found in the HAp-containing inks in comparison to the Newtonian behavior of the pure GM-ink (data not shown). This effect was also found by other authors  and is seen as a desired ink property for direct ink writing applications . Those results show that the processability of the inks by extrusion-based printing processes is improved by HAp particles and the viscosity lays in the range of 30–107 mPa s which is considered to be suitable for dispensing .
The influence of the added HAp on the mechanical properties of the gels was assessed by measuring the storage moduli G′ and the loss moduli G″ of the hydrogels. The results, depicted in Figure 1B, show that with increasing amounts of HAp the storage module of the hydrogels is significantly increased from 49.8±1.50 kPa in the pure GM-gels to 62.2±2.51 kPa and 69.6±1.47 kPa in the gels with 20% and 40% HAp, respectively. The addition of 60% HAp led to a storage module of 72.6±3.28 kPa. The respective loss moduli of the gels increased significantly from 1.5±0.13 kPa in the pure GM-gels to 5.3±0.46 kPa in the gels with 60% HAp, showing a similar trend as the storage moduli. Since several studies indicated a dependence of osteogenic differentiation from matrix stiffness, with high rates of osteogenesis being found on and in stiff materials which resemble the natural bone tissue , , HAp-modified materials are promising candidates for the use in bone TE.
To analyze the cytocompatibilty of the used photoinitiator, the irradiation parameters and the HAp nanoparticles in the hydrogel system, cytotoxicity assays according to DIN EN ISO 10993-5 were conducted. For the photopolymerization of the hydrogels in this study, a photoinitiator–lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) – was used that was described before as an alternative to the commonly used Irgacure® 2959 due to its better water-solubility, improvement in resulting polymerization kinetics and absorption spectrum at longer wavelengths , . For the assessment of its cytocompatibility, an experimental approach was used reflecting the irradiation process during hydrogel formation. For this purpose, hASCs were resuspended in a solution of unmodified gelatin, LAP was added in concentrations ranging from 0 to 0.8 mg mL−1 and UVA-irradiation took place for either 60 or 120 s. A control was left non-irradiated. As expected, metabolic activity of the cells decreased with increasing concentrations of LAP and increased irradiation time, as shown in Figure 2A. Acceptance criterion for cytocompatibility was set to >70% of the non-irradiated control without LAP, according to DIN EN ISO 10993-5, indicated by the green line in Figure 2A. For the non-irradiated control, cell vitality hardly fell below that value, even for the highest concentration of LAP at 0.8 mg mL−1. Irradiation of the samples led to a stronger decline in vitality, especially for LAP concentrations above 0.4 mg mL−1. UV exposure for 120 s led to lower vitality rates compared to 60 s. Concentrations of 0.2 mg mL−1 LAP and less resulted in a cell vitality of more than 70% for all conditions, thus meeting the requirements for cytocompatibility. For cells from all three donors tested, a concentration of 0.2 mg mL−1 was shown to be cytocompatible, even with the longer UV exposure of 120 s. Based on these findings, cytocompatible concentrations for the application in subsequent experiments were chosen, and the hydrogels were cured for 120 s. The results produced in this study using hASCs complement the findings of Fairbanks et al. who showed the photoinitiator LAP to be compatible with fibroblasts in similar ranges of LAP-concentration and under comparable crosslinking conditions .
The assessment of the HAp particles’ cytocompatibility was conducted similarly, also resembling an encapsulated state of the cells. hASCs from three donors with photoinitiator-concentrations and irradiation parameters proven to be cytocompatible beforehand were used. The cells were encapsulated in GM containing 0%–60% HAp particles, and cell viability was assessed after 24 h of culture. Figure 2B shows that the HAp-fractions of 20% and 40% of polymer mass resulted in mean viabilities of 77% and 86% of the control, respectively, and were thus above the threshold for cytocompatibilty that is set to 70% in DIN ISO 10993-5. However, upon increasing the HAp-amount to 60%, the mean viability of all donors dropped to 67%. One of the tested donors (donor 3), though, seemed to be more susceptible to the HAp, resulting in considerably lower values of viability for all tested HAp-concentrations, with only the hydrogels with 40% HAp reaching a viability higher than 70%. The cytocompatility of HAp was evaluated before with different cell types, and the published results were contradictory. Some studies found cytotoxic effects of HAp particles at concentrations in the range of the ones used in this study , while other authors found no significant cytotoxic effects when using up to 15-fold higher concentrations . Explanations for these differences in results might on the one hand be the influence of particle size on cytocompatibility of HAp particels , , and on the other hand there seems to be a correlation between the amount of particle uptake by the cells and the observed cytotoxicity , both parameters not being analyzed here.
Since in our studies the bioink with 40% HAp proved to be most suitable for the preparation of hydrogels with regard to cytocompatibility as well as mechanical gel properties, it was subsequently used for the building of cell-laden hydrogels via bioprinting. Multilayer constructs consisting of either pure GM-ink (15 wt%, control) or GM-ink+40% HAp were build up by deposition of six circular layers per construct (Figure 3A) and the use of irradiation parameters proven to be cytocompatible before, and were afterwards cultured for 5 days in stem cell culture medium. The constructs were analyzed with respect to the distribution of HAp particles, as well as for cell distribution directly after printing and on day 5. The printing process resulted in homogeneous round constructs, which showed a slightly concaved profile due to the surface tension of the ink (Figure 3B). The distribution of HAp-particles in the gels could be shown to be homogeneous over the whole height of the gel’s cross-section, with only few particle aggregates, depicted with black arrows (Figure 3C). Directly after printing the constructs, as well as after 5 days of culture (Figure 3D), cells could be detected in the gels, being distributed relatively homogenously in the gel, with few cell aggregates which could also be detected on day 1 (not shown).
In this study, we successfully modified a GM-based bioink for the bioprinting of bone tissue equivalents with HAp particles, and we could show improved properties of the bioink concerning processability with extrusion-based manufacturing methods, as well as of the mechanical properties of the resulting hydrogels. Additionally, the cytocompatibility of the used photopolymerization parameters and the used HAp were proven, and cytocompatible ranges were evaluated. The successful buildup of cell-laden hydrogels with bioprinting and their stability under physiologic conditions were shown. This now enables further work on the development of an actual bone tissue equivalent which requires the encapsulation of bone cell precursors and their osteogenic differentiation, which is expected to be strongly supported by the developed biomimetic matrix.
The authors thank the Fraunhofer-Gesellschaft (München), and the Carl Zeiss Stiftung (Stuttgart) for financial support.
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About the article
Published Online: 2016-05-06
Published in Print: 2016-09-01
Author’s statementConflict of interest: Authors state no conflict of interest.
Materials and methodsInformed consent: Informed consent has been obtained from all individuals included in this study.
Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the authors’ institutional review board or equivalent committee.