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Volume 17, Issue 3-4

Issues

Hydroxyapatite-modified gelatin bioinks for bone bioprinting

Annika Wenz
  • Institute of Interfacial Process Engineering and Plasma Technology IGVP, University of Stuttgart, Stuttgart, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Katharina Janke / Eva Hoch
  • Institute of Interfacial Process Engineering and Plasma Technology IGVP, University of Stuttgart, Stuttgart, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Günter E.M. Tovar
  • Institute of Interfacial Process Engineering and Plasma Technology IGVP, University of Stuttgart, Stuttgart, Germany
  • Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Kirsten Borchers / Petra J. Kluger
  • Corresponding author
  • Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany
  • Reutlingen University, Process Analysis and Technology, Reutlingen, Germany
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2016-05-06 | DOI: https://doi.org/10.1515/bnm-2015-0018

Abstract

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.

Keywords: additive manufacturing; mineralization; osteoblast differentiation; tissue engineering

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 [1]. 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) [2], [3], [4], [5], [6]. 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 [7]. 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 [8], [9]. 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 [10], 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 [11], [12]. 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 [13], [14], [15], and the introduction of mineral particles into the hydrogel solution, most often in the form of HAp [16], [17]. Investigated approaches range from the use of porous scaffolds made of HAp [18] or tricalciumphosphates (TCP) to the fabrication of composite scaffolds consisting of hydrogels and anorganic particles [reviewed in [19]).

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 [4], [20]. 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 [16] and is seen as a desired ink property for direct ink writing applications [21]. 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 [20].

Characterization of the bioinks and hydrogels. A GM derivative was synthesized with a five-fold molar excess of methacrylic anhydride as described by Hoch et al. [9]; a solution of 15 wt% GM in phosphate-buffered saline with divalent cations (PBS+) was made and different amounts of HAp particles (0%–60% of polymer mass; purchased from Sigma-Aldrich, Germany) were added. The suspensions’ viscosities were measured with a rotary concentric cylinder rheometer (Physica Modular Compact 5 MCR301 Rheometer, Anton Paar, Germany). Probes of HAp-containing GM hydrogels were prepared by dissolving GM (15 wt%) and the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, Fraunhofer IGB, Germany) (0.17% w/w to polymer mass) in PBS+ and adding different amounts of HAp (percent of dry weight of polymer). The GM-HAp-solutions were photo-crosslinked in an irradiation chamber (UVA, 365 nm; Hartmann Feinwerkbau GmbH, Germany) at 9.0 mW cm−2 for 120 s, and circular hydrogels with a radius of 3 mm were punched out. The gels were incubated in PBS at 37 °C for 48 h, and afterwards oscillatory dynamic measurements were conducted with a parallel plate model (Physica Modular Compact 5 MCR301 Rheometer, Anton Paar, Germany). (A) Mean viscosities of the different inks, measured at a constant shear rate of 500 s−1 at 25 °C (n=20 over 120 s). *Statistically significant differences between mean values (p≤0.001), calculated with one-way ANOVA and Tukey post hoc test (OriginPro 9.1). (B) Storage moduli G′ (dark gray) and loss moduli G″ (light gray) of the differently modified GM gels (5≤n≤7), measured at a normal force FN=10 N and a frequency f=1.5 Hz. *,#Statistically significant differences between mean values (p≤0.001), calculated with one-way ANOVA and Tukey post hoc test (OriginPro 9.1).
Figure 1:

Characterization of the bioinks and hydrogels.

A GM derivative was synthesized with a five-fold molar excess of methacrylic anhydride as described by Hoch et al. [9]; a solution of 15 wt% GM in phosphate-buffered saline with divalent cations (PBS+) was made and different amounts of HAp particles (0%–60% of polymer mass; purchased from Sigma-Aldrich, Germany) were added. The suspensions’ viscosities were measured with a rotary concentric cylinder rheometer (Physica Modular Compact 5 MCR301 Rheometer, Anton Paar, Germany). Probes of HAp-containing GM hydrogels were prepared by dissolving GM (15 wt%) and the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, Fraunhofer IGB, Germany) (0.17% w/w to polymer mass) in PBS+ and adding different amounts of HAp (percent of dry weight of polymer). The GM-HAp-solutions were photo-crosslinked in an irradiation chamber (UVA, 365 nm; Hartmann Feinwerkbau GmbH, Germany) at 9.0 mW cm−2 for 120 s, and circular hydrogels with a radius of 3 mm were punched out. The gels were incubated in PBS at 37 °C for 48 h, and afterwards oscillatory dynamic measurements were conducted with a parallel plate model (Physica Modular Compact 5 MCR301 Rheometer, Anton Paar, Germany). (A) Mean viscosities of the different inks, measured at a constant shear rate of 500 s−1 at 25 °C (n=20 over 120 s). *Statistically significant differences between mean values (p≤0.001), calculated with one-way ANOVA and Tukey post hoc test (OriginPro 9.1). (B) Storage moduli G′ (dark gray) and loss moduli G″ (light gray) of the differently modified GM gels (5≤n≤7), measured at a normal force FN=10 N and a frequency f=1.5 Hz. *,#Statistically significant differences between mean values (p≤0.001), calculated with one-way ANOVA and Tukey post hoc test (OriginPro 9.1).

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 [22], [23], 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 [24], [25]. 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 [25].

Cytocompatibility of photoinitiator and HAp particles. For cytotoxicity testing of the photoinitiator LAP, hASCs from subcutaneous fatty tissue (n=3 donors) were harvested in passage 2, resuspended in a solution of unmodified gelatin (5 wt%) at a concentration of 1.25×106 cells mL−1, and the suspension was pipetted into well plates. LAP resolved in PBS+ was added to the cells in triplicates, resulting in LAP-concentrations from 0 to 0.8 mg mL−1 in relation to the total volume. Afterwards, the plates were irradiated (365 nm; 9.0 mW cm−2) for 60 or 120 s. A control plate was not irradiated at all. After the addition of cell culture media to the wells, the cells were cultured for 24 h under standard culture conditions (37 °C, 5% CO2). After 24 h, the vitality of the cells was assessed with an MTS assay using the CellTiter 96® AQueous One Solution Reagent (Promega, Germany), and the resulting absorbance was measured with a microplate reader (Tecan infinite M200PRO, Tecan Deutschland GmbH, Germany). HAp-containing GM-solutions were prepared by dissolving GM (15 wt%) and LAP (0.17% w/w to polymeric content) in PBS+ and adding different amounts of HAp (Sigma-Aldrich; 0%–60% of dry weight of polymer; particle size <200 nm). hASCs from three different donors were harvested in passage 2 and resuspended in the solutions at a concentration of 2.0×106 cells mL−1. The suspensions were pipetted into wellplates in triplicates, and the hydrogels were polymerized by UVA irradiation (365 nm; 9.0 mW cm−2 for 2 min). The cells were cultured in DMEM+1% FSC at 37 °C for 24 h. Afterwards, an MTT assay was conducted and the formed MTT crystals were eluted with isopropanol and quantified with a microplate reader. (A) Results of the evaluation of photoinitiator cytocompatibility in dependence of irradiation time. The viability is shown as percent of non-irradiated control cells without LAP-addition. Green line: threshold for cytocompatibility at 70%. (B) Results of the cytocompatibility evaluation of the used HAp-particles. The cell viability is depicted as percentage of the control (pure GM). Green line: threshold for cytocompatibility at 70%.
Figure 2:

Cytocompatibility of photoinitiator and HAp particles.

For cytotoxicity testing of the photoinitiator LAP, hASCs from subcutaneous fatty tissue (n=3 donors) were harvested in passage 2, resuspended in a solution of unmodified gelatin (5 wt%) at a concentration of 1.25×106 cells mL−1, and the suspension was pipetted into well plates. LAP resolved in PBS+ was added to the cells in triplicates, resulting in LAP-concentrations from 0 to 0.8 mg mL−1 in relation to the total volume. Afterwards, the plates were irradiated (365 nm; 9.0 mW cm−2) for 60 or 120 s. A control plate was not irradiated at all. After the addition of cell culture media to the wells, the cells were cultured for 24 h under standard culture conditions (37 °C, 5% CO2). After 24 h, the vitality of the cells was assessed with an MTS assay using the CellTiter 96® AQueous One Solution Reagent (Promega, Germany), and the resulting absorbance was measured with a microplate reader (Tecan infinite M200PRO, Tecan Deutschland GmbH, Germany). HAp-containing GM-solutions were prepared by dissolving GM (15 wt%) and LAP (0.17% w/w to polymeric content) in PBS+ and adding different amounts of HAp (Sigma-Aldrich; 0%–60% of dry weight of polymer; particle size <200 nm). hASCs from three different donors were harvested in passage 2 and resuspended in the solutions at a concentration of 2.0×106 cells mL−1. The suspensions were pipetted into wellplates in triplicates, and the hydrogels were polymerized by UVA irradiation (365 nm; 9.0 mW cm−2 for 2 min). The cells were cultured in DMEM+1% FSC at 37 °C for 24 h. Afterwards, an MTT assay was conducted and the formed MTT crystals were eluted with isopropanol and quantified with a microplate reader. (A) Results of the evaluation of photoinitiator cytocompatibility in dependence of irradiation time. The viability is shown as percent of non-irradiated control cells without LAP-addition. Green line: threshold for cytocompatibility at 70%. (B) Results of the cytocompatibility evaluation of the used HAp-particles. The cell viability is depicted as percentage of the control (pure GM). Green line: threshold for cytocompatibility at 70%.

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 [26], while other authors found no significant cytotoxic effects when using up to 15-fold higher concentrations [17]. Explanations for these differences in results might on the one hand be the influence of particle size on cytocompatibility of HAp particels [27], [28], and on the other hand there seems to be a correlation between the amount of particle uptake by the cells and the observed cytotoxicity [26], 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).

Encapsulation of hASCs in HAp-containing hydrogels by bioprinting. hASCs from subcutaneous fatty tissue (n=3 donors) were harvested in passage 2 and resuspended at 5.0×106 cells mL−1 in HAp-containing GM-solutions prepared by dissolving GM (15 wt%) and LAP (0.17% w/w to polymeric content) in PBS+, and adding HAp (40% of dry weight of polymer). With an extrusion-based bioprinting system (Unitechnologies SA, Switzerland), hydrogels were fabricated layer by layer, using a needle with an inner diameter of 300 μm and a flow rate of 0.32 mm3 s−1. Each layer was irradiated with UVA light (365 nm) for 10 s with an additional 20 s irradiation step in the end. Afterwards, the cell-laden hydrogels were cultured in supplemented MSCGM medium (Lonza, Switzerland) for 5 days at 37 °C. For analysis on day 5, the gels were fixed, paraffin-embedded, sectioned, and the sections were stained with hematoxylin and eosin (H&E), as well as with Alizarin red S (Sigma, Germany). (A) Printing of cell-laden HAp-containing hydrogels with an extrusion-based approach. (B) Macroscopic pictures of printed HAp-containing (left) and pure GM hydrogels (right). Scale: 5 mm. (C) Histological cross-section of a HAp-containing hydrogel, stained for calcium-ions with Alizarin Red S (red). Arrows mark HAp-aggregates. (D) Histological cross-section of a HAp-containing hydrogel, cultured for 5 days and afterwards stained with H&E, showing cell nuclei (blue) and the cells’ cytoplasm (pink).
Figure 3:

Encapsulation of hASCs in HAp-containing hydrogels by bioprinting.

hASCs from subcutaneous fatty tissue (n=3 donors) were harvested in passage 2 and resuspended at 5.0×106 cells mL−1 in HAp-containing GM-solutions prepared by dissolving GM (15 wt%) and LAP (0.17% w/w to polymeric content) in PBS+, and adding HAp (40% of dry weight of polymer). With an extrusion-based bioprinting system (Unitechnologies SA, Switzerland), hydrogels were fabricated layer by layer, using a needle with an inner diameter of 300 μm and a flow rate of 0.32 mm3 s−1. Each layer was irradiated with UVA light (365 nm) for 10 s with an additional 20 s irradiation step in the end. Afterwards, the cell-laden hydrogels were cultured in supplemented MSCGM medium (Lonza, Switzerland) for 5 days at 37 °C. For analysis on day 5, the gels were fixed, paraffin-embedded, sectioned, and the sections were stained with hematoxylin and eosin (H&E), as well as with Alizarin red S (Sigma, Germany). (A) Printing of cell-laden HAp-containing hydrogels with an extrusion-based approach. (B) Macroscopic pictures of printed HAp-containing (left) and pure GM hydrogels (right). Scale: 5 mm. (C) Histological cross-section of a HAp-containing hydrogel, stained for calcium-ions with Alizarin Red S (red). Arrows mark HAp-aggregates. (D) Histological cross-section of a HAp-containing hydrogel, cultured for 5 days and afterwards stained with H&E, showing cell nuclei (blue) and the cells’ cytoplasm (pink).

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.

Acknowledgments

The authors thank the Fraunhofer-Gesellschaft (München), and the Carl Zeiss Stiftung (Stuttgart) for financial support.

References

  • 1.

    Wüst S, Müller R, Hofmann S. Controlled positioning of cells in biomaterials – approaches towards 3d tissue printing. J Funct Biomater. 2011;2:119. Google Scholar

  • 2.

    Nichol JW, Koshy ST, Bae H, Hwang CM, Yamanlar S, Khademhosseini A. Cell-laden microengineered gelatin methacrylate hydrogels. Biomaterials. 2010;31:5536–44. Google Scholar

  • 3.

    Chen Y-C, Lin R-Z, Qi H, Yang Y, Bae H, Melero-Martin JM, et al. Functional human vascular network generated in photocrosslinkable gelatin methacrylate hydrogels. Adv Funct Mater. 2012;22:2027–39. Google Scholar

  • 4.

    Bertassoni LE, Cardoso JC, Manoharan V, Cristino AL, Bhise NS, Araujo WA, et al. Direct-write bioprinting of cell-laden methacrylated gelatin hydrogels. Biofabrication. 2014;6:024105. Google Scholar

  • 5.

    Bertassoni LE, Cecconi M, Manoharan V, Nikkhah M, Hjortnaes J, Cristino AL, et al. Hydrogel bioprinted microchannel networks for vascularization of tissue engineering constructs. Lab Chip. 2014;14:2202–11. Google Scholar

  • 6.

    Billiet T, Gevaert E, De Schryver T, Cornelissen M, Dubruel P. The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability. Biomaterials. 2014;35:49–62. Google Scholar

  • 7.

    Van Den Bulcke AI, Bogdanov B, De Rooze N, Schacht EH, Cornelissen M, Berghmans H. Structural and rheological properties of methacrylamide modified gelatin hydrogels. Biomacromolecules. 2000;1:31–8. Google Scholar

  • 8.

    Hoch E, Schuh C, Hirth T, Tovar GE, Borchers K. Stiff gelatin hydrogels can be photo-chemically synthesized from low viscous gelatin solutions using molecularly functionalized gelatin with a high degree of methacrylation. J Mater Sci Mater Med. 2012;23:2607–17. Google Scholar

  • 9.

    Hoch E, Hirth T, Tovar GE, Borchers K. Chemical tailoring of gelatin to adjust its chemical and physical properties for functional bioprinting. J Mater Chem B. 2013;1:5675–85. Google Scholar

  • 10.

    Clarke B. Normal bone anatomy and physiology. Clin J Am Soc Nephrol. 2008;3(Suppl 3):S131–9. CrossrefGoogle Scholar

  • 11.

    Shu R, McMullen R, Baumann MJ, McCabe LR. Hydroxyapatite accelerates differentiation and suppresses growth of MC3T3-E1 osteoblasts. J Biomed Mater Res A. 2003;67:1196–204. Google Scholar

  • 12.

    Sahoo NG, Pan YZ, Li L, He CB. Nanocomposites for bone tissue regeneration. Nanomedicine (Lond). 2013;8:639–53. Google Scholar

  • 13.

    Chang MC, Ko C-C, Douglas WH. Preparation of hydroxyapatite-gelatin nanocomposite. Biomaterials. 2003;24:2853–62. Google Scholar

  • 14.

    Kim H-W, Kim H-E, Salih V. Stimulation of osteoblast responses to biomimetic nanocomposites of gelatin–hydroxyapatite for tissue engineering scaffolds. Biomaterials. 2005;26:5221–30. Google Scholar

  • 15.

    Kang H, Shih Y-R, Hwang Y, Wen C, Rao V, Seo T, et al. Mineralized gelatin methacrylate-based matrices induce osteogenic differentiation of human induced pluripotent stem cells. Acta Biomater. 2014;10:4961–70. Google Scholar

  • 16.

    Wust S, Godla ME, Muller R, Hofmann S. Tunable hydrogel composite with two-step processing in combination with innovative hardware upgrade for cell-based three-dimensional bioprinting. Acta Biomater. 2014;10:630–40. Google Scholar

  • 17.

    Sadat-Shojai M, Khorasani MT, Jamshidi A. 3-Dimensional cell-laden nano-hydroxyapatite/protein hydrogels for bone regeneration applications. Mater Sci Eng C Mater Biol Appl. 2015;49:835–43. Google Scholar

  • 18.

    Dessi M, Alvarez-Perez MA, De Santis R, Ginebra MP, Planell JA, Ambrosio L. Bioactivation of calcium deficient hydroxyapatite with foamed gelatin gel. A new injectable self-setting bone analogue. J Mater Sci Mater Med. 2014;25:283–95. Google Scholar

  • 19.

    Michel J, Penna M, Kochen J, Cheung H. Recent advances in hydroxyapatite scaffolds containing mesenchymal stem cells. Stem Cells Int. 2015;13:2015. Google Scholar

  • 20.

    Chang CC, Boland ED, Williams SK, Hoying JB. Direct-write bioprinting three-dimensional biohybrid systems for future regenerative therapies. J Biomed Mater Res B Appl Biomater. 2011;98:160–70. Google Scholar

  • 21.

    Barry RA, Shepherd RF, Hanson JN, Nuzzo RG, Wiltzius P, Lewis, JA. Direct-write assembly of 3D hydrogel scaffolds for guided cell growth. Adv. Mater. 2009;21:2407–10. Google Scholar

  • 22.

    Chatterjee K, Lin-Gibson S, Wallace WE, Parekh SH, Lee YJ, Cicerone MT, et al. The effect of 3D hydrogel scaffold modulus on osteoblast differentiation and mineralization revealed by combinatorial screening. Biomaterials. 2010;31:5051–62. Google Scholar

  • 23.

    Nii M, Lai JH, Keeney M, Han LH, Behn A, Imanbayev G, et al. The effects of interactive mechanical and biochemical niche signaling on osteogenic differentiation of adipose-derived stem cells using combinatorial hydrogels. Acta Biomater. 2013;9:5475–83. Google Scholar

  • 24.

    Majima T, Schnabel W, Weber W. Phenyl-2,4,6-trimethylbenzoylphosphinates as water-soluble photoinitiators. Generation and reactivity of O=Ṗ(C6H5)(O) radical anions. Die Makromolekulare Chemie. 1991;192:2307–15. Google Scholar

  • 25.

    Fairbanks BD, Schwartz MP, Bowman CN, Anseth KS. Photoinitiated polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility. Biomaterials. 2009;30:6702–7. Google Scholar

  • 26.

    Motskin M, Wright DM, Muller K, Kyle N, Gard TG, Porter AE, et al. Hydroxyapatite nano and microparticles: correlation of particle properties with cytotoxicity and biostability. Biomaterials. 2009;30:3307–17. Google Scholar

  • 27.

    Cai Y, Liu Y, Yan W, Hu Q, Tao J, Zhang M, et al. Role of hydroxyapatite nanoparticle size in bone cell proliferation. J Mater Chem. 2007;17:3780–7. Google Scholar

  • 28.

    Yuan Y, Liu C, Qian J, Wang J, Zhang Y. Size-mediated cytotoxicity and apoptosis of hydroxyapatite nanoparticles in human hepatoma HepG2 cells. Biomaterials. 2010;31:730–40. Google Scholar

About the article

Corresponding author: Prof. Dr. Petra J. Kluger, Fraunhofer Institute for Interfacial Engineering and Biotechnology, Nobelstrasse 12, 70569 Stuttgart, Germany, Phone: +49 711 970-4072, Fax: +49 711 970-4200 and Reutlingen University, Process Analysis and Technology, Reutlingen, Germany


Received: 2015-10-30

Accepted: 2016-04-09

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.


Citation Information: BioNanoMaterials, Volume 17, Issue 3-4, Pages 179–184, ISSN (Online) 2193-066X, ISSN (Print) 2193-0651, DOI: https://doi.org/10.1515/bnm-2015-0018.

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[1]
Nadeschda Schmidt, Jennifer Schulze, Dawid P. Warwas, Nina Ehlert, Thomas Lenarz, Athanasia Warnecke, Peter Behrens, and Hélder A. Santos
PLOS ONE, 2018, Volume 13, Number 3, Page e0194778
[2]
Christiane Claaßen, Marc H. Claaßen, Vincent Truffault, Lisa Sewald, Günter E. M. Tovar, Kirsten Borchers, and Alexander Southan
Biomacromolecules, 2017
[3]
Annika Wenz, Kirsten Borchers, Günter E M Tovar, and Petra J Kluger
Biofabrication, 2017, Volume 9, Number 4, Page 044103

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