Hydrogels are hydrophilic materials containing a high amount of water. They are attractive as scaffolds for bone tissue engineering (TE), in spite of their poor mechanical properties [1, 2]. One important aspect in using hydrogels in bone tissue engineering is their mineralizability [1, 2]. Mineralization of hydrogels is desirable for bone TE applications in order to promote bioactivity, i.e., the formation of a chemical bond with surrounding bone tissue after implantation . Potential further advantages of mineralization are promotion of osteoblastic differentiation through increased stiffness [4–6] and enhanced binding of growth factors which stimulate bone healing .
The use of hydrogels opens the possibility of encapsulation of not only cells, but also biologically active molecules such as enzymes. Hydrogel mineralization can be achieved by addition of enzymes such as alkaline phosphatase (ALP) , which causes mineralization of bone by cleavage of phosphate from organic phosphate, thereby increasing the local phosphate concentration and enabling precipitation of insoluble phosphate salts. ALP has been added to hydrogel materials to induce their mineralization with calcium phosphate (CaP) during incubation in solutions containing calcium and glycerophosphate (GP), which serves as a substrate for ALP [8–14].
One strategy to improve hydrogel mineralization for bone regeneration applications is the incorporation of calcium-binding groups such as carboxyl groups to function as nucleation sites for CaP nanocrystal growth . One hydrogel material, well-known for its calcium-binding groups is the polysaccharide alginate . Alginate is a block-copolymer consisting of (1,4)-linked β-D-mannuronate (M) and α-L-guluronate (G) residues. The G-groups participate in gelation of alginate by binding with the divalent cation Ca2+ according to the so-called egg-box model . The name egg-box comes from the molecular structure of the crosslinked areas of the hydrogel, which resembles an egg-box.
The calcium-binding residues fulfill a double purpose, firstly to aid mineral formation and secondly to enable crosslinking of the alginate chains by divalent cations, e.g., Ca2+ ions , to form hydrogels. Crosslinking by divalent cations is a mild gelation process, which requires no addition of heat, energy intensive radiation, or toxic chemicals, and enables encapsulation of cells or proteins in the hydrogel structure by mixing them into the pre-crosslinked solution .
Cells or proteins immobilized alginate solutions can be extruded through a needle to form droplets, which are crosslinked to form beads upon immersion in a solution of divalent cations . Cell-loaded hydrogels have been used to produce 3D scaffolds with a highly developed internal architecture and well-defined pore geometry by rapid prototyping techniques, whereby structures are built up layer-by-layer using computer-aided design (CAD) [1, 17, 18]. Porous scaffolds consisting of alginate struts have been produced using a plotting technique, whereby alginate solution is extruded to form fibers, which are crosslinked upon submersion in a CaCl2 solution and thus stabilized [18, 19].
3D bioplotting of a suspension of pre-fabricated alginate capsules in alginate solution enables integration of capsules within struts, an approach which has remained relatively unexplored. Different components desirable for bone regeneration, e.g., cells and bioactive proteins, can be incorporated in the capsules and struts. This enables compartmentalization of components, which facilitates greater flexibility in modification of the scaffold.
The aim of this study was to produce scaffolds for possible applications in bone tissue engineering consisting of alginate struts containing alginate capsules enriched with MG-63 osteoblast-like cells and ALP using the technique of 3D bioplotting. Two different compartmentalization strategies were compared, namely cells in the capsules and ALP in the struts and vice-versa, the rationale being that the osteoblastic cells proliferate and build up new bone tissue whereas the ALP enzyme induces mineralization of the hydrogel scaffold to improve the mechanical properties and the cell response. In contrast to a previous paper in which alginate and an pre-formed inorganic phase (hydroxyapatite) was combined to form composite scaffolds, where a phase separation process was used , in this study the inorganic phase was formed in the fabricated scaffold structure.
The present study aimed to fill the following gaps in the scientific literature: i) 3D bioplotting of biphasic capsule-strut porous alginate hydrogel scaffolds with MG-63 and ALP compartmentalized in capsules and struts and ii) evaluation of cytocompatibility of compartmentalized hydrogels and their ability to support MG-63 cell growth with a view to their suitability as scaffolds for applications in bone tissue regeneration.
Materials and methods
Unless stated otherwise, all materials, including sodium alginate (71238) and ALP (P7640), were obtained from Sigma-Aldrichh (Germany).
Preparation of pure alginate solution and alginate solution containing ALP
For the plotting experiments (see Figures 5 and 6) a stock solution of 1.4% (w/v) alginate was prepared by dissolving the alginate in phosphate buffered saline (PBS) pre-heated to 40°C under stirring for 1.5 h at 50°C. A stock solution of 10 mg/mL ALP was prepared by adding ALP into PBS in a vessel in an ultrasonication bath for 30 min. The ALP solution was pipetted into the alginate solution under stirring for 30 min to enable homogenization. The final alginate-ALP solution had a ratio of 3:1 alginate to ALP (volume ratio of stock solutions).
For the experiments considering mitochondrial activity and cell viability (see Figure 7) the concentration of the alginate strut solution was varied and thus a 3% (w/v) solution was used.
In the current study MG-63 osteoblast-like cells (Sigma-Aldrich) were used for encapsulation and bioplotting. As cell cultivation medium, DMEM (Dulbecco’s modified Eagle’s medium, Gibco, Germany) supplemented with 10% (w/v) fetal bovine serum (FBS, Sigma-Aldrich, Germany), 1% (w/v) penicillin/streptomycin and 10 mM Glycerophosphate (GP) was used. Cultivation took place at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were cultured for 2–3 d until confluence was reached. After detaching of the cells by adding trypsin, a final work-concentration of 1 million cells per milliliter of alginate solution was adjusted.
Alginate capsule fabrication
For the fabrication of the capsules an extrusion system connected with a precision fluid dispenser (Ultimus V, Nordson EFD, Germany) was used. The extrusion system was compatible with different kinds of needles having various diameters. For the capsules consisting of 1.5% (w/v) alginate solution a needle with a diameter of 200 μm was used while for capsules out of 1.0% (w/v) alginate solution, used for the plotting experiments, a 100 μm needle was used. In Figure 1 a flow diagram shows the preparation process of the alginate solution, the capsule fabrication process and the sieving.
The alginate was dissolved by stirring, to which ALP or cells were added. The resulted alginate solution was filled into a syringe and extruded through a needle using pneumatic pressure and collected in a beaker containing 0.1 M CaCl2 solution. The resulted alginate capsules were kept for 10 min in the CaCl2 solution to allow ionic crosslinking of the alginate. Subsequently capsules were sieved using a sterilized sieve of grid width 500 μm and washed in DMEM culture medium.
Prior to addition to the alginate solution intended for strut production (see section on Alginate scaffold plotting), capsules were washed thoroughly in a beaker with DMEM without additives and sieved to remove residual CaCl2 solution, in order to prevent crosslinking of strut matrix with residual CaCl2 solution during plotting.
Alginate scaffold plotting
A 3D BioPlotter (GeSIM mbH, Germany) was used which enables the formation of 3D structures by controlled moving of a syringe filled with the plotting solution and combined with a precision needle. Capsules were mixed with the strut solution using a spatula. The concentration of the capsules in the strut solution, which was optimized in preliminary tests (data not shown) ranged between 33.3% and 50% (w/v).
The plotting solution was extruded through the needle by using pneumatic pressure. The scaffolds were plotted into a standard 6-well culture plates. In Figure 2 this process is shown schematically. The parameters for plotting alginate capsules embedded in alginate strut solutions were empirically optimized. The geometrical parameters are: edge length of the scaffold, number of struts, space between struts, height of struts and number of layers. The plotter parameters are pressure, speed, needle diameter. In Table 1 the empirically optimized parameters are listed together with the corresponding values.
After the plotting process the scaffolds were gelled with 0.1 M CaCl2 solution for 15–20 min, then rinsed with pure DMEM without additives and serum and finally immersed in cell cultivation medium and cultivated in an incubator. Cultivation took place at 37°C in a humidified atmosphere of 95% relative humidity and 5% CO2.
The cell behavior was investigated by light microscopy at different time points. Different fluorescence stainings were used to label the cells and the mineral phase formed. The cells were labeled with DAPI (Life Technologies, Germany) for the cell nucleus and Vybrant (Life Technologies, Germany) for the cell cytoplasm. An OsteoImage™ Mineralization Assay kit (Lonza, USA) was used to investigate the development of the mineral phase in the scaffolds. A fluorescence microscope (FM, Scope.A1, Carl Zeiss, Germany) was used to investigate the stained scaffolds.
The cell viability was revealed by staining with calceinAM (Life Technlogies, Germany) for live cells and propidium iodide for dead cells (Life Technologies) after 1 day of incubation in a plotted scaffold. The cell viability was calculated as the percentage of the ratio of live cells to the total amount of cells.
Mitochondrial activity was evaluated through the enzymatic conversion of tetrazolium (WST-8 assay, Sigma-Aldrich) salt to formazan. A solution of 1% WST-8 in medium was added to each sample. Each sample was incubated for 4 h. The absorbance at 450 nm was measured with a plate reader (type Phomo, Anthos Mikrosysteme GmbH, Germany).
Results and discussion
Additive manufacturing is an attractive biomaterial processing technology that can be used for the fabrication of TE constructs. By layer by layer plotting, this technology enables the production of parts with complex undercuts or intricate external and internal geometries . The use of additive manufacturing techniques like bioplotting has the advantage of a relatively free design of the scaffold structure with regard to the geometry as well as the positioning of different materials, capsules, cell types and bioactive substances. With this technique printing on cell and tissue level seems to be a promising approach for biofabrication [22, 23]. Alginate is widely used in cell encapsulation and biofabrication because of its rapid ionic gelation with divalent cations. In the present study, the plotting of encapsulated cells and the enzyme ALP as a novel biofabrication strategy could be shown for the first time by fabricating simplified model structures, however, as discussed further below optimization of the process is required to translate this approach to constructs of clinically relevant size.
Influence of cells and ALP on alginate capsule fabrication and cytocompatibility
Light microscopy images of cell-free and cell-loaded alginate capsules are shown in Figure 3A, C and 3B, D, respectively. Cells were evenly distributed within capsules. In this study, microcapsules of alginate ranging from 550 to 700 μm in diameter (Figure 3E) were fabricated by the extrusion method shown in Figure 1. Neither the presence of cells nor ALP influenced capsule diameter appreciably (Figure 3E). This suggests that capsules can be loaded with cells or bioactive substances (e.g., enzymes, growth factors) without significantly altering capsule dimensions. This in turn aids comparability of studies performed using different types of cells or bioactive substances. Live-dead staining of cells in ALP-free and ALP-loaded capsules (Figure 4) revealed no obvious negative influence of ALP on cell vitality. In a previous study the authors could show that MG-63 cells were viable in ALP-free pure alginate capsules for more than 21 days of cultivation . During this period no obvious degradation was observed. ALP has been used to mineralize a range of natural and synthetic hydrogels with calciumphosphate [9–13, 25]. However, there is a lack of studies in literature on the biological effects of ALP on cells encapsulated in hydrogels. Several hydrogel materials, including collagen, gellan gum (GG), oligo[poly(ethylene glycol) fumarate] (OPF) and catechol-poly(ethylene glycol) (cPEG) have been mineralized using the ALP concentration used in this study, namely 2.5 mg ALP/mL hydrogel [9–11]. The data in this study suggest that this concentration is cytocompatible. This paves the way for addition of both ALP and bone-forming cells to other hydrogels. As alginate is a suitable hydrogel for cell plotting, different strategies to improve the application of this polysaccharide are under investigation. For example, the degradation and cell attachment can be controlled by blending alginate with gelatin . Furthermore, in vitro and in vivo experiments showed improved biocompatibility of modified alginate by oxidation to alginate di-aldehyde . The addition of cell loaded capsules in this plotting process enables completely new combinations of materials, cells and active agents for biofabrication.
3D bioplotting of capsule-strut porous scaffolds
A light microscopy image of a bioplotted scaffold consisting of ALP-loaded struts and cell-loaded capsules is shown in Figure 5A. It is clear that cells are only observed in the capsules and not in the struts, demonstrating successful compartmentalization. Cells were evenly distributed within capsules. Staining and fluorescence microscopy imaging of scaffolds after 10 days of incubation revealed that mineralization had occurred and vital cells were still present inside the capsules (Figure 5B). Regarding bioplotting, it has been shown that embedding various cell types in hydrogels is a promising strategy in the field of biofabrication. Landers et al. published in 2002 for the first time the fabrication of cell-containing alginate scaffolds and up to now a number of studies followed in this context .
Conversely, in Figure 6A a light microscopy image of a plotted scaffolds consisting of cell-loaded struts and ALP-loaded capsules revealed that capsules were devoid of cells, showing compartmentalization. Cells were evenly distributed within struts. In Figure 6B staining and fluorescence microscopy imaging of scaffolds after 10 days revealed the presence of vital cells (red color). Furthermore, mineralization (green color) of pieces of degraded hydrogel structures was observed. However, only the degradation of the strut material occurred, the capsules remained unchanged. Mineralization may be due to the enzymatic action of ALP and/or cellular activity. It remains unclear to what extent ALP and cells are responsible for mineral formation in this system.
Vitality of cells in bioplotted capsule-strut porous scaffolds
Live/dead staining of cells after 1 day of culture in scaffolds consisting of ALP-loaded or ALP-free alginate capsules and cell-loaded alginate struts revealed that ALP did not influence cell vitality appreciably (Figure 7A). The cell viability measurements suggest that only limited cell damage occurred during plotting. It could be assumed that after 24 h, cells were able to withstand the mechanical stress, or they have the capability to recover from it . Beside plotting pressure, nozzle diameter and plotting speed, the concentration of the applied alginate is the most influential parameter. Khalil et al. showed that an alginate concentration of 1.5% (w/v) led into a cell viability of 83% after cell plotting . Their study focused on the optimization of the cell viability. Other studies investigating alginate as a processing material with the focus more on the geometry of the constructs used higher alginate concentrations up to 3.5% (w/v), as the higher alginate concentration increases the shape stability after the plotting process . WST assay results after 2, 4 and 7 days of culture showed a positive effect of ALP on mitochondrial activity at all three time points (Figure 7B), suggesting higher proliferation. The reasons for the enhanced mitochondrial activity in the presence of ALP are unclear and discussion must remain speculative.
ALP is known to induce mineralization of hydrogels with calciumphosphate. Indeed, mineralization of alginate after 10 days was observed in this study (Figures 5 and 6). Mineralization of hydrogels of the calcium-binding polysaccharide GG led to superior osteoblast adhesion and proliferation , while incorporation of calciumphosphate particles into OPF hydrogels stimulated proliferation of rat mesenchymal stem cells .
With increasing incubation time, the strut diameter of the scaffolds reduced, which is a result of the material’s degradation (Figure 6B). In the hydrogel, gelation of alginate takes place by the formation of “egg box” structures in the presence of Ca2+. This gelling reaction can be reversed by removing these divalent cations. It is likely that the possible increase in hardness associated with mineralization may also influence proliferation. It should however be borne in mind that mineralization requires Ca2+ ions, which are also required for crosslinking of the alginate hydrogel. A situation may arise where mineral formation, strengthening the hydrogel, may necessitate removal of Ca2+ ions involved in alginate crosslinking, thus simultaneously weakening the hydrogel. On the other hand also the cell medium contains Ca2+ ions and thus it could be a possible source for this type of ions. One potential negative influence of mineralization is impediment of diffusion of nutrients, waste products and oxygen to cells inside the hydrogel. Mineralization is found to be greatest at the hydrogel surface in contact with osteogenic medium, leading to the formation of a highly mineralized outer layer, which impedes diffusion [9, 11]. A direct proliferative effect of ALP or the products of ALP-mediated cleaving of phosphate ions from glycerophosphate on MG-63 cells has not been reported but is not ruled out.
This study demonstrated the feasibility of bioplotting alginate scaffolds consisting of struts containing alginate capsules. This allowed compartmentalization of MG-63 osteoblast-like cells and the enzyme ALP within the scaffolds. ALP had no appreciable negative effect on cytocompatibility of MG-63 osteoblast-like cells. Mineralization was observed in all scaffolds containing ALP. However, further investigations are needed to investigate the stimulatory effect of this system on in vitro osteogenesis.
This work was supported by the Emerging Fields Initiative (EFI) of the University of Erlangen-Nuremberg (project TOPbiomat). Timothy E.L. Douglas acknowledges the Research Foundation Flanders (FWO) for support in the framework of a postdoctoral fellowship.
Conflict of interest and ethical approval statements: The authors have no conflict of interest. No ethical approval was required for this study
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