In vitro development and optimization of cell-laden injectable bioprinted gelatin methacryloyl (GelMA) microgels mineralized on the nanoscale

Abstract

Bone defects may occur in different sizes and shapes due to trauma, infections, and cancer resection. Autografts are still considered the primary treatment choice for bone regeneration. However, they are hard to source and often create donor-site morbidity. Injectable microgels have attracted much attention in tissue engineering and regenerative medicine due to their ability to replace inert implants with a minimally invasive delivery. Here, we developed novel cell-laden bioprinted gelatin methacrylate (GelMA) injectable microgels, with controllable shapes and sizes that can be controllably mineralized on the nanoscale, while stimulating the response of cells embedded within the matrix. The injectable microgels were mineralized using a calcium and phosphate-rich medium that resulted in nanoscale crystalline hydroxyapatite deposition and increased stiffness within the crosslinked matrix of bioprinted GelMA microparticles. Next, we studied the effect of mineralization in osteocytes, a key bone homeostasis regulator. Viability stains showed that osteocytes were maintained at 98 % viability after mineralization with elevated expression of sclerostin in mineralized compared to non-mineralized microgels, showing that mineralization can effectively enhances osteocyte maturation. Based on our findings, bioprinted mineralized GelMA microgels appear to be an efficient material to approximate the bone microarchitecture and composition with desirable control of sample injectability and polymerization. These bone-like bioprinted mineralized biomaterials are exciting platforms for potential minimally invasive translational methods in bone regenerative therapies.

Keywords: Bioprinting; Bone tissue engineering; GelMA; Microgels; Mineralization; Osteocytes; Sclerostin.

Figure 1 –

Optimization of different conditions for mineralizing microgels. (A) Microgels were mineralized with different conditions of photopolymerization time and media composition. (B) and (C) represent the mineralized matrix ratio and the crystallinity index of microgels cultivated in DMEM low and high glucose, as well as α-MEM media. (D-F) represent the mineralized matrix ratio, the crystallinity index, and the elastic moduli in kPa of microgels (GelMA 10%) that were exposed to 25, 50, or 100 seconds in the digital light printing (DLP) process. Statistical differences are represented by * (p<0.05), ** (p<0.01), and *** (p<0.001) after one-way or two-way ANOVA and a post-Tukey’s correction test.

Figure 2 –

Printing and mineralization of microgels shapes. (A) Schematic of microgel preparation using DLP printer and mineralization steps. (B – H) represent the printed shapes, including triangles, squares, circles, hearts, stars, and the OHSU logo. (C, F, J) show the printed microgels in phase contrast. (D, G, K) show microgels imaged in green fluorescence. (E, H, L) show the printed constructs stained with alizarin red. The scale bar in C-H represents 800 μm and In J-L represents 600 μm.

Figure 3:

Microgel characterization. The images show the morphology of mineralized and non-mineralized microgels in 4 and 10x of magnification by light microscopy. B and C show the differences in morphology and extrafibrillar mineralization between mineralized and non-mineralized microgels from top view and from a cross-section view by scanning electron (B) and transmission electron microscopy (C).

Figure 4 –

Characterization of non-mineralized and mineralized microgels. Mineralized microgels stained with Alizarin red (A) and von Kossa (B) show dark solid areas compared to the non-mineralized group after 3 days. By the energy diffraction x-ray analysis (EDAX), (C) the presence of Ca and P electron peaks were just observed in mineralized microgels. (D). FTIR analysis shows the absorbance of different organic (amides I-III) and inorganic (phosphate) chemical groups only on mineralized samples with the wavenumber expressed in cm⁻¹. The mineral matrix ratio (E), the crystallinity index (F) and the elastic modulus confirm higher stiffness (represented in kPa) and solid composition of mineralized microgels compared to the non-mineralized samples. Statistical differences are represented by ** p<0.01, and **** p<0.0001 after the One-way ANOVA test post-Turkey’s corrections.

Figure 5 –

Osteocyte morphology and functionality in mineralized and non-mineralized microgels. A-B and E-F represent the confocal images of non-mineralized (A-B) and mineralized (E-F) samples. Microgels stained with actin (green), DAPI (blue), and SOST (red) and SOST intensity (normalized fluorescence per cell) in mineralized microgels (A-H, K-L). Also, the dendrite length in μm (I) and the number of dendrites per cell (I-J) were not significantly different between mineralized and non-mineralized samples. Statistical differences are represented by **** p<0.0001 after the One-way ANOVA test post-Turkey’s corrections.

Figure 6 –

Microgels injectability. Microgels were mineralized for three days and injected from an 18 G needle (A-B) in PDMS defects (1mm of diameter) (C-F). The cellular viability of osteocytes in mineralized microgels was evaluated by the live and dead stain. The G Figure illustrates the percentage of live osteocytes on technical and biological triplicates before and after injection. There was no significant difference between the groups after t test analysis between the two groups.