Implementing BMP-7 Chemically Modified RNA for Bone Regeneration with 3D Printable Hyaluronic Acid-Collagen Granular Gels

Abstract

Chemically modified RNA (cmRNA) is emerging as a more effective alternative to protein delivery and DNA-based gene therapy. To implement this technology for bone regeneration, a suitable biomaterial functioning as scaffold and delivery system is necessary. This study introduces a 3D printable granular hydrogel consisting of hyaluronic acid and collagen (THA-Col) for the delivery of bone morphogenetic protein (BMP)-7 cmRNA as activated matrix to promote bone healing. Granular hydrogels are produced by mechanically fragmenting bulk THA-Col gels. Resulting microgels are 3D printable and are further investigated in comparison to bulk THA-Col gels for BMP-7 cmRNA transfection efficiency, cytotoxicity, and osteogenic differentiation of human mesenchymal stromal cells (hMSCs). Microgels showed higher cell viability than bulk gels, while both bulk and microgels could support transfection with BMP-7. During in vitro osteogenic differentiation, hMSCs on microgels showed higher production of alkaline phosphatase (ALP) compared to bulk gels. The combination of microgels loaded with BMP-7 cmRNA introduced in this work holds significant potential toward the development of patient-specific bone graft substitutes to replace autologous bone grafting and protein delivery.

Keywords: BMP‐7; bone regeneration; cmRNA; granular hydrogels; hMSCs; microgels; osteogenesis.

Figure 1

Schematic of THA‐Col microgel matrix fabrication.

Figure 2

3D Printed THA‐Col microgels matrix. a) Amplitude sweep after enzymatic crosslinking, n = 3. b) Viscosity curve of microgels, n = 3. c) Elastic recovery of microgels with alternating low (1%) and high (500%) strain, n = 3. d) Schematic evaluation of printability assessment (filament spreading and uniformity, filament fusion, and pore geometry). e) 3D printed THA‐Col microgels strands, filament fusion, lattice, and overhanging pillar, scale bar = 5 mm. Quantification of f) filament spreading, n = 3, g) filament uniformity, n = 3, h) filament fusion, n = 3, and i) pore geometry, n = 3. THA = Tyramine modified hyaluronic acid, Col = collagen.

Figure 3

THA‐Col bulk and microgel matrix characterization. a) Swelling ratio in PBS, n = 6. b) Degradation in 100 U mL⁻¹ hyaluronidase after 24 h of swelling, n = 6. c) Compressive modulus after 24 h of swelling, calculated between 0 and 10% strain, n = 6. One‐way ANOVA with Šidák correction was used for statistical analysis, ^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, ^∗∗∗∗ p < 0.0001, and ns = no significance. THA = Tyramine modified hyaluronic acid, Col = collagen.

Figure 4

Indirect cytotoxicity of THA‐Col bulk and microgel matrices tested with L929 cells. a) Schematic of in vitro indirect toxicity experimental set‐up. b) Metabolic activity after 24 h normalized to CCP – control, n = 4. c) Quantification of DNA content after 24 h, n = 4. d) Quantification of LDH release after 24 h, n = 4. e) Representative images of live (green) and dead (red) staining after 24 h, scale bar = 100 µm, n = 4. One‐way ANOVA with Šidák correction was used for statistical analysis, ^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, ^∗∗∗∗ p < 0.0001, and ns = no significance. CCP = Cell culture plastic, CCP + control = Cell culture plastic treated with 0.1% Triton, CCP – control = Cell culture plastic with unconditioned medium, THA = Tyramine modified hyaluronic acid, Col = collagen, LDH = Lactate dehydrogenase.

Figure 5

Transfection efficiency and cytotoxicity of BMP‐7 cmRNA with hMSCs on THA‐Col bulk and microgels. a) Schematic of in vitro transfection efficiency and cytotoxicity set‐up. b) Metridia luciferase expression after 1, 3, and 7 days, n = 3. c) BMP‐7 secretion in culture medium after 1, 3, and 7 days, n = 3. d) Quantification of LDH release after 1, 3, and 7 days, n = 3. e) Metabolic activity after 7 days normalized to CCP – control, n = 3. f) Representative images of live (green) and dead (red) staining after 7 days, scalebar = 100 µm, n = 3. One‐way ANOVA with Šidák correction was used for statistical analysis in figure c and two‐way ANOVA with Tukey's multiple comparison test was used for figure b,e,f, ^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, ^∗∗∗∗ p < 0.0001, and ns = no significance. THA = Tyramine modified hyaluronic acid, Col = collagen, MetLuc = Metridia luciferase.

Figure 6

Osteogenic differentiation of hMSCs on THA‐Col bulk and microgels matrices combined with BMP‐7 cmRNA. a) Schematic of in vitro osteogenic differentiation experimental set‐up. b) ALP activity after 14 days, n = 3. c) Quantification of DNA content after 14 days, n = 3. d) ALP activity normalized to DNA content after 14 days, n = 3. e) Images of ALP staining (blue) after 14 days, scalebar = 5 mm, n = 3. f) OPG secretion in culture medium pooled together per week, n = 3. g) OPG secretion at week 2 normalized to DNA content after 14 days, n = 3. One‐way ANOVA with Šidák correction was used for statistical analysis in figure b,c,d and two‐way ANOVA with Tukey's multiple comparison test was used for figure f, ^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, ^∗∗∗∗ p < 0.0001, and ns = no significance. THA = Tyramine modified hyaluronic acid, Col = collagen, ALP = Alkaline phosphatase, AR = Alizarin Red, OPG = Osteoprotegerin.