Bioactive injectable composites based on insulin-functionalized silica particles reinforced polymeric hydrogels for potential applications in bone tissue engineering

doi:10.1016/j.jmst.2021.08.003

Abstract

Abstract:

Novel bioactive injectable composites based on biopolymeric hydrogels reinforced with insulin-functionalized silica particles were synthesized. The insulin (INS) was immobilized on the surface of amine-modified silica particles employing covalent attachment by EDC/NHS chemistry and via electrostatic interaction. The resulting formulations were examined for the morphology (SEM), chemical composition (FTIR, XPS) as well as protein content. To facilitate the injectability and support the bone regeneration, developed particles were dispersed in biopolymeric sol composed of collagen, chitosan and lysine-modified hyaluronic acid and crosslinked with genipin. By means of rheological study, the sol-gel in situ transition of obtained systems was verified. It was found in vitro study that MG-63 cells cultured on the developed composites exhibit significantly higher alkaline phosphatase (ALP) activity, compared to the pristine hydrogel. Furthermore, the biomineralization ability in the simulated body fluid (SBF) model was also demonstrated. Our findings suggest that proposed herein novel hydrogel-based composites might be the promising formulation for regeneration of bone defects, especially as a less-cost effective support/alternative for BMP-2 systems.

Key words: Injectable composites, Biopolymers, Insulin-functionalized silica particles, Bone tissue engineering

Aleksandra Krajcer, Joanna Klara, Wojciech Horak, Joanna Lewandowska-Łańcucka. Bioactive injectable composites based on insulin-functionalized silica particles reinforced polymeric hydrogels for potential applications in bone tissue engineering[J]. J. Mater. Sci. Technol., 2022, 105: 153-163.

Figures/Tables 12

Table 1. Overview of synthesized materials with abbreviations employed.

Material	Abbreviation
Col:Ch:HA_mod+ SiO_2-NH₂-INS_{(electrostatic)}-C₁	Hyb-E-C₁
Col:Ch:HA_mod+ SiO_2-NH₂-INS_{(electrostatic)}-C₂	Hyb-E-C₂
Col:Ch:HA_mod+ SiO_2-NH₂-INS_{(electrostatic)}-C₃	Hyb-E-C₃
Col:Ch:HA_mod+ SiO_2-NH₂-INS_(covalent)-C₁	Hyb-C-C₁
Col:Ch:HA_mod+ SiO_2-NH₂-INS_(covalent)-C₂	Hyb-C-C₂
Col:Ch:HA_mod+ SiO_2-NH₂-INS_(covalent)-C₃	Hyb-C-C₃
Col:Ch:HA_mod (Col:Ch:HAmod = 50:20:30)	Hydrogel

Fig. 1. Schematic illustration of two approaches of insulin immobilization: covalent (on the left) and electrostatic (on the right).

Fig. 2. FTIR spectra for insulin, amino-functionalized silica particles (SiO2-NH2) and particles with biomolecules attached electrostatically (SiO2-NH2-INS-E) or covalently (SiO2-NH2-INS-C).

Fig. 3. XPS wide spectra of silica particles with amino groups (SiO2-NH2) and insulin immobilized using electrostatic interactions (SiO2-NH2-INS-E) or covalent bonds (SiO2-NH2--INS-C) with high-resolution results corresponding to O 1s, N 1s, C 1s, S 2p and Si 2p.

Table 2. Atomic composition of developed particles: SiO2-NH2; SiO2-NH2-INS-E; SiO2-NH2-INS-C.

Atomic composition (%)	O 1s	N 1s	C 1s	S 2p	Si 2p
SiO_2-NH₂	77.8	3.6	6.8	0	11.8
SiO_2-NH₂-INS-E	43.9	17.0	35.2	1.4	2.5
SiO_2-NH₂-INS-C	57.5	12.0	23.4	1.0	6.1

Fig. 4. High-resolution spectra deconvolution for O 1s, N 1s and C 1s of three types of particles: (SiO2-NH2), (SiO2-NH2-E), (SiO2-NH2-C).

Fig. 5. SEM microphotographs of three types of particles: SiO2-NH2 (A), SiO2-NH2-INS-E (B), SiO2-NH2-INS-C (C) with their size distributions (D), (E), (F), respectively.

Fig. 6. The values of the storage modulus (G’) measured in 10, 35 and 65 min after starting the experiment are presented on a logarithmic scale. Statistical significance was calculated using Student's t-test. A comparison between two means was analysed with statistical significance level set at p < 0.05; indicates statistical significance when compared with:* control after 65 min, # Hyb-E-C1 after 65 min, & Hyb-E-C2 after 65 min, $ Hyb-C-C1 after 65 min.

Fig. 7. SEM images of hybrid materials containing particles with electrostatically attached insulin: Hyb-E-C1, Hyb-E-C2, and Hyb-E-C3. Yellow arrows indicate the groups of silica carriers’ aggregates..

Fig. 8. TG profiles obtained in the oxidative atmosphere (air) for Hyb-E-C1, Hyb-E-C3 and Hyb-C-C3, respectively.

Fig. 9. SEM micrographs of the surface of pristine hydrogel and hybrid materials after 5 days of incubation in SBF. The insets are SEM images of higher magnification (5000 × ). The Ca/P ratios were determined by EDS analyses.

Fig. 10. (A) Cell number and (B) Alkaline phosphatase activity (ALP) of MG-63 cells grown on the surface of the materials studied on 1st, 3rd and 7th days of the culturing. Indicates statistical significance when compared with: (A)% Hyb-C-C1 day 7 (B) * control day 3; ** control day 7; # Hydrogel; $ Hyb-E-C1;% Hyb-C-C1. Cells cultured on the tissue culture plate (TCP) was considered as a control.

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