Coated electrospun alginate-containing fibers as novel delivery systems for regenerative purposes

Figures & data

Table 1 Composition of D formulations, expressed as % w/w

Formulation	DEX	ALG	P407	TX100	Solvent
D1	20	1	0	1.5	H2O
D2	25	1.5	1.5	0.5	H2O
D3	20	1	1.5	0	H2O
D4	20	1.5	2	0	H2O
D5	10	1.5	1.5	0.5	H2O
D6	25	1.5	1.5	0.5	Buffer
D7	10	1.5	1.5	0	H2O
D8	30	0.5	2	0	Buffer

Abbreviations: ALG, alginate; D, ALG/DEX-based solutions; DEX, dextran.

Table 2 Full factorial design experimental points

	Viscosity	Surface tension	Conductivity
D1	−1	−1	−1
D2	+1	−1	−1
D3	−1	+1	−1
D4	+1	+1	−1
D5	−1	−1	+1
D6	+1	−1	+1
D7	−1	+1	+1
D8	+1	+1	+1

Abbreviations: ALG, alginate; D, ALG/DEX-based solutions; DEX, dextran.

Figure 1 Factor space and experimenting points of a full factorial design.

Figure 2 ΔG′ values calculated for DEX 30% w/w/ALG mixtures containing increasing ALG concentrations (0.5%, 1%, and 1.5% w/w) (mean values ± SD; n=3). Abbreviations: DEX, dextran; ALG, alginate.

Table 3 Mean values (±SD; n=3) of viscosity at 1,000 s⁻¹, pseudoplasticity index, surface tension, and conductivity of each ALG/DEX formulation

	Viscosity at 1,000 s^−1 (Pa·s)	Pseudoplasticity index	Surface tension (mN/m)	Conductivity (μS/cm)	DoE score
D1	0.73±0.05	1.618±0.029	31.1±0.1	613±22	2.5
D2	1.88±0.05	1.972±0.079	31.0±0.7	833.8±2	10
D3	1.01±0.02	1.689±0.008	41.2±1.1	535±3	2.5
D4	1.32±0.14	2.140±0.012	38.3±1.8	990±14	5
D5	0.40±0.01	1.671±0.015	33.0±0.1	1,497±5	0
D6	1.68±0.04	2.164±0.017	31.3±1.0	2,487±7	7.5
D7	0.44±0.04	1.950±0.016	39.0±0.2	1,525±2	0
D8	1.80±0.22	1.486±0.006	40.4±2.0	1,462±10	7.5

Note: The score attributed to each formulation according to the morphology of the corresponding electrospun fibers is also reported.

Abbreviations: D, ALG/DEX-based solutions; DEX, dextran; ALG, alginate; DoE, Design of Experiments.

Figure 3 SEM micrographs of (A) D1, (B) D2, (C) D3, (D) D4, (E) D5, (F) D6; (G) D7, and (H) D8 products obtained from electrospinning.

Abbreviations: ALG, alginate; D, ALG/DEX-based solutions; DEX, dextran; SEM, scanning electron microscopy.

Figure 4 Standardized Pareto chart (A and C) and interaction plot (B and D) of the response variable fiber formation of the full factorial design.

Notes: Three factors, viscosity at high shear rates, surface tension, and conductivity were investigated in A and B, while pseudoplasticity index, surface tension, and conductivity were investigated in C and D.

Table 4 Composition (% w/w) and properties (mean values ± SD; n=3) of the polymer solutions D9–D12 prepared to verify the predictive power of the DoE approach

	D9	D10	D11	D12
Composition (% w/w)
DEX	30	22.5	20	20
ALG	0.5	1.5	1.5	1
P407	1.5	1.5	1.5	1.5
TX100	0.5	0.5	0.5	0.5
Polymer solution properties
Viscosity (Pa·s)	1.38±0.10	1.38±0.03	0.99±0.04	0.91±0.08
Pseudoplasticity index	1.791±0.002	1.923±0.054	1.790±0.101	1.589±0.051
Surface tension (mN/m)	32.7±0.8	31.9±0.6	31.7±0.4	32.6±0.5
Conductivity (mS/cm)	337±9	869±14	1,057±1	836±3

Abbreviations: D, ALG/DEX-based solutions; DEX, dextran; ALG, alginate; DoE, Design of Experiments; P407, poloxamer; TX100, triton.

Figure 5 SEM micrographs of (A) D9, (B) D10, (C) D11, and (D) D12 products obtained from electrospinning.

Abbreviations: ALG, alginate; D, ALG/DEX-based solutions; DEX, dextran; SEM, scanning electron microscopy.

Figure 6 SEM micrograph of the optimized ALG/PEO fibers (P1).

Abbreviations: ALG, alginate; PEO, polyethylene oxide; SEM, scanning electron microscopy.

Figure 7 Mechanical properties of both D9 and P1 fibers: maximum force of deformation (Fmax) and forces (F) measured at different deformation values are reported (mean values ± SE; n=3). *P<0.05 Student’s t-test.

Abbreviations: ALG, alginate; D, ALG/DEX-based solutions; P, ALG/PEO fibers; PEO, polyethylene oxide.

Figure 8 Characterization of D9 fibers after PLGA-based coating: (A) SEM micrographs; (B) fiber dimensions (mean values ± SD; n=30), one-way ANOVA, multiple range test (P<0.05): a vs b and c; (C) FTIR spectrographs.

Abbreviations: ALG, alginate; D, ALG/DEX-based solutions; DEX, dextran; FTIR, Fourier transform infrared; PLGA, polylactide-co-glycolide.

Figure 9 Mechanical properties of D9 as such and after coating with R 503H at 0.25% and 0.5% w/v: (A) maximum force of deformation (Fmax) and forces (F) measured at different deformation values (*P<0.05 compared to D9 Fmax and ^#P<0.05 compared to D9 F10%, Student’s t-test), (B) tensile work (*P<0.05 compared to D9, Student’s t-test), and (C) percentage of fiber elongation (*P<0.05 compared to D9) are reported (mean values ± SE; n=3).

Abbreviations: ALG, alginate; D, ALG/DEX-based solutions; DEX, dextran.

Table 5 Element content (mol %) of coated P1 fibers before and after hydration in deionized water

Sample	C	O	Ca	Cl	Other elements
P1	51.26	21.82	0	0	26.92
P1 coated	30.91	32	25.65	0.23	5.21
P1 coated after hydration in deionized H2O	35.02	41.41	17.88	0	5.69

Note: P1 fibers as such was reported as reference.

Abbreviations: ALG, alginate; P, ALG/PEO fibers; PEO, polyethylene oxide.

Figure 10 Characterization of P1 fibers after Ca²⁺ cross-linking and chitosan-based coating: (A) SEM micrographs; (B) fiber dimensions (mean ± SD; n=30), one-way ANOVA, multiple range test (P<0.05): a vs b and c; (C) FTIR spectrographs.

Abbreviations: ALG, alginate; D, ALG/DEX-based solutions; P, ALG/PEO fibers; PEO, polyethylene oxide; SEM, scanning electron microscopy; FTIR, Fourier transform infrared.

Figure 11 Mechanical properties of P1 fibers as such and after Ca²⁺ cross-linking and chitosan-based coating: (A) maximum force of deformation (one-way ANOVA, multiple range test [P<0.05]: a vs b and c; b vs c), (B) tensile work (one-way ANOVA, multiple range test [P<0.05]: a vs b and c; b vs c), and (C) percentage of fiber elongation are reported (mean values ± SE; n=3).

Abbreviations: ALG, alginate; P, ALG/PEO fibers; PEO, polyethylene oxide.

Figure 12 % Cell viability values observed for coated D9 and P1 fibers (mean values ± SD; n=6).

Abbreviations: ALG, alginate; CM, complete medium; D, ALG/DEX-based solutions; DEX, dextran; P, ALG/PEO fibers; PEO, polyethylene oxide.

Figure 13 SEM micrographs of the coated fibers: (A, B) coated D9 fibers after immersion for 7 days in complete medium in the absence (A) and in the presence (B) of fibroblasts; (C–E) coated P1 fibers after immersion for 7 days in complete medium in the presence of fibroblasts. The yellow arrows indicate cells.

Abbreviations: ALG, alginate; D, ALG/DEX-based solutions; P, ALG/PEO fibers; PEO, polyethylene oxide; SEM, scanning electron microscopy.

Figure S1 G′ vs frequency profiles of blends of 30% w/w DEX with different ALG concentrations and of the individual polymer solutions (mean values ± SD; n=3); (A) ALG 0.5% w/w–DEX 30% w/w, (B) ALG 1% w/w–DEX 30% w/w, (C) ALG 1.5% w/w–DEX 30% w/w.

Abbreviations: DEX, dextran; ALG, alginate.

Figure S2 Viscosity parameters (η₀ and η_∞) as a function of dextran concentration for solutions prepared in distilled water.

Note: Log–log profiles are reported in the inset.

Figure S3 Comparison of viscosity profiles, observed at 33°C, of DEX and PEO solutions, both prepared in deionized water at the concentration of 1% w/w (mean values ± SD; n=3).

Abbreviations: DEX, dextran; PEO, polyethylene oxide.