New injectable two-step forming hydrogel for delivery of bioactive substances in tissue regeneration

Abstract

A hydrogel based on chitosan, collagen, hydroxypropyl-γ-cyclodextrin and polyethylene glycol was developed and characterized. The incorporation of nano-hydroxyapatite and pre-encapsulated hydrophobic/hydrophilic model drugs diminished the porosity of hydrogel from 81.62 ± 2.25% to 69.98 ± 3.07%. Interactions between components of hydrogel, demonstrated by FTIR spectroscopy and rheology, generated a network that was able to trap bioactive components and delay the burst delivery. The thixotropic behavior of hydrogel provided adaptability to facilitate its implantation in a minimally invasive way. Release profiles from microspheres included or not in hydrogel revealed a two-phase behavior with a burst- and a controlled-release period. The same release rate for microspheres included or not in the hydrogel in the controlled-release period demonstrated that mass transfer process was controlled by internal diffusion. Effective diffusion coefficients, D _eff, that describe internal diffusion inside microspheres, and mass transfer coefficients, h, i.e. the contribution of hydrogel to mass transfer, were determined using 'genetic algorithms', obtaining values between 2.64·10^-15 and 6.67·10^-15 m²/s for D _eff and 8.50·10^-10 to 3.04·10^-9 m/s for h. The proposed model fits experimental data, obtaining an R ²-value ranged between 95.41 and 98.87%. In vitro culture of mesenchymal stem cells in hydrogel showed no manifestations of intolerance or toxicity, observing an intense proliferation of the cells after 7 days, being most of the scaffold surface occupied by living cells.

Keywords: FITC-dextran; collagen–cyclodextrin–chitosan; estradiol; hydrogel; mass transfer; rheology.

Figure 1

(A) XRD pattern of carbonated apatite prepared by thermal decomplexing of Ca/citrate/phosphate/carbonated solutions at pH = 8.5 at 80°C. (B) FTIR spectrum in transmittance mode of carbonated apatite prepared by thermal decomplexing of Ca/citrate/phosphate/carbonated solutions at pH = 8.5 at 80°C

Figure 2

(A) SEM image of lyophilized PLGA microspheres. (B) SEM image showing the internal structure of hydrogel

Figure 3

Variation of viscosity of collagen solution (10 mg/ml), before (COL) and after cyclodextrin (COL+CD), chitosan (COL+CD+CS) and PEG (COL+CD+CS+PEG) incorporation, with shear rate at 37°C. The upper box shows the viscosity versus shear rate of the complete gel, before (COL+CD+CS+PEG) and after the incorporation of microspheres and nano-hydroxyapatite (COL+CD+CS+PEG + spheres)

Figure 4

Evolution of the viscoelastic behavior of collagen with the incorporation of cyclodextrin, chitosan and PEG. Variation of (A) viscosity with shear rate and (B) elastic and viscous moduli with frequency in collagen (COL) versus a mixture of collagen and cyclodextrin (COL+CD) with a mass ratio equal to that in the final gel, maintaining the collagen concentration in both samples (COL) and (COL+CD). Variation of (C) viscosity with shear rate and (D) elastic and viscous moduli with frequency in collagen–cyclodextrin mixture (COL+CD) versus a collagen–cyclodextrin–chitosan mixture (COL+CD+CS) with a mass ratio equal to that in the final gel, maintaining the collagen and cyclodextrin concentration in both samples (COL+CD) and (COL+CD+CS). Variation of (E) viscosity with shear rate and (F) elastic and viscous moduli with frequency in collagen–cyclodextrin–chitosan mixture (COL+CD+CS) versus the complete gel (COL+CD+CS+PEG) with a mass ratio equal to that in the final gel, maintaining the collagen, cyclodextrin and chitosan concentrations in both samples (COL+CD+CS) and (COL+CD+CS+PEG)

Figure 5

FT-IR spectra in transmittance mode for (A) collagen, cyclodextrin and the mixture of both; (B) chitosan, collagen/cyclodextrin and collagen/cyclodextrin/chitosan mixtures; (C) PEG, collagen/cyclodextrin/chitosan and collagen/cyclodextrin/chitosan/PEG mixtures

Figure 6

In vitro release profile of β-estradiol from microspheres and microspheres-loaded hydrogel. In the upper right part of the figure is shown a magnification of the first hours. (A) Semi-continuous sampling method; (B) batch sampling method

Figure 7

In vitro and in vivo release profiles of RITC-dextran from microspheres and microspheres-loaded hydrogel. (A) Semi-continuous sampling method; (B) batch sampling method

Figure 8

Location of the predicted D_eff and h within the residual sum of squares (RSS) level curves. (A) 17-β-Estradiol, batch sampling method; (B) 17-β-estradiol, semi-continuous sampling method; (C) RITC-dextran, batch sampling method; (D) RITC-dextran, semi-continuous sampling method

Figure 9

Comparison of the experimental released fractions, M/M_∞ (points) versus the simulated ones (lines) for: (A) 17-β-estradiol, batch sampling method; (B) 17-β-estradiol, semi-continuous sampling method; (C) RITC-dextran, batch sampling method; (D) RITC-dextran, semi-continuous sampling method

Figure 10

rMSC seeded on the hydrogel scaffold. Representative photomicrographs (10×) of cell viability by calcein-AM staining after (A) 1.5 h (time for cellular adhesion); (B) 1 day; (C) 3 days and (D) 7 days of incubation in complete culture medium at 37°C and 5% CO₂