Antibacterial Crosslinker for Ternary PCL-Reinforced Hydrogels Based on Chitosan, Polyvinyl Alcohol, and Gelatin for Tissue Engineering

Abstract

Current hydrogels used for cartilage tissue engineering often lack the mechanical strength and structural integrity required to mimic native human cartilage. This study addresses this limitation by developing reinforced hydrogels based on a ternary polymer blend of poly(vinyl) alcohol (PVA), gelatin (GL), and chitosan (CH), with gentamicin sulfate (GS) as an antimicrobial agent and a crosslinker. The hydrogels were produced using two crosslinking methods, the freeze/thaw and heated cycles, and reinforced with forcespun polycaprolactone (PCL) nanofiber to improve mechanical performance. Chemical characterization revealed that GS forms weak hydrogen bonds with the ternary polymers, leading to esterification with PVA, and covalent bonds are formed as the result of the free amino group (-NH₂) of chitosan that reacts with the carboxylic acid group (-COOH) of gelatin. SEM images help us to see how the hydrogels are reinforced with polycaprolactone (PCL) fibers produced via force spinning technology, while mechanical properties were evaluated via uniaxial tensile and compressive tests. Water retention measurements were performed to examine the crosslinking process's influence on the hydrogel's water retention, while the hydrogel surface roughness was obtained via confocal microscopy images. A constitutive model based on non-Gaussian strain energy density was introduced to predict experimental mechanical behavior data of the hydrogel, considering a non-monotonous softening function. Loading and unloading tests demonstrated that GS enhanced crosslinking without compromising water retention or biocompatibility because of the reaction between the free amino group of CH and the carboxylic group of gelatin. The PCL-reinforced PVA/GL/CH hydrogel shows strong potential for cartilage repair and tissue engineering applications.

Keywords: articular cartilage replacement; forcespinning polycaprolactone fibers; gentamicin sulfate; hydrogels; non-Gaussian constitutive material model.

Figure 1

Spun PCL fibers by the Forcespinning technique. The inset figure marked with a red circle shows the spinneret that contains the solution for fabricating the PCL fibers.

Figure 2

Hypothetical mechanism reaction of PVA/GL/CH Hydrogel crosslinked with gentamicin sulfate. Dashed lines indicate the chemical interactions of functional groups between the components.

Figure 3

(a) XRD patterns of precursors of hydrogel: gelatin, chitosan polyvinyl alcohol, and (b) hydrogels crosslinked with gentamicin at low temperature (LT-AB), high temperature (HT-AB), and with no gentamicin (HT-C).

Figure 4

(a) FTIR spectra of the hydrogel precursors, including gelatin, chitosan, polyvinyl alcohol, and gentamicin sulfate. The spectra also show the hydrogels that were crosslinked at high temperature without gentamicin (HT-C), with gentamicin (HT-AB), and those crosslinked at low temperature without gentamicin (LT-C), and with gentamicin (LT-AB). (b) A zoomed-in view of the region from 3500 to 2500 cm⁻¹ of (a). (c) A zoomed-in view of the region from 1800 to 1000 cm⁻¹.

Figure 5

(a) Thermograms of chitosan, gelatin, and PVA in powder and the hydrogels crosslinked at high and low temperatures. (b) Derivative TGA results.

Figure 6

Morphological analysis of (a,b) LT-AB hydrogel and (c,d) HT-AB hydrogel.

Figure 7

SEM images for hydrogels reinforced with PCL fibers with gentamicin to different volumes (a) 1 mL, (b) 1.5 mL, (c) 2 mL at 1000x magnification. (d) 1 mL, (e) 1.5 mL, (f) 2 mL at 2500x magnification.

Figure 8

Surface morphology of hydrogel. (a) LT-AB hydrogel and (b) HT-AB hydrogel.

Figure 9

Stress versus stretch curves the patellofemoral groove of an equine [65] and the knee joint and human chondral cartilage knee layer [127]. Symbols (circle and triangle) indicate experimental data, and dashed lines indicate predicted results from the constitutive model given by Equation (7). Here, the material parameters found to fit data using Equation (7) are for equine cartilage: μ = 0.2 Mpa, N₈ = 5.5, f = 0.16, A₁ = −1.35 MPa, A₂ = 2 MPa, and for human cartilage: μ = 0.2 MPa, N₈ = 5.5, f = 0.195, A₁ = −1.1 MPa, and A₂ = 4.05 MPa.

Figure 10

Uniaxial extension stress–stretch curves of hydrogels crosslinked at (a) high temperature (HT) and (b) low temperature (LT). Symbols are experimental data, while dashed lines are simulation results obtained from Equation (7). The material parameter values used to fit data with simulations results computed from Equation (7) were for (a) HT-C-AB and HT-C: μ = (0.25, 0.2) MPa, N₈ = (2.5, 10.5), f = (0.075, 0.035), A₁ = (14.5, 18.5) MPa, and A₂ = (−4.5, −0.5) MPa, for (b) LT-AB and LT-C: μ = (0.195, 0.205) MPa, N₈ = (60, 30.5), f = (0.0145, 0.019), A₁ = (−15.5, −14) MPa, and A₂ = (2.45, 1.5) MPa. Here, HT-AB and LT-AB are the hydrogel samples reinforced with GS.

Figure 11

Compressive strength data of hydrogels crosslinked at (a) high temperature (HT), (b) low temperature (LT), and (c) native cartilage. Symbols are experimental data, while dashed lines are simulation results obtained from Equation (7). The material parameter values used to fit data with simulations results computed from Equation (7) were for HT-AB and HT-C: μ = 0.2 MPa, N₈ = 3.5, f = (0.16, 0.09), A₁ = (4.15, 1.25) MPa, A₂ = (−3.5,−0.75) MPa, for LT-AB and LT-C: μ = 0.2 MPa, N₈ = 3.5, f = 0.16, A₁ = (−1.35,−1.5) MPa, and A₂ = (−0.195, −0.3) MPa, for HD and the human native cartilage: μ = 0.2 MPa, N₈ = (3.5, 5.5), f = (0.16, 0.195), A₁ = (7.25, −1.1) MPa, and A₂ = (0, 4.05) MPa.

Figure 12

Contact angle images of (a) LT-hydrogel and (c) HT-hydrogel crosslinked with gentamicin sulfate. (b) and (d) images of hydrogels evaluated in (a) and (c), respectively.

Figure 13

Water absorption evaluation of hydrogels crosslinked at low and high temperatures with and without gentamicin sulfate.

Figure 14

(a) LT-AB hydrogels incubated in microplates with 24 holes and 20,000 stem cells seeded per hole. (b) Cell growth of (a) after the incubation period.

Figure 15

Loading and unloading engineering stress vs. stretch curves. (a) Hydrogels without fibers. (b) Hydrogels reinforced with fibers. Color lines represent theoretical predictions obtained from Equations (7) and (8). Dots are experimental data collected from compressive tests.