Evaluation of multifunctional polysaccharide hydrogels with varying stiffness for bone tissue engineering

Abstract

The use of hydrogels for bone regeneration has been limited due to their inherent low modulus to support cell adhesion and proliferation as well as their susceptibility to bacterial infections at the wound site. To overcome these limitations, we evaluated multifunctional polysaccharide hydrogels of varying stiffness to obtain the optimum stiffness at which the gels (1) induce proliferation of human dermal fibroblasts, human umbilical vascular endothelial cells (HUVECs), and murine preosteoblasts (MC3T3-E1), (2) induce osteoblast differentiation and mineralization, and (3) exhibit an antibacterial activity. Rheological studies demonstrated that the stiffness of hydrogels made of a polysaccharide blend of methylcellulose, chitosan, and agarose was increased by crosslinking the chitosan component to different extents with increasing amounts of genipin. The gelation time decreased (from 210 to 60 min) with increasing genipin concentrations. Proliferation of HUVECs decreased by 10.7 times with increasing gel stiffness, in contrast to fibroblasts and osteoblasts, where it increased with gel stiffness by 6.37 and 7.8 times, respectively. At day 14 up to day 24, osteoblast expression of differentiation markers-osteocalcin, osteopontin-and early mineralization marker-alkaline phosphatase, were significantly enhanced in the 0.5% (w/v) crosslinked gel, which also demonstrated enhanced mineralization by day 25. The antibacterial efficacy of the hydrogels decreased with the increasing degree of crosslinking as demonstrated by biofilm formation experiments, but gels crosslinked with 0.5% (w/v) genipin still demonstrated significant bacterial inhibition. Based on these results, gels crosslinked with 0.5% (w/v) genipin, where 33% of available groups on chitosan were crosslinked, exhibited a stiffness of 502±64.5 Pa and demonstrated the optimal characteristics to support bone regeneration.

FIG. 1.

(A–L) Graphical representation of change in G′ and G′′ with increasing genipin concentrations. To determine the linear viscoelastic region of the hydrogel, strain sweep (A–D) and frequency sweep (E–H) were run on the gels. Based on the data from strain sweep and frequency sweep, a time sweep at 0.5 Hz and 1% strain was run to monitor the gel behavior. (M–P) Scanning electron micrographs of all the sample hydrogels. Due to the complex nature of the interaction of the hydrogel constituents and irregular surface morphology, the actual size of the pores could not be determined.

FIG. 2.

(A) Graphical representation of increasing degree of crosslinking with increasing genipin concentrations. The hydrogels were crosslinked to up to 60% with 1% (w/v) genipin concentrations. The hydrogel with 1% (w/v) genipin concentration was significantly different from control, 0.1% and 0.5% gel (p<0.05, n=3). (B) Gelation times increased with the control gel showing significantly higher (p<0.05, n=3) gelation time compared to crosslinked samples (C) Storage modulus was found to be significantly higher in all the groups with increasing genipin concentrations (p<0.05, n=3). Groups with different alphabetical characters are significantly different from each other at p<0.05.

FIG. 3.

(A) Cells that do not attach due to improper hydrogel stiffness do not spread out and thus appear smaller and round. A decrease in attachment and proliferation of HUVE cells is found to correspond to an increase in the storage modulus of the underlying gels. The opposite is found with fibroblasts in which, decreased attachment with increasing stiffness was demonstrated. (B–D) Proliferation of endothelial cells, human dermal fibroblasts, and MC3T3-E1 osteoblast cells on hydrogels with varying storage modulus at day 7. Groups with different alphabetical characters are significantly different from each other at p<0.05. HUVE, human umbilical vascular endothelial.

FIG. 4.

(A) Quantification of mineralization using a threshold on the fluorescence level in MATLAB (at 10×magnification). (B) Graphical representation of percentage mineralization. (C) Quantification of mineralization by atomic absorption spectroscopy groups with different alphabetical characters is significantly different from each other at p<0.05. (D) ALP detection assay for detecting the ALP activity of the cells cultured on the hydrogel scaffolds for 25 days. The ALP activity in total cell lysates were assayed and expressed as the ratio of fluorescence readings of ALP in cells growing on 0.1, 0.5, and 1 wt% hydrogels as compared to the total protein content isolated from the cells and quantified using Bio-Rad protein assay at n=3. ALP, alkaline phosphatase.

FIG. 5.

As can been seen from the graphs, there is a significant upregulation of several osteogenic marker genes like OCN, OPN, and OPG (p<0.05) with increasing stiffness of the hydrogel scaffolds. (A) Relative fold expression of all the major genes is highest at 0.5% genipin concentration at day 7 with OCN and OPG showing two- to threefold increase relative to the control (p<0.05). (B) Relative fold expression of osteogenic markers for osteoblasts at day 15. ALP increased significantly with increasing stiffness to up to 20-fold (p<0.05, n=3) on hydrogel with 0.5% w/v crosslinker concentration after 15 days of culture. Osteocalcin showed a significantly uniform increase with stiffness to up to 21 times from 0.1% to 1% genipin concentration (p<0.05, n=3) and osteopontin showed highest expression in the 0.5% hydrogel group. Osteoprotegerin was found to be significantly higher in the 0.5% hydrogel group showing up to an ∼33-fold increase in expression relative to the control non-crosslinked hydrogels. (C) For day 24, all the genes were significantly upregulated compared to day 7 and day 15 expression levels of the genes. The 0.5% genipin concentration hydrogel showed significant upregulation of OCN to up to 40–50-fold, BSP to 40-fold, and ALP to 45-fold at p<0.05 compared to 0.1% and 1% genipin concentration hydrogels. Groups with different alphabetical characters are significantly different from each other at p<0.05.

FIG. 6.

Graphical representation of biofilm on gels with varying levels of crosslinked (amine) groups. Since amino groups on chitosan are responsible for the antibacterial effect of the hydrogels, crosslinking them using genipin would lead to a decrease in the antibacterial effect. It was seen that hydrogels with 1% (w/v) crosslinker concentrations had a significantly higher biofilm formation (therefore, a lesser antibacterial efficacy) compared to the non-crosslinked and crosslinked groups (p<0.05; n=3). Groups with different alphabetical characters are significantly different from each other at p<0.05.