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.

Agarose Hydrogel Characterization for Regenerative Medicine Applications: Focus on Engineering Cartilage

“…The Campos group has used a fibrin-agarose material for engineering a variety of tissues including peripheral nerves, skin, oral mucosa, and corneas [40][41][42][43][44][45]. Others are investigating collagen-agarose gels for skin tissue engineering [46] or in combination with methylcellulose and chitosan for bone tissue engineering [47].…”

Engineered decellularized matrices to instruct bone regeneration processes

“…The mechanical properties of engineered substrates also play a key role, as the osteogenic phenotype of mesenchymal progenitors is induced by stiff substrates with an elasticity ranging from 25 to 40 kPa, corresponding to stiffness in physiological osteoid [34]. More recent studies highlight the complexity of the underlying mechanisms, since material stiffness can differentially regulate (i) osteogenic cell phenotypes, depending on the specific stage of differentiation [35,36] and (ii) polarization of macrophages, in turn regulating the process of bone healing [37].…”

When 1 + 1 > 2: Nanostructured composites for hard tissue engineering applications

“…Inorganic nanoparticles incorporated to a polymeric network have thus been shown to improve an array of properties of the pure polymer, including stiffness, resistance to wear and crack propagation, compressive load-bearing capacity, overall stability and even tensile strength 83 . Studies have shown that osteoblasts proliferate better on surfaces stiffer than most polymers 84 as well as that mesenchymal stem cells (MSCs) differentiate into neurons on soft surfaces and osteoblasts on the stiff ones 85 , which explains how come the dispersion of CAP particles throughout a polymeric matrix leads to an increased Young’s modulus and increased bioactivity of the composite material at the same time 86 . Moreover, it is a rule of thumb that biomaterials in general should be rough so as to promote cell attachment 87,88 , except in a few cases, including joint and some soft tissue implants for which smooth surfaces are more desirable; impregnation of polymers with inorganic nanoparticles contributes to this surface roughness and makes additional processing steps such as etching or sandblasting unnecessary.…”