Dual Function of Glucosamine in Gelatin/Hyaluronic Acid Cryogel to Modulate Scaffold Mechanical Properties and to Maintain Chondrogenic Phenotype for Cartilage Tissue Engineering

Abstract

Glucosamine (GlcN) fulfills many of the requirements as an ideal component in scaffolds used in cartilage tissue engineering. The incorporation of GlcN in a gelatin/hyaluronic acid (GH) cryogel scaffold could provide biological cues in maintaining the phenotype of chondrocytes. Nonetheless, substituting gelatin with GlcN may also decrease the crosslinking density and modulate the mechanical properties of the cryogel scaffold, which may be beneficial as physical cues for chondrocytes in the scaffold. Thus, we prepared cryogel scaffolds containing 9% GlcN (GH-GlcN9) and 16% GlcN (GH-GlcN16) by carbodiimide-mediated crosslinking reactions at −16 °C. The crosslinking density and the mechanical properties of the cryogel matrix could be tuned by adjusting the content of GlcN used during cryogel preparation. In general, incorporation of GlcN did not influence scaffold pore size and ultimate compressive strain but increased porosity. The GH-GlcN16 cryogel showed the highest swelling ratio and degradation rate in hyaluronidase and collagenase solutions. On the contrary, the Young’s modulus, storage modulus, ultimate compressive stress, energy dissipation level, and rate of stress relaxation decreased by increasing the GlcN content in the cryogel. The release of GlcN from the scaffolds in the culture medium of chondrocytes could be sustained for 21 days for GH-GlcN16 in contrast to only 7 days for GH-GlcN9. In vitro cell culture experiments using rabbit articular chondrocytes revealed that GlcN incorporation affected cell proliferation, morphology, and maintenance of chondrogenic phenotype. Overall, GH-GlcN16 showed the best performance in maintaining chondrogenic phenotype with reduced cell proliferation rate but enhanced glycosaminoglycans (GAGs) and type II collagen (COL II) secretion. Quantitative real-time polymerase chain reaction also showed time-dependent up-regulation of cartilage-specific marker genes (COL II, aggrecan and Sox9) for GH-GlcN16. Implantation of chondrocytes/GH-GlcN16 constructs into full-thickness articular cartilage defects of rabbits could regenerate neocartilage with positive staining for GAGs and COL II. The GH-GlcN16 cryogel will be suitable as a scaffold for the treatment of articular cartilage defects.

Preparation of Gelatin and Gelatin/Hyaluronic Acid Cryogel Scaffolds for the 3D Culture of Mesothelial Cells and Mesothelium Tissue Regeneration

“…The microstructure of the scaffold of cryogel samples was examined with an S-3000N scanning electron microscope (SEM) from Hitachi (Tokyo, Japan), after gold sputter coating. The porosity of the cryogel was calculated using the ethanol displacement method [68]. To study in vitro degradation of cryogel, a collagenase (30 units/mL) solution was prepared in PBS (pH 7.4).…”

Osteogenic potential of induced pluripotent stem cells from human adipose-derived stem cells

“…Gelatin is a water-soluble multipeptide polymer that is obtained by hydrolyzing collagen from animal skin and tissues. The arginine-glycine-aspartic acid (RGD) sequence of gelatin allows for better cell attachment and bioactivity [5, 17, 26, 27]. Moreover, different materials and growth factors could be fabricated additionally into cryogel scaffolds to alter physical characteristics and cellular stimulation.…”

“…The cryogel also possesses the advantage of plasticity and tailoring especially in areas like craniofacial regions which required dedicated manipulations. Previous studies demonstrated a great potential of cryogel scaffolds in tissue engineering of the bone [5, 16, 17, 25, 28], adipose tissue [6], and cartilage [26].…”

Maintenance of Multipotent Mesenchymal Stem Cells of Farm Animals in Cryogels Based on Naturally-Derived Polymers

“…Доклинические испытания с использованием свиней демонстрировали биосовместимость желатиновых криогелей, их нетоксичность для организма животных. Имеются данные о добавлении к желатину других компонентов, которые повышают прочность желатиновых криогелей, например введении в исходный состав хитозана, который способствует поддержанию клеточной адгезии и пролиферации (32)(33)(34)(35).…”