Homogeneous hydroxyapatite/alginate composite hydrogel promotes calcified cartilage matrix deposition with potential for three-dimensional bioprinting

Abstract

Calcified cartilage regeneration plays an important role in successful osteochondral repair, since it provides a biological and mechanical transition from the unmineralized cartilage at the articulating surface to the underlying mineralized bone. To biomimic native calcified cartilage in engineered constructs, here we test the hypothesis that hydroxyapatite (HAP) stimulates chondrocytes to secrete the characteristic matrix of calcified cartilage. Sodium citrate (SC) was added as a dispersant of HAP within alginate (ALG), and homogeneous dispersal of HAP within ALG hydrogel was confirmed using sedimentation tests, electron microscopy, and energy dispersive spectroscopy. To examine the biological performance of ALG/HAP composites, chondrocyte survival and proliferation, extracellular matrix production, and mineralization potential were evaluated in the presence or absence of the HAP phase. Chondrocytes in ALG/HAP constructs survived well and proliferated, but also expressed higher levels of calcified cartilage markers compared to controls, including Collagen type X secretion, alkaline phosphatase (ALP) activity, and mineral deposition. Compared to controls, ALG/HAP constructs also showed an elevated level of mineralized matrix in vivo when implanted subcutaneously in mice. The printability of ALG/HAP composite hydrogel precursors was verified by 3D printing of ALG/HAP hydrogel scaffolds with a porous structure. In summary, these results confirm the hypothesis that HAP in ALG hydrogel stimulates chondrocytes to secrete calcified matrix in vitro and in vivo and reveal that ALG/HAP composites have the potential for 3D bioprinting and osteochondral regeneration.

Osteochondral Regeneration with 3D‐Printed Biodegradable High‐Strength Supramolecular Polymer Reinforced‐Gelatin Hydrogel Scaffolds

“…[5][6][7][8] Thus, the success of osteochondral regeneration is largely dependent upon constructing a scaffold with an ability to recapitulate niche cues. [14][15][16][17] Among a wide array of hydrogels for tissue engineering, gelatin hydrogel has been developed as a variety of bioinks for 3D printing due to its better biocompatibility, biodegradability, bioactivity, and abundance from diverse sources. [9][10][11][12][13] Recently, with rapid development of 3D printing technology, increasing studies, including ours, are focused on fabricating scaffolds by 3D printing of hydrogel bioinks for osteochondral repair.…”

“…Hydrogels are emerging as a promising class of biomaterials for both soft and hard tissue regeneration . Recently, with rapid development of 3D printing technology, increasing studies, including ours, are focused on fabricating scaffolds by 3D printing of hydrogel bioinks for osteochondral repair . Among a wide array of hydrogels for tissue engineering, gelatin hydrogel has been developed as a variety of bioinks for 3D printing due to its better biocompatibility, biodegradability, bioactivity, and abundance from diverse sources.…”

From Shape to Function: The Next Step in Bioprinting

“…Recreating this structure (and therefore the associated mechanical function) holds great promise for the creation of durable cartilage implants to treat degenerative diseases like osteoarthritis. On the one hand, both bioprinted cartilage‐specific cells and stem cells demonstrated some ability to deposit hyaline cartilage‐like ECM in vivo, as well as calcified cartilage . Within the field of biofabrication, technologies are being developed to print zone‐specific bioinks, for instance by depositing subsequent layers of inks laden with a gradient in cell concentration, or with different cartilage‐derived and bone‐marrow derived specific progenitor cells, to recreate the native gradient of glycosaminoglycans (GAGs) and the expression of zonal markers .…”

“…On the one hand, both bioprinted cartilage-specific cells and stem cells demonstrated some ability to deposit hyaline cartilage-like ECM in vivo, [ 266 ] as well as calcified cartilage. [ 267 ] Within the field of biofabrication, technologies are being developed to print zone-specific bioinks, for instance by depositing subsequent layers of inks laden with a gradient in cell concentration, [ 268 ] or with different cartilagederived and bone-marrow derived specific progenitor cells, to recreate the native gradient of glycosaminoglycans (GAGs) and the expression of zonal markers. [ 269 ] Further building on these strategies, layering of multiple materials can allow to create osteochondral graft, comprising of closely interconnected bone and cartilage units.…”

Advancements and Frontiers in the High Performance of Natural Hydrogels for Cartilage Tissue Engineering

“…Sodium citrate was added into ALG as the dispersant of hydroxyapatite (HAP). Eames et al found that the ALG/HAP complex could trigger the chondrocytes to secrete a calcified matrix, which was testified by the favorable survival and proliferation of chondrocytes in the ALG/HAP structure and high expression level of calcified cartilage markers (You et al, 2019). Embedding bone marrow-derived mesenchymal stem cells (bMSCs) in RGD (arginine/glycine/aspartic acid)-functionalized, γ-ray alginate hydrogels could enhance the osteochondral regeneration and promote the development of a more mechanically functional repair tissue (Critchley et al, 2019).…”