Bioinspired Collagen/Glycosaminoglycan-Based Cellular Microenvironments for Tuning Osteoclastogenesis

Abstract

Replicating the biocomplexity of native extracellular matrices (ECM) is critical for a deeper understanding of biochemical signals influencing bone homeostasis. This will foster the development of bioinspired biomaterials with adjustable bone-inducing properties. Collagen-based coatings containing single HA derivatives have previously been reported to promote osteogenic differentiation and modulate osteoclastogenesis and resorption depending on their sulfation degree. However, the potential impact of different GAG concentrations as well as the interplay of multiple GAGs in these coatings is not characterized in detail to date. These aspects were addressed in the current study by integrating HA and different sulfate-modified HA derivatives (sHA) during collagen in vitro fibrillogenesis. Besides cellular microenvironments with systematically altered single-GAG concentrations, matrices containing both low and high sHA (sHA1, sHA4) were characterized by biochemical analysis such as agarose gel electrophoresis, performed for the first time with sHA derivatives. The morphology and composition of the collagen coatings were altered in a GAG sulfation- and concentration-dependent manner. In multi-GAG microenvironments, atomic force microscopy revealed intermediate collagen fibril structures with thin fibrils and microfibrils. GAG sulfation altered the surface charge of the coatings as demonstrated by ζ-potential measurements revealed for the first time as well. This highlights the prospect of GAG-containing matrices to adjust defined surface charge properties. The sHA4- and the multi-GAG coatings alike significantly enhanced the viability of murine osteoclast-precursor-like RAW264.7 cells. Although in single-GAG matrices there was no dose-dependent effect on cell viability, osteoclastogenesis was significantly suppressed only on sHA4-coatings in a dose-dependent fashion. The multi-GAG coatings led to an antiosteoclastogenic effect in-between those with single-GAGs which cannot simply be attributed to the overall content of sulfate groups. These data suggest that the interplay of sGAGs influences bone cell behavior. Whether these findings translate into favorable biomaterial properties needs to be validated in vivo.

Sulfated hyaluronan alters fibronectin matrix assembly and promotes osteogenic differentiation of human bone marrow stromal cells

“…A degraded, low-molecular weight HA, was prepared as previously described to serve as reference material for in vitro experiments since the molecular weight of natural HA precursor is decreased during sulfation45545556 HA derivatives with a degree of sulfation (D.S., number of sulfate residues per disaccharide unit) of 1.0–1.5 referred to as low-sulfated HA derivative sHA1 (Table 1). …”

“…7, Table 1)5657. The double-labeled ATTO565-ATTO655-sHA1 derivative was prepared by coupling ATTO565-NH 2 to the reducing end of sHA1 via an azomethine bond and subsequent reduction of the C=N double bond (end-on functionalization), followed by coupling of ATTO655-NH 2 at the carboxyl group of the GAG using EDC/NHS (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxy succinimide).…”

Collagen: a network for regenerative medicine

“…In mixtures of collagen and hyaluronic acid, it was found that additions of sulphate groups to the hyaluronic acid led to a decrease in fiber size after fibrillogenesis. 54 The extra sulphation also correlated to a decrease in osteoclastogenesis. 54 However, organic materials are not the only molecules which can be incorporated into growing collagen fibers.…”

“…37,39 These molecules can be incorporated into growing fibrils during in vitro fibrillogenesis, and their properties influence the final structure. 54 In mixtures of collagen and hyaluronic acid, it was found that the sulphation state of the GAG altered the isoelectric point of the final gels formed, and higher sulphation decreased the fibril size. 54 Silicates can also can interact with collagen fibrils to control nucleation.…”

“…54 In mixtures of collagen and hyaluronic acid, it was found that the sulphation state of the GAG altered the isoelectric point of the final gels formed, and higher sulphation decreased the fibril size. 54 Silicates can also can interact with collagen fibrils to control nucleation. When silica nanoparticles were negatively charged, positively charged collagen fibrils associated with the particles, and formed fibers as the pH was lowered.…”