Hydroxyapatite-decorated Fmoc-hydrogel as a bone-mimicking substrate for osteoclast differentiation and culture

Abstract

Hydrogels are water-swollen networks with great potential for tissue engineering applications. However, their use in bone regeneration is often hampered due to a lack of materials’ mineralization and poor mechanical properties. Moreover, most studies are focused on osteoblasts (OBs) for bone formation, while osteoclasts (OCs), cells involved in bone resorption, are often overlooked. Yet, the role of OCs is pivotal for bone homeostasis and aberrant OC activity has been reported in several pathological diseases, such as osteoporosis and bone cancer. For these reasons, the aim of this work is to develop customised, reinforced hydrogels to be used as material platform to study cell function, cell-material interactions and ultimately to provide a substrate for OC differentiation and culture. Here, Fmoc-based RGD-functionalised peptide hydrogels have been modified with hydroxyapatite nanopowder (Hap) as nanofiller, to create nanocomposite hydrogels. Atomic force microscopy showed that Hap nanoparticles decorate the peptide nanofibres with a repeating pattern, resulting in stiffer hydrogels with improved mechanical properties compared to Hap- and RGD-free controls. Furthermore, these nanocomposites supported adhesion of Raw 264.7 macrophages and their differentiation in 2D to mature OCs, as defined by the adoption of a typical OC morphology (presence of an actin ring, multinucleation, and ruffled plasma membrane). Finally, after 7 days of culture OCs showed an increased expression of TRAP, a typical OC differentiation marker. Collectively, the results suggest that the Hap/Fmoc-RGD hydrogel has a potential for bone tissue engineering, as a 2D model to study impairment or upregulation of OC differentiation. Statement of significance Altered osteoclasts (OC) function is one of the major cause of bone fracture in the most commonly skeletal disorders (e.g. osteoporosis). Peptide hydrogels can be used as a platform to mimic the bone microenvironment and provide a tool to assess OC differentiation and function. Moreover, hydrogels can incorporate different nanofillers to yield hybrid biomaterials with enhanced mechanical properties and improved cytocompatibility. Herein, Fmoc-based RGD-functionalised peptide hydrogels were decorated with hydroxyapatite (Hap) nanoparticles to generate a hydrogel with improved rheological properties. Furthermore, they are able to support osteoclastogenesis of Raw264.7 cells in vitro as confirmed by morphology changes and expression of OC-markers. Therefore, this Hap-decorated hydrogel can be used as a template to successfully differentiate OC and potentially study OC dysfunction.

Translating Material Science into Bone Regenerative Medicine Applications: State-of-The Art Methods and Protocols

“…Several papers also aim to evaluate in vitro the important prerequisite of resorbability of bone graft substitutes, defined as the capability of biomaterials to disappear from the site of implantation over time [ 129 ]. Standard protocols are usually based on the cultivation of primary monocytes/macrophages isolated from blood samples [ 130 , 131 , 132 ] or from bone marrow [ 106 , 113 , 133 ], or of the RAW 264.7 murine macrophage cell line [ 133 , 134 , 135 ] on the scaffolds of interest, followed by the analysis of monocyte adhesion, proliferation, and osteoclast formation. The osteoclastogenesis process can be studied using different protocols.…”

“…The presence of tartrate resistant acidic phosphate (TRAP)-positive cells, representing mature osteoclasts, is usually evaluated [ 106 , 113 , 131 , 132 , 133 , 134 , 135 ]. The measure of the enzymatic activity of TRAP5b and cathepsin K (CTSK) in the medium supernatant of scaffold-seeded cells, as well as of their expression levels, are also reported [ 92 , 130 , 133 ].…”

“…The expression levels of key markers involved in osteoclast differentiation such as TRAP, TNF receptor superfamily member 11a (TNFRSF11A/RANK), TNF superfamily member 11 (TNFSF11/RANKL), nuclear factor of activated T cells 1 (NFATC1), matrix metallopeptidase 9 (MMP9), osteoclast stimulatory transmembrane protein (OCSTAMP), and dendrocyte expressed seven transmembrane protein (DCSTAMP) are also analyzed at both transcript and protein level [ 72 , 92 , 106 , 133 , 134 , 135 ].…”

Hydrogel-based delivery system applied in the local anti-osteoporotic bone defects

“…However, chitosan-based hydrogels often fail to meet the mechanical strength requirements of bone fillers. The poor mechanical strength of chitosan-based hydrogels can be compensated by incorporating several types of nanofillers, i.e., carbon nanotubes and cellulose nanocrystals, which is particularly important for treating managing osteoporotic vertebral compression fractures (Cancian et al, 2016;Ghavimi et al, 2019;Vitale et al, 2022). Frontiers in Bioengineering and Biotechnology frontiersin.org…”

Hydroxyapatite-Tethered Peptide Hydrogel Promotes Osteogenesis

“…Wang et al [ 23 ] introduced a cell adhesive ligand Fmoc-arginyl-glycyl-aspartate (FmocRGD) and developed FmocFF/FmocRGD hydrogel for three-dimensional (3D) culturing of mesenchymal stem cells. Recently, Vitale et al [ 24 ] developed a three-component assembled hydrogel composed of FmocFF/FmocS/FmocRGD, where FmocS was Fmoc-serine. This hydrogel enabled HAp-binding and cell adhesion, and promoted osteoclast differentiation of RAW264.7 macrophages, showing potential in bone tissue engineering.…”