Customised hydrogel matrices for osteoclast differentiation and culture

  • Mattia Vitale

Student thesis: Phd


Bone is a highly dynamic tissue that constantly undergoes remodelling to ensure correct turnover over time. Bone homeostasis is finely balanced by osteoclasts, that resorb bone, and osteoblasts that lay down new bone matrix. Most studies are focused on the osteoblasts role in bone formation while osteoclasts are often overlooked. Yet, the role of osteoclasts is pivotal for bone homeostasis and aberrant osteoclast activity has been reported in several pathological diseases, such as osteoporosis and bone cancer. Therefore, it is important to develop cell culture platforms for the study of osteoclast function and their interactions with the surrounding matrix. As such the overall aim of this work is to develop customised hydrogel-based materials for use as substrates to study osteoclast differentiation and function in vitro. Hydrogels are water-swollen networks with great potential for tissue engineering applications. However, their use in bone regeneration has often been hampered by insufficient mechanical properties and lack of mineralization. Here are presented data on the modification of self-assembling peptide hydrogels with both minerals and matrix proteins which demonstrate the improved performance of the modified hydrogels as platforms for cell culture and differentiation. Hydroxyapatite was used in order to create a nanocomposite hydrogel with enhanced mechanical properties as well as improved bioactivity. Atomic force microscopy confirmed that hydroxyapatite nanoparticles were successfully incorporated within Fmoc-based hydrogels without disrupting the self-assembling mechanism while providing a superior scaffold with improved mechanical properties. Interestingly, the newly developed nanocomposite supported the viability and differentiation of pre-osteoclasts in vitro. This was confirmed by the presence of typical mature osteoclasts features (e.g. multinucleation and actin ring) and expression of typical osteoclast genes. A new protocol to incorporate collagen intro self-assembly peptide hydrogels was also developed. Hydrogels were modified through a passive diffusion protocol in which collagen molecules of different sizes were successfully incorporated and retained over time. SDS-PAGE showed that these collagens interact with the hydrogel fibres without affecting the overall mechanical properties of the composite hydrogels. Furthermore, the collagen molecules incorporated into the hydrogels were still biologically active and provided sites for adhesion and spreading of human fibrosarcoma cells through interaction with the α2β1 integrin. Finally, the effect of collagen as costimulatory pathway during osteoclastogenesis via OSCAR, a well-known osteoclast associated receptor, was investigated. Different proteins containing the OSCAR-collagen binding domain and their binding to OSCAR, were characterised by circular dichroism and surface plasmon resonance, respectively. OSCAR-peptides were incorporated into the previously developed hydroxyapatite-decorated hydrogel. Resulting scaffolds were tested to assess their capability of trigger the differentiation of osteoclast compared to the unmodified hydrogels. As expected, collagen peptides containing the OSCAR-binding domain, that were incorporated into hydroxyapatite modified hydrogels, enhanced the differentiation of osteoclast precursors compared to the unmodified hydrogels. This work laid the foundation for generating new bone-mimicking substrates that can be used as a platform to generate mature osteoclasts which can be progressed to help study the pathological mechanisms associated with their over activation during bone disease.
Date of Award31 Dec 2022
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorJudith Hoyland (Supervisor), Stephen Richardson (Supervisor) & Jordi Bella (Supervisor)


  • Fmoc
  • hydroxyapatite
  • nanocomposite
  • collagen
  • osteoclasts
  • hydrogel
  • biomaterials

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