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Ti-Graphene Bone Tissue Template Engineering

Periodic Reporting for period 1 - BonE-GraphT (Ti-Graphene Bone Tissue Template Engineering)

Reporting period: 2017-07-01 to 2019-06-30

Bone is a regenerative tissue, with potential for healing provided certain physiological conditions are met for: supporting angiogenesis for tissue restoration; environment osteogenesis for remineralisation by using growth factors/bone morphogenic proteins; supporting bio-mechanical function by load transmission for preventing bone non-union. Absence of one or more factors become apparent when damaged bone stops healing after surgery. During healing processes, overall osteogenicity may be explained by osteoinduction, conduction and integration. BonE-GraphT focussed on promoting osteoinduction/conduction in a new materials engineering concept, where bone-forming mineral is grown with an electrically conducting graphene on a titanium surface, for inducing osteoblast cell response for remineralisation. Deposition of calcium phosphate minerals using ultrafast femtosecond pulsed laser on titanium metal substrate, with/without graphene as an electrically conducting medium, was investigated. Deposited thin film composite materials were analysed for cell proliferation tests to ascertain osteogenic potential for surface modified materials.

Training summary: This fellowship offered opportunity for research training in: advanced biomaterials processing/characterisation, osteogenic characterisation; opportunity for interaction with a Clinician, Prof Giannoudis, trauma/orthopaedic surgeon. Cell characterisation training was supported by Dr Raif, who trained the Fellow in osteoblast cell growth. Materials characterisation support was provided by host institute Research team. Original research findings were presented at conferences/published in peer-reviewed journals.

Emerging Societal Importance: Probability of bone related injury increases with age due to falls, osteoporosis and bone disease. Healing potential of the human body also diminishes with age, demanding research in bone materials for promoting bone healing. Bone failure data from the International Osteoporotic Society is quite compelling in demonstrating risk of bone failure in 50-60 age group (female/males). Main cause of bone failure remains loss of bone mineral density for supporting load-bearing capacity of the body. Combined with increasing obesity, bone-failure related morbidity continues to increase not only within the EU but also in the rest of the developed and emerging economies. Chance of full recovery of a 60+ year old patient suffering from acute osteoporosis, after trauma reduces dramatically to less than 25%. Over 300,000 hospital admissions occurred in 2017 in UK due to poor bone stock in frail patients. Frailty based bone injury may be prevented via tissue augmentation and via increased osteoinduction and conduction for remineralisation which has been investigated in BonE-GraphT as a solution for bone-compromised patients. The research is continuing since the BonE-GraphT ended for developing a new paradigm for treating osteoporotic patients.

Main research training objectives for realising research impact were:
1. To develop osteoconductive/inductive surface on titanium alloys, widely used for implant/post-surgery fracture fixation. It is essential that such materials are non-toxic, mechanically robust in the human body. Thus, studying coating of Ti-alloys with osteogenic surface via novel cost-compatible techniques will help to develop implants for compromised patients.
2. Research training demonstrated that pulsed laser deposition of HAp films on Ti-alloy & formation of graphene as a compatible surface is possible for surface engineering of implant materials. Such materials were successfully tested for osteo-induction/conduction.
3. Research also offered opportunity for bringing together novel tools for materials characterisation/understanding of 2D-materials growth on metal & inorganic glass surface for range of sensing/light-generation device applications, which may be compatible with bio-implants. Deposition of 2D-MoS2 materials were studied for light emission properties. A long-term objective is to design osteoconductive/inductive bone implants with integrated light-based sensor for monitoring healing processes
Research training activities:
• Literature review - Ti-alloy bone implant integration/current failure rates. Patients with 10+-year old implants were more likely to have implant failure due to age, loss of bone stock or implant weakening.
• Research ethics form was completed as a deliverable and relevant ethics/lab safety training were completed before experimental work began. Fellow was monitored for progress in their training activities, fortnightly.
• RTA activities commenced, after laser safety course/training were successfully completed.
• Ti-alloy based graphene/HAp materials processing using fs-PLD with graphene as strengthening and osteoconducting component for bone-template engineering was studied. We investigated graphene synthesis using fs-PLD and grew graphene layers using amorphous carbon films of finite thicknesses after annealing in the presence of nickel catalyst, which was then removed and HAp layer was grown using PLD.
• Characterisation of Graphene and HA-p-G on porous Ti-alloys: Second part of training activities focussed on thin film materials characterisation after PLD. We used FTIR, X-ray diffraction, Raman spectroscopy, TEM/SEM/XPS and fluorescence spectroscopy for the deposited materials analysis.
• Cell cultivation, cytotoxicity, cell attachment & cyto-mineralization: Fellow received training on cell culture experimental methodologies in Oral Biology at St James Hospital. Once deposition condition for calcium phosphate and carbon was optimised, osteoblast cell culture tests were performed for supporting mineralization. Cell proliferation/viability tests were carried out using laser confocal microscopy, and the mineral samples were analysed using Raman spectroscopy. Nanoscale toxicity effects were also investigated.
• BonE-GraphT also investigated the application of 2D-MoS2 films on glass surface for measuring bone healing by characterising vitality using the mineralisation and blood circulation using near-IR photon emission technique. The growth of MoS2 thin films using PLD silica was demonstrated and light-emission properties were characterised for implantable medical device engineering.
Leeds University continues the research, via new Prof Giannoudis-led EU (SBR starts in Feb 2020) project in School of Medicine with 3 concurrent PhD projects. In SBR the Leeds team is developing a novel sensor methodology for bone healing. Also, via support of MRC-CiC project, animal model studies are also being planned, which will complete in June 2020.
Researcher training supervisors
Confocal mage graphene oxide-calcium phosphate composite structure showing cell attachment
Bone cross-section
Cross-sectional TEM image of 2D-MoS2.
Group Photo with Marie-Curie and PDRF Fellows, PhD, MSc, and UG students
Cell attachment/proliferation G292 cells on CaP & a-C thin films deposited using fs-PLD on Ti substr
Graphene/CaP composite growth using femtosec pulsed laser deposition/design of final bone structure
Raman spectra and TEM image of graphene deposited using Fs-PLD