Production of Compositional Analogues of Bone Based on Electrophoretically Deposited Mineralised Collagen Films

The mineralisation of collagen fibrils in bone at the nanoscale has a significant role in determining the properties of the tissue at the macroscale. The ability to reproduce this mineralisation in vitro offers the potential to provide insights into the highly complex process in vivo and has implications for the production of successful bone graft materials in the tissue engineering field. Recently, a new bone mineral model has been proposed such that the bone mineral platelets are composed of an apatitic core surrounded by a disordered surface environment, whose shape is maintained by citrate molecules which bridge the mineral platelets in the hydrated, disordered regions. The surface regions can be modelled by octacalcium phosphate-citrate (OCP-CIT), where citrate molecules are found within the hydrated layers of the octacalcium phosphate (OCP) crystal structure. This thesis describes work undertaken to fabricate mineralised collagen films using a “bottom-up” development approach, starting with the optimisation of OCP synthesis, followed by mineralisation of collagen suspensions and then evaluation of the collagen-mineral interface. Initially, the suitability of two OCP-carboxylate synthesis routes for collagen mineralisation was investigated. The synthesis parameters of the hydrolysis of α-tricalcium phosphate (α-TCP) and calcium carbonate were optimised and the phase purity and crystallinity of the synthesis products were evaluated using X-ray diffraction (XRD) and nuclear magnetic resonance (NMR) analysis. Reducing the temperature of calcium carbonate hydrolysis from 60◦C to 37◦C was found to decrease the OCP crystallinity. However, the crystallinity of OCP produced via the hydrolysis of α-TCP, conventionally conducted at 37◦C, increased when the pH was maintained at 6.50 throughout the synthesis, and significantly improved the proportion of OCP in the reaction product. These results suggested that the hydrolysis of α-TCP synthesis route was most appropriate for the next stage of the investigation. The mineral synthesis procedure was then conducted in collagen suspensions. Electrophoretic deposition (EPD) was successfully used to produce defect-free, free-standing mineralised collagen films of at least 20 µm in thickness from mineralised collagen suspensions under an applied voltage of 10 V. The rate of deposition was increased by decreasing the zeta potential of the mineralised collagen particles from -7.7 ± 2.9 to -25.9 ± 3.7 mV by adding hyaluronic acid (0.5 mg/mL). The rate of electrolysis was suppressed by reducing the conductivity of the suspension 100 fold via dialysis against deionised water, and by moderating the deposition and EPD cell parameters. The impact of citrate ions and hyaluronic acid on the collagen mineralisation process was evaluated using XRD and NMR analysis of the mineralised collagen films. The data obtained revealed a similar composition to the proposed bone mineral model when the collagen suspensions were mineralised using the OCP-CIT synthesis, but failed to achieve a bonded collagen-mineral interface. However, when mineralisation was conducted in the presence of both citrate ions and hyaluronic acid, the procedure was found to recapitulate both the bone mineral model composition and the collagen-mineral interface in bone. Preliminary work to test the biological response revealed that the films were too delicate to handle in cell culture. Film stabilisation was required through collagen crosslinking and therefore an assessment of glutaraldehyde, EDC-NHS, and genipin crosslinkers on collagen films produced using EPD was undertaken. The data revealed no significant differences in the morphology and proliferation of human osteoblasts between crosslinking conditions. However, the alkaline phosphatase production and cell clustering was highest on collagen films crosslinked with a relatively low level of EDC-NHS crosslinking, suggesting that these films offered the highest osteogenic potential. In summary, compositional analogues of bone based on electrophoretically deposited mineralised collagen films were produced and characterised, and these offered the composition and collagen-mineral interface predicted from bone models. This work highlights a novel in vitro system to evaluate collagen mineralisation processes and the collagen-mineral interface. This work delivers an important step towards improved bone grafting materials and provides a potential model system for bone disease investigations.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Staunton, Katrina

Advisor dc:contributor.advisor

• Best, Serena

• Collagen mineralisation
• Electrophoretic deposition
• Calcium phosphate
• Synthesis

Licence

• https://www.repository.cam.ac.uk/bitstreams/dbc30c65-76e5-4e39-bf00-e2a980615e26/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.110742

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/371609