Production of Multi-layer and Cell-Containing Collagen Membranes Using Direct Electrophoretic Deposition

Mammalian soft tissues generally comprise cells embedded in a collagen-based extracellular matrix (ECM). The most abundant form of collagen is type I, which is therefore a logical choice for tissue repair applications, such as wound healing and vascular structures. Collagen membranes can be produced using electrophoretic deposition (EPD). A voltage is applied to an aqueous suspension between two electrodes, causing movement of charged particles to form a dense membrane in a variety of shapes and sizes. The films need to be removed easily from their electrode and their strength and tear resistance optimised to create suturable components. This work investigates the production of collagen membranes via EPD, characterises the physical and mechanical properties of multi-layered structures and explores the feasibility of incorporating live cells directly from suspension. Collagen films produced using EPD may suffer from adhesion effects at the electrode and bubbles from electrolysis. A dialysed sacrificial layer of gelatin was used circumvent these issues and effects were tested for samples deposited at 30 V for 10 minutes. Dialysis decreased the conductivity of the gelatin and reduced the current measured significantly, and observations using microscopy revealed that bubble nucleation was suppressed. However, deposited collagen membranes were found to tear easily when handled. Therefore, multiple collagen depositions on the gelatin-coated substrates were investigated. Multilayer collagen films were deposited using a direct voltage of 30 V for 10 minutes per layer. The mass, thickness and density of the membranes increased as multiple layers were deposited. The tensile strength and trouser tear resistance of the multi-layer collagen membranes were tested at 37 °C in-aqua. Increasing the number of layers from 1 to 6, resulted in increases in the tensile modulus from 0.43 to 0.78 MPa, the ultimate tensile strength (UTS) from 0.282 to 0.434 MPa, and the tear resistance from 280 to 450 J/m2. These changes were related to densification of individual layers on increased exposure to electric field. The mechanical property values were lower than for cast collagen membranes in the literature and therefore the effect of aligning the collagen fibres in multi-layer membranes was investigated. Aligned collagen membranes with a coherency index of 0.43 were deposited using 30 V direct voltage and a bespoke EPD flow-rig. The effect of collagen fibre alignment and orientation on the tensile and tear resistance of membranes was investigated for multilayer samples. The tensile modulus and UTS of the membranes increased and the tear resistance decreased with increasing alignment of the fibres to the loading direction. For a 1-layer membrane, the tensile modulus for films in-aqua ranged from 0.73 – 1.53 MPa, the UTS from 0.46 – 0.88 MPa, and the tear resistance from 384 – 832 J/m2. For a 3-layer membrane with orientations varying at 120 between layers, the tensile modulus was 1.79 MPa, UTS was 1.15 MPa, and tear resistance was 799 J/m2 and these values compared more favourably with those found in the literature. To investigate the feasibility of co-depositing human dermal fibroblast (HDF) cells with collagen, experiments were conducted using 30 V direct voltage with cell-containing mixture of cell media and sucrose. Using two photon microscopy, HDF cells were observed to be distributed throughout the deposited collagen membranes. Cell viability decreased with increasing deposition time. For experiments conducted at 30 V, a deposition time of 6 minutes provided the optimal combination of collagen deposition and cell concentration, yielding a 55% cell viability. It was hypothesised that reducing applied voltage and pulsing the voltage might improve the viability of HDF cells. Direct and pulsed voltages (10, 20 and 30 V) were investigated for deposition times from 3 to 15 minutes. Cell viability increased with decreasing deposition time and applied voltage. HDF cells experienced necrosis close to the depositing electrode at higher deposition voltages and at longer deposition times, potentially due the presence of an acidic pH or the release of metal ions next to the electrode. Pulsing the voltage improved cell viability by up to 15% compared with the same deposition times and voltages applied with a continuous voltage. Under optimum conditions it was possible to achieve 76% cell viability. In summary, free-standing multilayer membranes with aligned and oriented collagen fibrils were produced with mechanical properties that offer potential for surgical suturing. By controlling deposition parameters and electrolyte environment, it was possible to co-deposit live HDF cells within the collagen membrane as a possible solution for tissue repair and wound healing applications.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Bonfield, Jack

Advisor dc:contributor.advisor

• Best, Serena

• Collagen
• Electrophoretic deposition
• Membrane
• Biomaterials
• Medical materials
• Polymer
• Fibroblasts

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/9b258a9c-76c6-449f-a4c8-e7aa7d9083a8/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.117921

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/383616