Development of a Point of Care 3D Bioprinting System for Wound Healing Applications
In the quest to improve both aesthetic and functional outcomes for patients, the clinical care of full-thickness cutaneous wounds has undergone significant development over the past decade. A shift from replacement to regeneration has prompted the development of skin substitute products, however, inaccurate replication of host tissue properties continues to stand in the way of realising the ultimate goal of scar-free healing. Advances in three-dimensional (3D) bioprinting and biomaterials used for tissue engineering have converged in recent years to present opportunities to progress this field. However, many of the proposed bioprinting strategies for wound healing involve lengthy in-vitro cell culture and construct maturation periods, employ complex deposition technologies, and lack credible point of care (POC) delivery methods. To survive the journey to bedside, printing protocols must be curated, and biomaterials/cells selected which minimise treatment time and facilitate rapid, single-stage wound closure.
In this thesis, we present a novel approach to this clinical challenge by developing a fit-for-purpose in-situ extrusion bioprinting system for full-thickness wound treatment at POC. Our approach was to additively fabricate constructs laden with ReCell™; a clinically regulated autologous cell source which can be rapidly harvested at POC. We hypothesised that the heterogeneous mixture of uncultured dermal and epidermal skin cells would self-assemble in response to endogenous cues in-vivo, thereby facilitating full-thickness wound healing.
In Chapter 3, bioprinting hardware components, including a custom colinear extrusion nozzle and handheld delivery device dubbed the ‘Dermifix’ were designed and developed. The colinear extrusion nozzle was characterised to confirm side-by-side ink deposition, competitive print resolution capabilities and flexibility to alter construct composition by modulating individual ink contributions. The ergonomic design of the Dermifix was showcased through proof-of-concept manual extrusion and precise multi-layer deposition in-vitro. Additionally, hardware components for the spray delivery of an exogenous crosslinking solution were characterised, highlighting the ability of the ReCell™ kit nozzle to deliver crosslinkers in a controlled manner in-situ using optimised operating pressure settings.
The bioprinting hardware developed in Chapter 3 was then employed in Chapter 4 to fabricate multi-material constructs using a novel colinear extrusion method. Ink formulations were optimised to enable the controlled fabrication of multi-layer cross-hatched constructs laden with macropores and rich in cell attachment sites, serving to facilitate the retention of cells and their subsequent migration during self-assembly. A ReCell™ mimic cell suspension containing human dermal fibroblasts (HDFs) and human epidermal keratinocytes (HKs) was developed and subsequently incorporated into the printing process to show that bioprinted constructs supported the survival, attachment and proliferation of both cell types in-vitro.
Finally, in Chapter 5, the proposed handheld bioprinting system was evaluated in-vivo for the treatment of full-thickness porcine wounds at POC. During this investigation, the effect of utilising an autologous ReCell™ suspension in place of the ReCell™ mimic developed in Chapter 4 was highlighted. Specifically, it was shown that residual enzyme molecules in the ReCell™ suspension acted to prematurely degrade the carrier bioink formulation, in turn, diminishing its printability. This challenge was addressed in a follow-up in-vivo experiment by supplementing the bioink formulation with human platelet lysate (HPL). In-situ bioprinted constructs were shown to accelerate wound re-epithelialisation compared to a popular POC treatment method (Integra®). The incorporation of ReCell™ to the bioink formulation did not have a significant impact on the wound healing outcomes assessed, nor did we observe the hypothesised self-assembly of ReCell™ in-vivo. Nevertheless, feasibility of the system at POC was demonstrated for the timely treatment of wounds up to 4 cm2 in size.
Taken together, the proposed 3D bioprinting system holds significant promise as a tool for POC full-thickness wound treatment. The data presented in this thesis establishes a foundation for the further refinement of hardware and ink compositions, aiming to realise improved patient outcomes in the future.
History
Year
2024Thesis type
- Doctoral thesis