Droplet-based bioprinting for craniomaxillofacial skin reconstruction

Open Access
- Author:
- Gudapati, Hemanth
- Graduate Program:
- Engineering Science and Mechanics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 12, 2020
- Committee Members:
- Ibrahim Tarik Ozbolat, Dissertation Advisor/Co-Advisor
Ibrahim Tarik Ozbolat, Committee Chair/Co-Chair
Bruce Gluckman, Committee Member
Larry Cheng, Committee Member
Justin Lee Brown, Outside Member
Ralph H Colby, Outside Member
Judith Todd Copley, Program Head/Chair - Keywords:
- Protein Rheology
Protein Inkjet
Rodent Skin Reconstruction
Droplet-based Bioprinting
Microvalve-based Bioprinting
Collagen
Fibrinogen
Thrombin
Interfacial Rheology
3D Bioprinting
Craniomaxillofacial Tissue Reconstruction
Interfacial Protein Aggregation - Abstract:
- Craniomaxillofacial (CMF) anomalies affect 7% of the newborns and 3 million children and adults every year in the United States. These anomalies arise because of birth defects, accidents (trauma), removal of tumors, and degenerative diseases. The anomalies not only physically handicap their victims but also psychologically affect them. Repair of the anomalies requires the reconstruction of underlying hard and soft tissues such as bone and skin. Reconstruction of bone, which is superior to demineralized bone (or bone devoid of calcium) has been demonstrated. Although skin is a very simple organ, reconstruction of skin which is closer to native (healthy) skin has not been demonstrated. At present, damaged skin is reconstructed by transplanting healthy skin from the patients themselves and/or donors. However, this causes a new injury at the donor site which is analogous to a second-degree burn. Tissue engineering aims solves this by engineering skin artificially. Traditional top-down tissue engineering approaches rely on porous scaffolds to reconstruct damaged tissues. The scaffolds act as a template and guides the growth of new tissue. However, migration of specific cells at predetermined locations is difficult to control. Accordingly, regeneration of blood vessels and recapitulation native skin architecture is difficult. Bottom-up tissue engineering aims to solve this by assembling skin modularly with microscale building blocks. Droplet-based bioprinting (DBB) is such a bottom-up tissue engineering approach which uses droplets of 10 – 500 µm in diameter as the building blocks. Collagen type I and fibrin hydrogels are widely used as structural and functional biomaterials in tissue engineering applications because of their non-toxic polymerization mechanism. Also, they closely mimic the natural microenvironments of living cells and are biodegradable. The main objective of this dissertation is to study the reconstruction of skin with droplet-based bioprinting of collagen, fibrinogen and thrombin aqueous-buffered protein solutions, which are the precursors of collagen and fibrin hydrogels. This was investigated with bulk and interfacial rheology of the protein solutions to understand their flow behavior, inkjet printing experiments to understand the dynamics of droplet formation and breakup of the solutions, and direct deposition of droplets of the protein solutions containing skin cells and physiological signaling molecules into the critical size full-thickness skin wounds in the CMF area of rodent models. Direct deposition of collagen, fibrinogen, and thrombin solutions into the skin wounds accelerates wound healing. In addition, fibrous proteins collagen and fibrinogen and globular protein thrombin adsorb and aggregate at the solution/air interface and form a viscoelastic solid layer at the interface. The viscoelastic film corrupts the bulk rheological measurements in rotational rheometers by contributing to an apparent yield stress. The addition of a non-ionic surfactant, such as polysorbate 80 (PS80) in small amounts between 0.001 and 0.1 v/v%, prevents the formation of the interfacial layer, allowing the estimation of true bulk viscosity of the solutions. Further, dilute protein solutions form stable droplets during inkjet printing. Conversely, semidilute unentangled protein solutions, the solutions in which protein molecules begin to interpenetrate each other, form undesirable satellite droplets because of constant presence of aggregates or sub visible particles near the nozzle orifice. The volume of cylindrical jets or ligaments decreases with increasing protein concentration during microvalve-based bioprinting. Ligament volume does not decrease with increasing number of delivered jets for collagen solutions, suggesting absence of aggregation of collagen at the solution/solid interface of microvalve devices. Ligament volume decreases with increasing number of delivered jets for fibrinogen and thrombin solutions, suggesting aggregation of the proteins at the solution/solid interface of channels of the devices.