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Fabrication of Tissue Engineering Scaffolds Using Stereolithography Benita M. Comeau 226 Pages Directed by Dr. Clifford L. Henderson New methods and materials for the fabrication of hierarchically structured, 3D tissue scaffolds using stereolithography (SL) are presented. The ability to chemically modify selected areas on a scaffold is one way to direct cell growth in deliberate patterns; which is necessary for the engineering of complex, functioning tissues. SL will allow for the building of complex 3D structures with well defined geometries, and a second level of order is created by subsequent modification of chemical groups via catalyzing a de-protection event through exposure to another wavelength of light. The investigated system utilizes an acid-catalyzed de-protection event to change the surface chemistry of an SL-made polymer, analogous to conventional chemically amplified photoresists. The chemical modification alters the surface energy, affecting how proteins interact with the material. This allows selective areas to be more favorable towards cell adhesion. The results of this work include the identification of cytocompatible photo-acid generators that are necessary for the acid-catalyzed de-protection, the demonstration that traditional photolithographic materials may be used for cell patterning, quartz crystal microbalance studies which illuminate why these patterning methods work, the design and performance of a mirror array based stereolithographic apparatus capable of multi-wavelength exposures, and the synthesis and formulation of a novel stereolithographic resin for use in this system. The findings suggest that this system has great potential for use in cell and tissue studies, and possibilities for future use and research are discussed.

Thesis (M.S.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed Jan. 29, 2010). Includes bibliographical references (p. 66-70).

The field of tissue engineering has grown immensely since its inception in the late 1980s. However, currently commercialized tissue engineered products are simple in structure. This is due to a pre-clinical bottleneck in which complex tissues are unable to be fabricated. 3D bioprinting has become a versatile tool in engineering complex tissues and offers a solution to this bottleneck. Characterizing the mechanical properties of engineered tissue constructs provides powerful insight into the viability of engineered tissues for their desired application. Current methods of mechanical characterization of soft hydrogel materials used in tissue engineering destroy the sample and ignore the effect of 3D bioprinting on the overall mechanical properties of a construct. Herein, this work reports on the novel use of a non-destructive method of viscoelastic analysis to demonstrate the influence of 3D bioprinting strategy on mechanical properties of hydrogel tissue scaffolds. 3D bioprinting is demonstrated as a versatile tool with the ability to control mechanical and physical properties. Structure-function relationships are developed for common 3D bioprinting parameters such as printed fiber size, printed scaffold pattern, and bioink formulation. Further studies include effective real-time monitoring of crosslinking, and mechanical characterization of multi-material scaffolds. We envision this method of characterization opening a new wave of understanding and strategy in tissue engineering. / Graduate

A thin layer of calcified cartilage at the native cartilage-to-bone junction facilitates integration between deep zone articular cartilage and subchondral bone, while maintaining the integrity of the two distinct tissue regions. Regeneration of this interface remains a significant clinical challenge for long-term and functional cartilage repair. The strategy for osteochondral interface formation discussed in this thesis focuses on the design and optimization of a biomimetic scaffold for stable calcified cartilage formation. The ideal interface scaffold supports chondrocyte biosynthesis and the formation of calcified cartilage with physiologically-relevant mechanical properties. Furthermore, the interface scaffold allows for osteointegration and the maintenance of the calcified cartilage matrix. It is hypothesized that ceramic presence and zonal chondrocyte interactions regulate cell biosynthesis and mineralization, and these cell-matrix and cell-cell interactions are essential for calcified cartilage formation and maintenance. Biomimetic design parameters for an interface scaffold were determined by characterizing the native interface in terms of mineral and matrix distribution. A composite hydrogel-hydroxyapatite scaffold was then designed to support formation of a functional calcified cartilage matrix. The hydrogel phase maintains the chondrocyte phenotype and allows for incorporation of ceramic particles, while the biomimetic ceramic phase is osteointegrative and decreases the need for cell-mediated mineralization. This scaffold was optimized <italic>in vitro</italic> based on hydrogel type, chondrocyte population, and ceramic particle size. The collective findings from these cell-ceramic interaction studies determined that hypertrophic chondrocytes, cultured in the presence of micron-sized hydroxyapatite particles, exhibit enhanced hypertrophy and matrix deposition. Scaffold ceramic dose and seeding density were also optimized for promoting calcified cartilage formation <italic>in vitro</italic>. In order to implement the scaffold for integrative cartilage repair, a scaffold was designed to regenerate both uncalcified and calcified cartilage on a bilayered hydrogel scaffold. Furthermore, a polymer-ceramic nanofiber component was added to augment the original design for <italic>in vivo</italic> implementation. The hydrogel-nanofiber composite scaffold was evaluated <italic>in vivo</italic> and found to support mineralization and osteointegration within the bone region while preventing endochondral ossification within the repair tissue. Finally, inspired by the stratified organization of zonal chondrocyte populations above the calcified cartilage interface, the layered hydrogel model was used to determine the role of zonal chondrocyte organization on calcified cartilage stability. This thesis collectively explores cell-ceramic and cell-cell interactions, and their ramifications for calcified cartilage formation and maintenance. Specifically, ceramic presence promotes the deposition of a calcified cartilage matrix by hypertrophic chondrocytes in a dose-dependent manner, and furthermore, communication between surface zone and deep zone chondrocyte populations suppresses mineralization within articular cartilage above the calcified cartilage interface. It is anticipated that the scaffold design strategy developed in this thesis can also be applied to the regeneration of other complex interfaces where there are transitions from soft-to-hard tissue.

Thesis (M.S.)--University of Alabama at Birmingham, 2008. / Description based on contents viewed Feb. 5, 2008; title from title screen. Includes bibliographical references (p. 65-68).

Thesis (Ph. D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed Sept. 2, 2009). Additional advisors: Uday Vaidya, Burton Patterson, Susan Bellis, Mark Weaver, Vinoy Thomas. Includes bibliographical references.

The goal of this work was to design and build a mask projection microstereolithography (MPμSL) 3D printing system to characterize, process, and quantify the performance of novel photopolymers. MPμSL is an Additive Manufacturing process that uses DLP technology to digitally pattern UV light and selectively cure entire layers of photopolymer resin and fabricate a three dimensional part. For the MPμSL system designed in this body of work, a process was defined to introduce novel photopolymers and characterize their performance. The characterization process first determines the curing characteristics of the photopolymer, namely the Critical Exposure (Ec) and Depth of Penetration (Dp). Performance of the photopolymer is identified via the fabrication of a benchmark test part, designed to determine the minimum feature size, XY plane accuracy, Z-axis minimum feature size, and Z-axis accuracy of each photopolymer with the system. The first characterized photopolymer was poly (propylene glycol) diacrylate, which was used to benchmark the designed MPμSL system. This included the achievable XY resolution (212 micrometers), minimum layer thickness (20 micrometers), vertical build rate (360 layers/hr), and maximum build volume (6x8x36mm3). This system benchmarking process revealed two areas of underperformance when compared to systems of similar design, which lead to the development of the first two research questions: (i) 'How does minimum feature size vary with exposure energy?' and (ii) 'How does Z-axis accuracy vary with increasing Tinuvin 400 concentration in the prepolymer?' The experiment for research question (i) revealed that achievable feature size decreases by 67% with a 420% increase in exposure energy. Introducing 0.25wt% of the photo-inhibitor Tinuvin 400 demonstrated depth of penetration reduction from 398.5 micrometers to 119.7 micrometers. This corresponds to a decrease in Z-axis error from 119% (no Tinuvin 400) to 9% Z-axis error (0.25% Tinuvin 400). Two novel photopolymers were introduced to the system and characterized. Research question (iii) asks 'What are the curing characteristics of Pluronic L-31 how does it perform in the MPμSL system?' while Research Question 4 similarly queries 'What are the curing characteristics of Phosphonium Ionic Liquid and how does it perform in the MPμSL system?' The Pluronic L-31 with 2wt% photo-initiator had an Ec of 17.2 mJ/cm2 and a Dp of 288.8 micrometers, with a minimum feature size of 57.3 ± 5.7 micrometers, with XY plane error of 6% and a Z-axis error of 83%. Phosphonium Ionic Liquid was mixed in various concentrations into two base polymers, Butyl Diacrylate (0% PIL and 10% PIL) and Poly Ethylene Dimethacrylate (5% PIL, 15% PIL, 25% PIL). Introducing PIL into either base polymer caused the Ec to increase in all samples, while there is no significant trend between increasing concentrations of IL in either PEGDMA or BDA and depth of penetration. Any trends previously identified between penetration depth and Z accuracy do not seem to extend from one resin to another. This means that overall, among all resins, depth of penetration is not an accurate way to predict the Z axis accuracy of a part. Furthermore, increasing concentrations of PIL caused increasing % error in both XY plane and Z-axis accuracy . / Master of Science

Functional integration of bone with soft tissues such as tendon is essential for joint motion and musculoskeletal function. This is evident in the rotator cuff of the shoulder, which consists of four muscles and their associated tendons that connect the humerus and scapula. The cuff functions to stabilize the shoulder joint, and actively controls shoulder kinematics. Rotator cuff injuries often occur as a result of tendon avulsion at the tendon-bone interface, with more than 250,000 cuff repair surgeries performed annually in the United States. However, these procedures are associated with a high failure rate, as re-tears often occur due to the lack of biological fixation of the tendon to bone post-surgery. Instead of regenerating the tendon-bone interface, current repair techniques and augmentation grafts focus on improving the load bearing capability of the repaired rotator cuff. Biologically, the supraspinatus tendon inserts into bone via a biphasic fibrocartilaginous transition, exhibiting region-dependent changes in its compositional, structural and mechanical properties, which enables efficient load transfer from tendon to bone as well as multi-tissue homeostasis. Inspired by the native tendon-bone interface, we have designed and evaluated a biomimetic bilayer scaffold, comprised of electrospun poly (lactide-co-glycolide) (PLGA) nanofibers seamlessly integrated with PLGA-hydroxyapatite (HA) fibers, in order to engineer tendon-bone integration. The objective of this thesis is to explore the key design parameters that are critical for integrative tendon-bone repair using this biphasic scaffold as a model. Specifically, intrinsic to the scaffold, effects of fiber alignment, fiber diameter, mineral distribution, and polymer composition on integrative rotator cuff tendon-bone healing were evaluated in vivo using a rat model. Results indicated that an aligned, nanofiber-based scaffold with a distinct order of non-mineralized and mineralized regions will lead to insertion regeneration and integrative tendon-bone repair. Additional tissue engineering design parameters such as healing time and animal model were also tested. It was observed that the biphasic scaffold exhibited a stable long term response, as the mechanical properties of rat shoulders repaired by this scaffold remained comparable to that of the control at 20 weeks post-surgery. This scaffold was also evaluated in a large animal model (sheep), in which a clinically-relevant rotator cuff repair procedure was implemented with the biphasic scaffold. Results demonstrated the scaffold lead to integrative rotator cuff repair through the regeneration of the enthesis in both small and large animal models. In summary, through a series of in vivo studies, the work of this thesis has identified the critical tissue engineering parameters for integrative and functional rotator cuff tendon repair. More importantly, the design principles elucidated here are anticipated to have a broader impact in the field of tissue engineering, as they can be readily applied towards the regeneration of other soft-hard tissue interfaces.