Indiana University-Purdue University Indianapolis (IUPUI) / EFFECTS OF DYNAMATRIX® ON ANGIOGENIC CYTOKINE EXPRESSION FROM HUMAN DENTAL PULP FIBROBLASTS: AN IN VITRO STUDY by Joseph Benjamin Adams Indiana University School of Dentistry Indianapolis, IN Introduction: An exogenous scaffold may lead to more predictable pulp tissue regeneration and continued root formation in a regenerative endodontic procedure. DynaMatrix® is a natural membrane scaffold made of porcine small intestine, currently used in periodontal regenerative surgeries. Objective: The purpose of this study was to investigate if human dental pulp fibroblasts (HDPFs) seeded on DynaMatrix® membrane would result in an increase in the expression of angiogenic cytokines. Materials and Methods: HDPFs (75,000 per well) were seeded in 6-well plates. Three groups were tested: Group 1 (C): HDPFs in 70 media only; Group 2 (M): DynaMatrix® (Cook Biotech, Indianapolis, IN) alone in media; and Group 3 (C+M): HDPFs seeded on DynaMatrix® membranes. After 72 hours of incubation in serum positive, the conditioned media were collected and analyzed for the expression of 20 angiogenic cytokines utilizing RayBiotech Inc., arrays per the manufacturer’s instruction. The data were analyzed by ANOVA. Results: Group M was significantly higher than C for bFGF (p = 0.0023). C+M was significantly higher than M for ANG (p = 0.0104); GRO (p = 0.0003); IFN-γ (p = 0.0023); IL-6 (p = 0.0003); IL-8 (p = 0.0003); Leptin (p = 0.0003); MCP-1 (p = 0.0104); TIMP-1 (p = 0.0190); TIMP-2 (0.0123). C was significantly higher than C+M for ANG (p = 0.0104); MCP-1 (p = 0.0104); and THPO (p = 0.0308). Cytokines such as b-FGF, ANG, and leptin promote angiogenesis, and stimulate migration and proliferation of cells. Conclusion: The cytokine expression profile from the cells seeded on DynaMatrix® suggests that it might be a suitable scaffold for regenerative endodontic procedures. It could improve vascularization by increasing angiogenic cytokines in the microenvironment of the treated root canal and supporting tissue regeneration.

For small diameter (<6 mm) blood vessel replacements, lack of collaterals and vascular disease preclude homografts; while synthetic analogs, ePTFE, expanded polytetrafluoroethylene, and PET, polyethyleneterephathalate, are prone to acute thrombosis and restenosis. It is postulated that the hierarchical assembly of cell populated matrices fabricated from protein analogs provides a new design strategy for generating a structurally viable tissue engineered vascular graft. To this end, synthetic elastin and collagen fiber analogs offer a novel strategy for creating tissue engineered vascular grafts with mechanical and biological properties that match or exceed those of native vessels. This work details techniques developed for the fabrication of prosthetic vascular grafts from a series of extracellular matrix analogs composed of nanofibrous collagen matrices and elastin-mimetic proteins, with and without cells, and subsequent evaluation of their biocompatibility and mechanical properties. The work details the fabrication and mechanical analysis of vascular grafts made from aforementioned protein analogs. Subesequent studies detail seeding and proliferation of rodent mesenchymal stem cells on protein-based composites to recapitulate the media of native vasculature. Finally detailing in vivo biocompatibility and stability of tissue engineered vascular grafts.

This research centers on the fabrication, characterization, and engineering of inverse opal scaffolds, a novel class of three-dimensional (3D) porous scaffolds made of biocompatible and biodegradable polymers, for applications in tissue engineering and regenerative medicine. The unique features of an inverse opal scaffold include a highly ordered array of pores, uniform and finely tunable pore sizes, high interconnectivity, and great reproducibility. The first part of this work focuses on the fabrication and functionalization of inverse opal scaffolds based on poly(D,L-lactic-co-glycolic acid) (PLGA), a biodegradable material approved by the U.S. Food and Drug Administration (FDA). The advantages of the PLGA inverse opal scaffolds are also demonstrated by comparing with their counterparts with spherical but non-uniform pores and poor interconnectivity. The second part of this work shows two examples where the PLGA inverse opal scaffolds were successfully used as a well-defined system to investigate the effect of pore size of a 3D porous scaffold on the behavior of cell and tissue growth. Specifically, I have demonstrated that i) the differentiation of progenitor cells in vitro was dependent on the pore size of PLGA-based scaffolds and the behavior of the cells was determined by the size of individual pores where the cells resided in, and ii) the neovascularization process in vivo could be directly manipulated by controlling a combination of pore and window sizes when they were applied to a mouse model. The last part of this work deals with the novel application of photoacoustic microscopy (PAM), a volumetric imaging modality recently developed, to tissue engineering and regenerative medicine, in the context of non-invasive imaging and quantification of cells and tissues grown in PLGA inverse opal scaffolds, both in vitro and in vivo. Furthermore, the capability of PAM to monitor and quantitatively analyze the degradation of the scaffolds themselves was also demonstrated.

Biodegradable hydrogels have become very promising materials for a number of biomedical applications, including tissue engineering and drug delivery. For optimal tissue engineering design, the mechanical properties of hydrogels should match those of native tissues as closely as possible because these properties are known to affect the behavior and function of cells seeded in the hydrogels. At the same time, high water-contents, large mesh sizes and well-tuned degradation rates are favorable for the controlled release of growth factors and for adequate transport of nutrients through the hydrogel during tissue regeneration. With these factors in mind, the goal of this research was to develop and investigate the behavior of injectable, biodegradable hydrogels with enhanced stiffness properties that persist even at high degrees of swelling. In order to do this, degradable functionalities were incorporated into photo-crosslinkable poly(ethylene glycol) and poly(acrylic acid) hydrogels, and these two components were used to make a series of double-network hydrogels. Synthesis of the precursor macromers, photopolymerization of the hydrogels, and structural parameters of the hydrogels were analyzed. The composition and the molecular weight between crosslinks (Mc) of the hydrogel components were varied, and the degradation, swelling, thermal and mechanical properties of the hydrogels were characterized over various time scales. These properties were compared to corresponding properties of the component single-network hydrogels.

Thesis (Ph.D)--University of Tennessee Health Science Center, 2007. / Title from title page screen (viewed on October 8, 2007). Research advisor: Joo L. Ong, Ph.D. Document formatted into pages (xiii, 128 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 106-114).

Over 600,000 bone-grafting operations are performed each year in the United States. The majority of the bone used for these surgeries comes from autografts that are limited in quantity or allografts with high failure rates. Current synthetic bone grafting materials have poor mechanical properties, handling characteristics, and bioactivity. The goal of this dissertation was to develop a clinically translatable bone tissue engineering scaffold with improved handling characteristics, bioactivity, and smart delivery modalities. We hypothesized that this could be achieved through the rational selection of Food and Drug Administration (FDA) approved materials that blend favorably with hydroxyapatite (HA), the principle mineral component in bone. This dissertation describes the development of smart bone tissue engineering scaffolds composed of the biodegradable amphiphilic polymer poly(D,L-lactic acid-co-ethylene glycol-co- D,L-lactic acid) (PELA) and HA. Electrospun nanofibrous HA-PELA scaffolds exhibited improved handling characteristics and bioactivity over conventional HApoly( D,L-lactic acid) composites. Electrospun HA-PELA was hydrophilic, elastic, stiffened upon hydration, and supported the attachment and osteogenic differentiation of rat bone marrow stromal cells (MSCs). These in vitro properties translated into robust bone formation in vivo using a critical-size femoral defect model in rats. Spiral-wrapped HA-PELA scaffolds, loaded with MSCs or a lowdose of recombinant human bone morphogenetic protein-2, templated bone formation along the defect. As an alternate approach, PELA and HA-PELA were viii rapid prototyped into three-dimensional (3-D) macroporous scaffolds using a consumer-grade 3-D printer. These 3-D scaffolds have differential cell adhesion characteristics, swell and stiffen upon hydration, and exhibit hydration-induced self-fixation in a simulated confined defect. HA-PELA also exhibits thermal shape memory behavior, enabling the minimally invasive delivery and rapid (>3 sec) shape recovery of 3-D scaffolds at physiologically safe temperatures (~ 50ºC). Overall, this dissertation demonstrates how the rational selection of FDA approved materials with synergistic interactions results in smart biomaterials with high potential for clinical translation.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydrogels prepared from photopolymerization have been widely used in many biomedical applications. Ultraviolet (200-400 nm) or visible (400-800 nm) light can interact with light-sensitive compounds called photoinitiators to form radical species that trigger photopolylmerization. Since UV light has potential to cause cell damage, visible light-mediated photopolymerization has attracted much attention. The conventional method to fabricate hydrogels under visible light exposure requires usage of co-initiator triethanolamine (TEA) at high concentration (∼200 mM), which reduces cell viability. Therefore, the first objective of this thesis was to develop a new method to form poly(ethylene glycol)-diacrylate (PEGDA) hydrogel without using TEA. Specifically, thiol-containing molecules (e.g. dithiothreitol or cysteine-containing peptides) were used to replace TEA as both co-initiator and crosslinker. Co-monomer 1-vinyl-2-pyrrolidinone (NVP) was used to accelerate gelation kinetics. The gelation rate could be tuned by changing the concentration of eosinY or NVP. Variation of thiol concentration affected degradation rate of hydrogels. Many bioactive motifs have been immobilized into hydrogels to enhance cell attachment and adhesion in previous studies. In this thesis, pendant peptide RGDS was incorporated via two methods with high incorporation efficiency. The stiffness of hydrogels decreased when incorporating RGDS. The second objective of this thesis was to fabricate hydrogels using poly(ethylene glycol)-tetra-acrylate (PEG4A) macromer instead of PEGDA via the same step-and-chain-growth mixed mode mechanism. Formation of hydrogels using PEGDA in this thesis required high concentration of macromer (∼10 wt.%). Since PEG4A had two more functional acrylate groups than PEGDA, hydrogels could be fabricated using lower concentration of PEG4A (∼4 wt.%). The effects of NVP concentration and thiol content on hydrogel properties were similar to those on PEGDA hydrogels. In addition, the functionality and chemistry of thiol could also affect hydrogel properties.

Thesis (Ph. D.)--University of South Florida, 2008. / Title from PDF of title page. Document formatted into pages; contains 133 pages. Includes vita. Includes bibliographical references.

Indiana University-Purdue University Indianapolis (IUPUI) / Dicalcium phosphate dihydrate (DCPD) cements have been used for bone repair due to its excellent biocompatibility and resorbability. However, DCPD cements are typically weak and brittle. To overcome these limitations, the sodium citrate used as a setting regulator and the coating of poly-L-lactide acid (PLLA) technique have been proposed in this study. The first purpose of this thesis is to develop composite PLLA/DCPD scaffolds with enhanced toughness by PLLA coating. The second purpose is to examine the biocompatibility of the scaffolds. The final purpose is to investigate the degradation behaviors of DCPD and PLLA/DCPD scaffolds. In this experiment, DCPD cements were synthesized from monocalcium phosphate monohydrate (MCPM) and 𝛽-tricalcium phosphate (𝛽 –TCP) by using deionized water and sodium citrate as liquid components. The samples were prepared with powder to liquid ratio (P/L) at 1.00, 1.25 and 1.50. To fabricate the PLLA/DCPD composite samples, DCPD samples were coated with 5 % PLLA. The samples were characterized mechanical properties, such as porosity, diametral tensile strength, and fracture energy. The mechanical properties of DCPD scaffolds with and without PLLA coating after the in vitro static degradation (day 1, week1, 4, and 6) and in vitro dynamic degradation (day 1, week 1, 2, 4, 6, and 8) were investigated by measuring their weight loss, fracture energy, and pH of phosphate buffer solution. In addition, the dog bone marrow stromal stem cells (dBMSCs) adhesion on DCPD and PLLA/DCPD composite samples were examined by scanning electron microscopy. The cell proliferation and differentiation in the medium conditioned with DCPD and PLLA/DCPD composite samples were studied by XTT (2,3-Bis(2-methoxy-4- nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt), and alkaline phosphatase (ALP) assay, respectively. The addition of sodium citrate and PLLA coating played a crucial role in improving the mechanical properties of the samples by increasing the diametral tensile strength from 0.50 ± 0.15 MPa to 2.70 ± 0.54 MPa and increasing the fracture energy from 0.76 ± 0.18 N-mm to 12.67 ± 4.97 N-mm. The DCPD and PLLA/DCPD composite samples were compatible with dBMSCs and the cells were able to proliferate and differentiate in the conditioned medium. The degradation rate of DCPD and PLLA/DCPD samples were not significant different (p > 0.05). However, the DCPD and PLLA/DCPD composite samples those used sodium citrate as a liquid component was found to degrade faster than the groups that use deionized water as liquid component