Defense Date

2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biomedical Engineering

First Advisor

Gary L. Bowlin

Abstract

Macrophages (MФ) and mast cells are important cell types in the context of tissue remodeling and regeneration. Mast cells participate in the early stages of wound healing and modulate the acute inflammatory responses to biomaterials. Mast cells can secrete a myriad of different cytokines by the process of degranulation; the process of regulated secretion in which preformed contents stored in their granules are rapidly released by exocytosis. Some of these cytokines such as IL-4, IL-13 and TNF-α can modulate the MФ phenotype. Macrophages (MΦ) are innate immune cells, crucial for tissue homeostasis, presentation of foreign and self-antigens following infection/injury, pathogen clearance, inflammation resolution, angiogenesis, and wound healing. MΦ display plasticity and can acquire pro-inflammatory (M1) or angiogenic/wound healing (M2) phenotypes depending upon the environmental stimuli. The phenotypic profile of MФ as M1 or M2 following exposure to the biomaterial can dictate the downstream processes of tissue remodeling and angiogenesis. An analysis of how these two cell types interact with electrospun biomaterials and how different properties of an electrospun biomaterial impacts the MΦ phenotype is the focus of this thesis. Mast cells synthesize several potent angiogenic factors and can also stimulate fibroblasts, endothelial cells and macrophages. An understanding of how they participate in wound healing and angiogenesis is important to further our knowledge about in situ vascular prosthetic regeneration. The adhesion, proliferation and cytokine secretion of bone marrow derived murine mast cells (BMMC) on electrospun polydioxanone (PDO), polycaprolactone (PCL) and silk scaffolds, as well as tissue culture plastic (TCP) has been investigated in the presence or absence of IL-3, SCF, IgE and IgE with a crosslinking antigen, dinitrophenol-conjugated albumin (DNP). It was previously believed that only activated BMMCs exhibit adhesion and cytokine secretion. However, this study shows non-activated BMMC adhesion to electrospun scaffolds. Silk scaffold was not found to be conducive for mast cell adhesion and cytokine secretion. Activation by IgE and DNP significantly enhanced mast cell adhesion, proliferation, migration and secretion of TNF-α, MIP-1α and IL-13. This indicates that mast cells might play a role in MФ polarization (M1/M2), biomaterial integration into the host tissue, regeneration, and possibly angiogenesis. In the next study, bone marrow derived murine macrophages (BMMΦs, 106 cells) were seeded on TCP (24 well plate) and PDO scaffolds (15 mm discs) electrospun from varying polymer concentrations (60, 100, and 140 mg/ml). Scaffold evaluation showed that large polymer concentrations led to larger fiber diameters, which in turn led to larger pore-sizes and porosity but a smaller surface area to volume ratio. After 24 hrs of culture, the cell lysates were analyzed for Arginase (Arg1) and inducible nitric oxide synthase (iNOS) expression by western blot and cell culture supernatants were analyzed for Nitric oxide (NO2-), Tumor Necrosis Factor – alpha (TNF-α), Interleukin-6 (IL-6), Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor – beta1 (TGF-β1) and basic fibroblast growth factor (bFGF) levels. The results indicated a correlation between Arg1 expression and increasing fiber/pore-size, indicating that the larger fiber/pore-sizes polarize towards a M2 phenotype. Also, the expression of iNOS was downregulated on the larger fiber/pore-size. The levels of NO2- were significantly higher on the lower fiber/pore-sizes indicating an M1 phenotype. The levels of VEGF, TGF-β1 and bFGF increased with increasing fiber/pore-sizes. The results showed higher Arg1 expression in M2s on the 60 mg/ml scaffold created by the air-flow impedance method compared to the 60 mg/ml scaffold created on the solid mandrel created by traditional electrospinning. The Arg1 expression was reduced on the compressed 140 mg/ml PDO scaffold compared to the normal 140 mg/ml scaffold. This result indicates that pore-size might be playing a greater role compared to fiber diameter in BMMФ phenotype modulation. In order to assess the angiogenic potential of BMMΦs cultured on PDO scaffolds, a 3D angiogenesis bead assay was performed using conditioned media from the BMMΦ:PDO interaction. The results of the 3D angiogenesis bead assay showed that the conditioned media from BMMΦs of M0 and M2 phenotypes cultured on the 140 mg/ml PDO scaffold induced larger sprouting and higher percentage density of sprouts when compared to the 60 mg/ml PDO scaffold and TCP. To investigate the signaling mechanism involved in this phenotypic switch, BMMΦs were isolated from the bone marrow of MyD88 knockout (KO) mice (Jackson Laboratories) and cultured on PDO (60 and 140 mg/ml) scaffolds (106 /disc) and TCP for 24 hrs and their Arg1 and iNOS expression was analyzed by western blot. The expression of Arg1 and iNOS was severely impaired on the BMMΦs from MyD88-/- mice cultured on the 140 mg/ml scaffold when compared to the 60 mg/ml PDO scaffold and TCP. This result indicates that scaffolds with different fiber/pore-sizes signal differently. A subcutaneous mouse model (described in Chapter 6) was used to evaluate the angiogenic and regenerative potential of PDO scaffolds in vivo. The DIVAA assay showed statistically higher FITC-dextran signal intensity for the 140 mg/ml scaffold compared to the 60 mg/ml scaffold indicating greater angiogenic response in the 140 mg/ml tube. However, problems of high background were observed in this assay with the use of electrospun PDO. The observed high background was probably due to the formation of complexes between dextran and adsorbed plasma proteins on the surface of the PDO. More studies are needed to optimize this assay for use with biomaterials such as PDO. H&E staining of the harvested PDO tubes (60 mg/ml and 140 mg/ml) was also performed. The cross-sections of these tubes showed greater cell recruitment and infiltration into the fibrous structures of the 140 mg/ml tube compared to the 60 mg/ml tube. This result corroborates the in vitro result of BMMФ infiltrating deeper into the structures of the 140 mg/ml scaffold compared to the 60 mg/ml scaffold. The scaffolds were also analyzed by immunostaining for iNOS (indicative of M1 phenotype of MФs). The results showed statistically higher ratios of iNOS positive:negative areas on the 60 mg/ml scaffold compared to the 140 mg/ml scaffold. Overall, these studies indicate that 140 mg/ml scaffold supports greater cell recruitment and cell infiltration in vivo but a smaller ratio of iNOS positive:negative areas compared to the 60 mg/ml scaffold, which supports a predominately M1 MФ phenotype. The studies indicate that varying properties of PDO can alter both the phenotype and function of BMMΦs in vitro and in vivo. We have also shown that the 140 mg/ml scaffold signal BMMΦs through MyD88-dependent mechanisms. A complete understanding of the way materials signal would allow us to control or modulate undesirable immune reactions to biomaterials in vivo. These studies would also help engineer biomaterials that promote angiogenesis and regeneration.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

8-11-2012

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