Access Type

Open Access Dissertation

Date of Award

January 2017

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemical Engineering and Materials Science

First Advisor

Howard W. Matthew

Abstract

The gold standard for bone regeneration requires harvesting a piece of healthy bone from the patient through a painful procedure, but this piece of autologous bone graft contains the bone and endothelial cells required to rapidly regenerate bone in a defect. We have developed a modular bone regeneration platform, composed of mesenchymal stem cells (MSCs) and hydroxyapatite (HAP) microgranules encapsulated in microcapsules, to replace autologous graft harvesting. The microcapsules are composed of a polyelectrolyte membrane formed by the ionic-complex reaction between chondroitin 4-sulfate (C4S) and chitosan. The specific aims of this thesis were to 1) examine the ability of C4S/HAP/chitosan microcapsules to support osteogenesis of encapsulated MSCs, 2) characterize how microcapsule mineralization influences mechanical properties of fused microcapsule constructs, and 3) analyze how endothelial progenitors (EPs) attached to the microcapsule exterior influence the vascularization of fused constructs in vivo.

The microcapsules supported the osteogenesis of encapsulated MSCs, and an in vitro analysis showed enhanced alkaline phosphatase (ALP), osteocalcin and osteopontin expression. Furthermore, biochemical assays and Scanning Electron Microscopy (SEM) confirmed that osteoinduced MSCs deposited a calcium and collagen rich mineralized extracellular matrix (ECM) in the microcapsule interior after 4 weeks osteoinduction in vitro. Hydrated, compressive mechanical testing demonstrated that fused constructs composed of mineralized microcapsules exhibited significant resistance to compression, up to a yield strength of 10.4 + 4.4 MPa. Analysis of the yield strength and elastic moduli demonstrated that the compressive mechanical properties depend primarily on active mineralization of the microcapsules by differentiating MSCs, and to a lesser extent on HAP microgranules. Micro computed tomography (MicroCT) and SEM analysis of fused constructs showed that the organization and architecture of the mineral within the microcapsules determined the overall mechanical properties of fused constructs. EPs or MSCs were cultured on the microcapsule exterior, and fused constructs were fabricated with these microcapsules, so that the EPs/MSCs were localized to the intercapsule pore space. Fused constructs containing EPs or MSCs in the pore space were evaluated for their vascularization and tissue regeneration in a rat subcutaneous model. Doppler Ultrasound (US) analysis of blood flow through implanted constructs revealed that microcapsules with EPs or MSCs in the construct pore space had enhanced vascularization 4 weeks post-surgery, compared to fused constructs with only encapsulated osteoprogenitors and acellular constructs. Results indicate that the C4S/HAP/Chitosan microcapsules can function as the basis of a bone regeneration platform, and that culture of either EPs or undifferentiated MSCs can enhance the vascularization of fused microcapsule constructs in vivo. Our studies warrant further development and optimization of the C4S/HAP/Chitosan microcapsules as a replacement for painful autologous bone graft harvesting.

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