Date of Award

4-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cell Biology and Physiology

Research Advisor

Y. James Kang, Ph.D.

Committee

Adebowale Adebiyi, PhD; Gary L. Bowlin, PhD; James Carson, PhD; Kaushik Parthasarathi, PhD; Gabor J. Tigyi, PhD

Keywords

Bio-Patch, Hydrogel Spheres, Ischemic Heart Disease; Octyl-Cyanoacrylate, Stem Cell Therapy, Survival and Retention

Abstract

Introduction. Ischemic heart disease (IHD) is a major concern of human health issue. The structural repair and functional recovery of injured hearts is a major challenge in the clinical setting. Preclinical studies over last 10 years have demonstrated the potential of using stem cells to treat IHD, but the efficacy of this therapy is jeopardized by uncontrollable migration and low survival of the injected stem cells. An approach based on tissue engineering enabling target-specific stem cell delivery such as cardiac patches has emerged as an alternative solution for stem cell therapy for IHD. It employs scaffold materials to engulf various stem cells and growth factors, forming patches implantable to the injured tissue. It has been proven to be a promising method for tissue repair and regeneration, however, it remains challenging to retain a sufficient survival of the stem cells attached to the scaffold materials to fulfill the therapeutic requirement. Therefore, the purpose of this study was to develop and characterize a bio-patch combining stem cells, hydrogel, and scaffolds to improve cell survival and retention for improvement of stem cell therapy for IHD. The focus of this study was to develop an ideal bio-patch that should have suitable mechanical properties maintaining the stemness of stem cells, promoting cell adhesion, and ensuring cell proliferation and differentiation under appropriate conditions. Methods. In this study, adipose-derived stem/stromal cells (ADSC) were chosen as seed cells, and bovine type I collagen was employed to engulf ADSC to produce hydrogel spheres. To fully characterize the behavior of ADSC in the hydrogel, spheres of varying sizes (~200-1000 µm for microspheres, and ~1000-2000 µm for macrogels) were generated. To determine the optimal formulation, cell proliferation, viability, migration, and differentiation capacity of the seed cells were assessed under the conditions of varying concentrations of collagen I (2, 4, 6, 8 mg/mL) and different cell densities (1, 5, 10, 15 x 106 /mL cells). To enhance the mechanical stability of collagen spheres, an additional Poly-L-Lysis (PLL) shell material with different concentrations (0.01, 0.05, 0.1, 0.25, 0.5 mg/mL), and crosslink times (5, 10, 20, 30 minutes) were evaluated to define the best formulation for the optimal condition for cell viability. Electrospun collagen patch was fabricated to serve as flat scaffold, and an octyl-cyanoacrylate (OCA) adhesive was employed to strengthen the adhesion between ADSC microspheres and the scaffold. To define the ideal OCA concentration for ADSC survival, the cell cytotoxicity of various brands of OCA was assessed in both direct and indirect contact on a dosedependent manner. Additionally, the adipogenesis and osteogenesis of ADSC were assessed following OCA treatment to further determine the effect of OCA on ADSC. To maintain ADSC physiological performance, ADSC macrogels were immediately attached to the scaffold and immersed in the growth medium. After bio-patch produced, the cells were released by 0.1% type I collagenase after different days of culturing (1, 3, 5 days), and cell proliferation, adipogenesis, osteogenesis ability were evaluated to check the properties of ADSC. The growth factors released situation (VEGF) under hypoxia conditions were evaluated by RT-PCR. Finally, in the in vivo study, a rat model of IHD was employed to assess the effect of the bio-patch on the injured heart by examining changes in infarcted area and cardiac contractile function after bio-patch implantation. v Results. The ADSC exhibited reduced migration, adhesion, viability, and proliferation ability with high concentrations of collagen, specifically, cell viability decreased when collagen concentration higher than 4 mg/mL. Furthermore, cell viability decreased with high cell density engulfed in macrogel. The author found that 5x106 cells/mL in collagen solution was the optimal concentration for cell survival. The ADSC’s viability decreased with high concentrations as well as prolonged assembly time of PLL; The author found the concentration at 0.05 mg/mL and the assembling time of 10 min was the best formulation for cell survival and structure integrity. Characterizations of ADSC released from macrogels by flow cytometry showed good preservation of stem cell surface marker expression. Trilineage differentiation analyses further confirmed the preservation of ADSC released from microspheres. The author found OCA affects ADSC in a dosedependent manner, OCA at concentrations below 14 µM did not affect the morphology, proliferation, and viability of the tested cells (50000 cells). OCA from different sources displayed variable adverse effects on the cells but overall, the concentration over 23 µM showed a significant cytotoxic effect. Furthermore, the author also observed that OCA at its concentration of 5 µM did not alter the trilineage differentiation of ADSC. Under this condition, the adhesive capacity of OCA for the bio-patch was well maintained. The expression of VEGF was significantly increased under hypoxic conditions that was regulated by increased expression of Hif-1α. Finally, the bio-patch’s function in a rat model of IHD was validated. Improved heart function was observed in the ischemic heart after treatment with the bio-patch for 7 days. Histological observation exhibited a decreased TNF- expression and improved IL10 expression, with less fibrosis and collagen deposition. Conclusion. An implantable bio-patch as an effective delivery system for ADSC was developed in this study. Specifically, the author worked out the formulation of each component: the optimal collagen/PLL concentrations, cell density in different spheroid size, and the best OCA to ADSC ratio under different conditions. Animal studies using a rat model of IHD validated the potential of the bio-patch in prevention of acute ischemic cardiac injury. This protection by ADSC patch most likely resulted from a paracrine mechanism and anti-inflammatory effect.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

https://orcid.org/0000-0003-3389-5333

DOI

10.21007/etd.cghs.2023.0619

Additional Files
2023-006-Zhang-DOA.pdf (163 kB)
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