Translating Human Adipose Stem Cells for Musculoskeletal Applications: A Tissue Engineering, Mechanistic, and Electrical Cell-Substrate Impedance Spectroscopy Approach

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URL: https://repository.lib.ncsu.edu/handle/1840.20/33542

OCLC: 978351933

PROQUEST: 1914681820

Abstract

Human adipose stem cells (hASC) are an attractive cell source for tissue engineering, regenerative medicine and immunomodulatory applications due to their relative ease of harvest, proliferative capacity, and multipotent differentiation potential. However, in order to translate hASC into widespread clinical use, there are a number of obstacles that must be addressed. This body of work describes novel tissue engineering techniques for musculoskeletal repair using hASC, elucidates relevant mechanisms in cartilage and bone biology, and studies age-grouped hASC donor-to-donor variability in osteogenic differentiation using electrical cell-substrate impedance spectroscopy.

Within our first study to advance the translation of hASC therapy, we describe a new method for enhanced cellular infiltration in meniscal allografts. Medial menisci were decellularized and a needle-punched method was used to enhance porosity. After 28 days of in vitro culture, we demonstrate needle-punching enhances hASC infiltration, which could improve long-term efficacy of meniscal transplantation procedures by helping to maintain the meniscus in vivo.

With the understanding gained in the meniscal study, we used cartilage extracellular matrix to drive differentiation in a 3D bioplotted scaffold that induces site-specific hASC differentiation. Our scaffold was fabricated using 3D bioprinting of biodegradable polycaprolactone (PCL) with either 20%TCP tricalcium phosphate (TCP) or decellularized cartilage extracellular matrix (dECM) to induce site-specific osteogenesis and chondrogenesis, respectively. In addition, histological analyses of full osteochondral scaffolds showed site-specific tissue characterization using a single adult stem cell source. In future in vivo studies, this approach holds great potential to treat OA patients in a highly personalized manner using a patient’s own hASC donor cells.

In order to better understand the signaling mechanisms that drive hASC differentiation and musculoskeletal development within our scaffold system, we next investigated relevant musculoskeletal mechanisms. The goal of the first mechanistic study was to determine how LRP4, LRP5, and LRP6 within canonical Wnt-signaling are regulated in simulated microgravity and cyclic hydrostatic pressure in order to elucidate the mechanisms of cartilage degeneration. LRP5 is demonstrated to be upregulated in both simulated microgravity and hydrostatic pressure and in the articular cartilage of hind limb unloaded mice. Further elucidation of this mechanism could provide significant clinical benefit for the identification of pharmaceutical targets for the maintenance of cartilage health.

After identifying an important mechanism for regulating cartilage homeostasis we next studied a signaling mechanism relevant to hASC osteogenic differentiation. We show that Corin is highly upregulated throughout osteogenic differentiation and demonstrate that calcium accretion and metabolic activity are decreased when Corin is knocked down via siRNA. Interestingly, Corin knockdown also significantly increased VEGF mRNA expression during osteogenesis, suggesting that Corin is also involved in the regulation of angiogenic mechanisms. Overall this study suggests that Corin is a key regulator of osteogenesis in hASC, likely through crosstalk with vascular pathways.

One of the major commonalities throughout all hASC studies is that there is major donor-to-donor variability. In order to better understand this variability, we investigated the use of electrical cell-substrate impedance spectroscopy (ECIS) to track complex bioimpedance patterns of hASC throughout proliferation and osteogenic differentiation. Superlots comprised of hASC from young, middle-aged, and elderly donors were seeded on gold electrode arrays to track complex impedance measurements throughout proliferation and osteogenic differentiation. We show that stages of osteogenic differentiation can be tracked via ECIS. In addition, hASC from younger donors require longer time to differentiate than hASC from older donors. This is the first study to use ECIS to predict osteogenic potential of multiple hASC populations and to show that donor age may temporally control onset of osteogenesis. Overall, the findings presented in this dissertation are critical for the translation of safe and effective hASC therapies for musculoskeletal repair.

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