Modulating human connective tissue progenitor cell behavior on cellulose acetate scaffolds by surface microtextures

Abstract

Soft lithography techniques are used to fabricate cellulose acetate (CA) scaffolds with surface microtextures to observe growth characteristics of the progeny of human marrow-derived connective tissue progenitor cells (CTPs). Human CTPs were collected and cultured on CA scaffolds comprised postmicrotextures and smooth surfaces for up to 30 days. Cells on the smooth surfaces migrated without any preferred orientation for up to 30 days. On microtextures, cells tended to direct their processes toward posts and other cells on day 9. By day 30, cells on microtextures covered the surface with extracellular matrix. DNA quantification revealed approximately threefold more cells on microtextures than on the smooth surfaces. The alkaline phosphatase (AP) mRNA expression was slightly higher on smooth surfaces on day 9. However, by day 30, AP mRNA showed higher expression on microtextures. The mRNA expression of collagen type I was increased on microtextures by day 30, whereas smooth surfaces demonstrated similar expression. The osteocalcin mRNA expression was increased on postmicrotextures relative to smooth surfaces by day 30.

Figure 1

Fabrication of CA postmicrotextures by soft lithography. The cross-sectional schematic diagrams depict (a) starting substrate, which is a 100-mm diameter, 500-μm thick silicon (Si) wafer; (b) a 6-μm thick layer of SU-8 2010 photoresist was spin-coated on top of the Si wafer; (c) using ultraviolet (UV) photolithography, a 10-μm diameter texture pattern was transferred from a photomask onto the photoresist; (d) developed photoresist (pattern); (e) molding of CA by casting; and (f) release of CA cast from SU-8 mold.

Figure 2

SEM images of CA substrates. (a) CA postmicrotextures with posts that were 6 μm in height, 10 μm in diameter, and with 10-μm separation between posts; and (b) smooth CA surface (scale bar = 10 μm).

Figure 3

SEM images of CTP progeny on CA postmicrotextures, smooth CA, and control surfaces on days 9 and 30. CTPs attached to CA postmicrotextures, smooth CA, and control surfaces with varying cell morphology. On postmicrotextures, CTPs mostly tended to attach next to the posts and spread between them while directing their processes toward posts and other cells on day 9 (solid arrows and inset). On day 30, numerous cells grew and spread over the top of the CA postmicrotextures and covered most of the surface with ECM (dashed arrows and inset). In contrast, cells on the smooth and control surfaces exhibited arbitrary flattened shapes and migrated without any preferred orientation for up to 30 days.

Figure 4

CTP number on CA postmicrotextures and corresponding smooth surfaces. The PicoGreen DNA quantification was repeated thrice. CA postmicrotextures exhibited a greater number of CTPs than smooth CA and control surfaces. On days 9 and 30, there were a greater number of cells on CA postmicrotextures than on smooth CA surfaces, when compared with the control. Numerical values denote original cell number and standard errors. * denotes statistical significance compared to smooth CA surfaces and control on days 9 and 30, respectively (p < 0.05).

Figure 5

Fluorescent and phase contrast microscopy images of CTPs on CA postmicrotextures, smooth CA, and control surfaces on (a) day 9 and (b) day 30. Fluorescent images show cells stained with DAPI and AP, whereas phase contrast images show von Kossa stain. Cell nuclei were stained with DAPI to qualitatively reveal more cells on postmicrotextures than smooth and control surfaces. CTPs on the CA postmicrotextures stained more intensely for AP, a marker of osteoblastic phenotype, compared to smooth CA and control surfaces on day 9, and AP increased on all scaffolds by day 30. Furthermore, the coverage and intensity of von Kossa stain on CA postmicrotextures was increased compared to the smooth CA and control surfaces. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 6

mRNA expression of: (a) AP, (b) collagen type I (Col I), and (c) osteocalcin (OC) from CTPs after 9 and 30 days on CA postmicrotextures and smooth CA surfaces. The real-time RT-PCR was repeated four times, and the data are normalized to corresponding control surfaces (set to 1.0) within a particular experiment to account for differences in CTPs among individual donors. AP mRNA expressed slightly higher on day 9 compared to day 30 on smooth surfaces. In contrast, the mRNA expression of AP has increased by day 30 on postmicrotextures. On day 9, Col I mRNA expression on both postmicrotextures and smooth surfaces is similar. The mRNA expression of Col I was increased on postmicrotextures on day 30, but there was similar expression of Col I mRNA on smooth surfaces. Even though OC mRNA expression was almost the same on day 9 for both smooth and posttextured surfaces, it expressed more strongly on postmicrotextures on day 30. Numerical values denote mean and standard errors. * denotes statistical significance (p < 0.05).