Micrometer scale guidance of mesenchymal stem cells to form structurally oriented cartilage extracellular matrix

Abstract

Tissue engineering is a possible method for long-term repair of cartilage lesions, but current tissue-engineered cartilage constructs have inferior mechanical properties compared to native cartilage. This problem may be due to the lack of an oriented structure in the constructs at the microscale that is present in the native tissue. In this study, we utilize contact guidance to develop constructs with microscale architecture for improved chondrogenesis and function. Stable channels of varying microscale dimensions were formed in collagen-based and polydimethylsiloxane membranes via a combination of microfabrication and soft-lithography. Human mesenchymal stem cells (MSCs) were selectively seeded in these channels. The chondrogenic potential of MSCs seeded in these channels was investigated by culturing them for 3 weeks under differentiating conditions, and then evaluating the subsequent synthesized tissue for mechanical function and by type II collagen immunohistochemistry. We demonstrate selective seeding of viable MSCs within the channels. MSC aligned and produced mature collagen fibrils along the length of the channel in smaller linear channels of widths 25-100 μm compared to larger linear channels of widths 500-1000 μm. Further, substrates with microchannels that led to cell alignment also led to superior mechanical properties compared to constructs with randomly seeded cells or selectively seeded cells in larger channels. The ultimate stress and modulus of elasticity of constructs with cells seeded in smaller channels increased by as much as fourfolds. We conclude that microscale guidance is useful to produce oriented cartilage structures with improved mechanical properties. These findings can be used to fabricate large clinically useful MSC-cartilage constructs with superior mechanical properties.

FIG. 1.

Schematic of ultrastructure of articular cartilage (adapted from Hunzikeret al.): Blue circular shapes are chondrocytes and the black bands are collagen fibrils. The figure shows orthogonal arrangement of collagen fibrils in the central and near the subchondral bone regions of the cartilage and a parallel arrangement near the superficial region of the cartilage. This ultrastructure has been shown to play a role in cartilage mechanical properties. Color images available online at www.liebertpub.com/tea

FIG. 2.

(A) Microchannel design created in AutoCAD. The channels ranged from a minimum width of 25 μm to a maximum width of 1000 μm. The spacing between channels of the same width was varied from 50 to 250 μm for different designs to allow us to test the effect of channel density in future experiments. (B) A schematic of the process used to obtain patterns in collagen–glycosaminoglycan (GAG) membranes. (C–F) Scanning electron micrographs of linear channels of width 25 (C), 50 (D), 100 (E), and 500 (F) μm formed in collagen-GAG membranes. Arrows point to the channels and arrowheads point to spaces between the channels. (G) Accuracy of channel reproduction in collagen-GAG membranes. Template width indicates the width of channels in the mask used for photolithography. Mean values of measured width±standard deviations are shown. Ten channels from three different membranes for each channel size were used. EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Color images available online at www.liebertpub.com/tea

FIG. 3.

A schematic of the technique used to obtain selective seeding of mesenchymal stem cells (MSCs). The steps are uniform coating of F108 on a glass coverslip, selective transfer of F108 onto nonchannel surfaces through contact with F108-coated glass, and selective seeding of MSCs in channels. Color images available online at www.liebertpub.com/tea

FIG. 4.

Effect of F108 on selective seeding of human MSCs in polydimethylsiloxane (PDMS) microchannels. MSCs prestained with Vybrant^® were seeded on to F108-treated PDMS linear channels overnight. Fluorescent images of cells in linear channels of widths 25 μm (A), 50 μm (B), and 100 μm (C) show selective seeding compared to untreated channel surfaces (D). Scale bars: 50 μm (A, D), 100 μm (B, C). Color images available online at www.liebertpub.com/tea

FIG. 5.

Effect of F108 selective seeding of human MSCs in collagen microchannels. MSCs prestained with Vybrant were seeded onto F108-treated collagen linear channels overnight. Fluorescent images of cells in linear channels of widths 25 μm (A), 50 μm (B), 100 μm (C), 500 μm (D), and 1000 μm (E) show selective seeding compared to untreated channel surfaces (F). Insets in (D) and (E) show magnified portions of gaps (50 μm) between the channels devoid of cells. Scale bars: 50 μm (A, B, F), 100 μm (C), 200 μm (D), 400 μm (E). Color images available online at www.liebertpub.com/tea

FIG. 6.

Chondrogenesis under microscale guidance. Fluorescent images of linear channels of widths 25 μm (A), 50 μm (B), 100 μm (C), 500 μm (D), and 1000 μm (E) show alignment in smaller 25–100 μm channels. Cell nuclei alignment (blue) is correlated with actin (red) alignment in smaller channels. Insets in (C) and (D) show magnified portions of channels with cells. [F, H(i)] show actin alignment in linear channels of width 25 μm, which was contrasted with random alignment of actin filaments in linear channels of width 500 μm [G, H(ii)]. Confocal fluorescent images (green is type II collagen, blue is DAPI, and red is DiI) of 25 (I) and 1000 μm (J) channels show mature aligned extracellular matrix (arrowheads) in the smaller channel. Scale bars: 10 μm [H(i), H(ii), I, J], 25 μm (F, G), 50 μm (A–C), 100 μm (D), 200 μm (E). Color images available online at www.liebertpub.com/tea

FIG. 7.

Cell orientation under microscale guidance. Distribution of angles of orientation of human MSCs formed against the longer dimension of the PDMS (A) and collagen (B) channels of widths 25–1000 μm. For smaller channels (25, 50, 100 μm), six channels from three different samples were used. For larger channels (500, 1000 μm), three channels from three different samples were used. Cells from three donors were used.

FIG. 8.

Effect of microscale guidance on mechanical properties. Collagen membranes were subject to tensile testing until failure. Mean values of elasticity modulus (A), a typical force–displacement curve (A: upper left corner), and ultimate stress (B) are shown. BK and EDC represent, respectively, uncrosslinked and EDC crosslinked collagen membranes without microchannels or MSCs. C25 and C100 represent collagen membranes consisting of 25- and 100-μm linear channels only, without MSCs. All other channels were seeded with MSCs. Ran: EDC crosslinked collagen membranes without channels; 25, 50, 100, 250, 500, and 1000 represent widths (μm) of linear channels in the EDC crosslinked collagen membranes. n=4 for 1000, n=5 for Ran, C25 and C100 and n=6 for all other conditions. (A) *Statistically significant difference (p<0.05) compared to data with no channels (RAN). ^#Statistically significant difference (p<0.05) compared to data with 1000-μm channels (1000). (B) *Statistically significant difference (p<0.05) compared to data with no channels (RAN), 250-μm (250), 500-μm (500), and 1000-μm (1000)-wide channels. Cells from two donors used in the above experiments.