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 mm compared to larger linear channels of widths 500-1000 mm. 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.

Evaluation of the Early In Vivo Response of a Functionally Graded Macroporous Scaffold in an Osteochondral Defect in a Rabbit Model

“…This correlates well with other studies, where cartilage repair was attributed to cell homing, engraftment and repair due to pore architecture of the construct used. 4,9,19 Despite that collagen type I staining was observed in all defects in the present study, chondrocytes adjacent to the cell-free construct did not stain positive. Although, collagen type I is associated with fibrocartilage repair, spatial and temporal patterns of collagen type I expression have been observed during cartilage development, 26 at early time points during in vitro chondrogenesis of MSC 14 and after ACI in humans.…”

“…1A illustrateds the generation of a biomimetic pore structure, with an open tunnel system, created by the customdesigned layer structure. In addition to creating an open functionally-graded pore template for cells that complements the work of Chou et al 4 and Lee et al, 19 the compressive modulus values were orders of magnitude higher (10 MPa) than previous composite constructs 27 (0.005-0.10 MPa) and represent a step towards mechanical property values proposed for hyaline cartilage regeneration in the region 1-12MPa. 12 However, it must be noted that compression testing of the macroporous PLCL scaffolds was performed on dry samples at room temperature and that future studies will be conducted to examine the mechanical properties of constructs stored 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 Previous studies have successfully employed bone marrow-derived MSCs for cartilage repair applications.…”

The Effect of Biomolecular Gradients on Mesenchymal Stem Cell Chondrogenesis under Shear Stress

“…Further, exposure to TGF-β1 gradients led to chondrogenesis. These results, together with a previous study that showed that aligned MSCs produce aligned collagen, suggest that oriented cartilage tissue structures with superior mechanical properties can be obtained by using flow and chondrogenic factor gradients formed by microfluidics [19].…”

“…The primary antibody host species is mouse and the antigen species is chicken. It has been shown to have cross-reactivity with human type II collagen [19,31] synthesized by human MSCs. The samples were then washed in PBS for 1 h. FITC-conjugated goat anti-mouse IgG, diluted 1:500 in 1% NGS, was then applied to the aggregates for 45 min.…”

“…This study investigated the use of a gradient generating microfluidic device to study the effects of fluid flow-induced shear stress on MSC alignment and biomolecular gradients on MSC chondrogenesis. In a previous study, aligned MSCs undergoing chondrogenesis synthesized aligned type II collagen and resulted in tissue constructs with enhanced mechanical properties [19]. Previous studies have also indicated that exposure of cells, including endothelial cells and MSCs, to fluid flow induced shear stress results in cellular alignment along the flow direction [20][21][22].…”