Tenogenic differentiation of human MSCs induced by the topography of electrochemically aligned collagen threads - PubMed (original) (raw)

Tenogenic differentiation of human MSCs induced by the topography of electrochemically aligned collagen threads

Vipuil Kishore et al. Biomaterials. 2012 Mar.

Abstract

Topographical cues from the extracellular microenvironment can influence cellular activity including proliferation and differentiation. Information on the effects of material topography on tenogenic differentiation of human mesenchymal stem cells (human MSCs) is limited. A methodology using the principles of isoelectric focusing has previously been developed in our laboratory to synthesize electrochemically aligned collagen (ELAC) threads that mimics the packing density, alignment and strength of collagen dense connective tissues. In the current study, human MSCs were cultured on ELAC and randomly oriented collagen threads and the effect of collagen orientation on cell morphology, proliferation and tenogenic differentiation was investigated. The results indicate that higher rates of proliferation were observed on randomly oriented collagen threads compared to ELAC threads. On the other hand, tendon specific markers such as scleraxis and tenomodulin, were significantly increased on ELAC threads compared to randomly oriented collagen threads. Additionally, osteocalcin, a specific marker of bone differentiation was suppressed on ELAC threads. Previous studies have reported that BMP-12 is a key growth factor to induce tenogenic differentiation of MSCs. To evaluate the synergistic effect of BMP-12 and collagen orientation, human MSCs were cultured on ELAC threads in culture medium supplemented with and without BMP-12. The results revealed that BMP-12 did not have an additional effect on the tenogenic differentiation of human MSCs on ELAC threads. Together, these results suggest that ELAC induces tenogenic differentiation of human MSCs by presenting an aligned and dense collagen substrate, akin to the tendon itself. In conclusion, ELAC has a significant potential to be used as a tendon replacement and in the development of an osteotendinous construct towards the regeneration of bone-tendon interfaces.

Copyright © 2011 Elsevier Ltd. All rights reserved.

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Figures

Figure 1

Figure 1

Validation of the Stemness of Human MSCs. (A) Day 14 – Control (No induction), (B) Day 14 - Alizarin red S staining after induction of osteogenic differentiation of human MSCs, (C) Day 14 – Oil red O staining after induction of adipogenic differentiation of human MSCs. Scale Bar: 200 µm.

Figure 2

Figure 2

Visual Examination of Cell Morphology and Distribution by Actin Staining. Actin stained images of Random thread at day 1 (A), ELAC thread at day 1 (B), Random thread at day 14 (C), ELAC thread at day 14 (D). Scale Bar: 0.5 mm.

Figure 3

Figure 3

Effect of Collagen Orientation on Cell Adhesion and Proliferation. (A) Cell Adhesion - Cell adhesion was two-fold higher on ELAC threads compared to random collagen threads. (B) Cell Proliferation – Higher rates of cell proliferation was observed on random collagen threads compared to ELAC threads. (* indicates statistical significance between ELAC and random collagen threads).

Figure 4

Figure 4

Effect of Collagen Orientation on Tenogenic Differentiation of Human MSCs. (A) Scleraxis, (B) Tenomodulin, (C) Tendon ECM Genes (Tenascin-C and Collagen-III), (D) Bone Differentiation Markers (Runx2 and Osteocalcin). Increase in the expression of tendon specific (scleraxis and tenomodulin) and tendon related (tenascin-D and collagen-III) genes indicates that the oriented collagen topography of ELAC threads stimulates the tenogenic differentiation of human MSCs. Data are normalized to β-actin and relative to the random collagen control. . (* indicates statistical significance between ELAC and random collagen threads).

Figure 5

Figure 5

Effect of Collagen Orientation on Cell Morphology, Alignment and ECM Deposition by Scanning Electron Microscopy. (A) Random – day 2, (B) ELAC – day 2, (C) Random – day 14, (D) ELAC – day 14, (E) Random – day 14 (High Mag), (F) ELAC – day 14 (High Mag). Cells have been highlighted in blue for clarity.

Figure 6

Figure 6

Effect of BMP-12 on Tenogenic Differentiation of Human MSCs on ELAC Threads. . (A) Scleraxis, (B) Tenomodulin, (C) Tendon ECM Genes (Tenascin-C and Collagen-III), (D) Bone Differentiation Markers (Runx2 and Osteocalcin). The expression of tendon specific genes (scleraxis and tenomodulin) was comparable with or without BMP-12 treatment indicating that BMP-12 did not have an additive effect over collagen orientation on the tenogenic differentiation of human MSCs. Data are normalized to β-actin and relative to ELAC without BMP-12 control. (* indicates statistical significance between ELAC and random collagen threads).

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References

    1. Squier CA, Magnes C. Spatial relationships between fibroblasts during the growth of rat-tail tendon. Cell Tissue Res. 1983;234:17–29. - PubMed
    1. Fischer LP, Carret JP, Gonon GP, Sayfi Y. Arterial vascularization of the patellar ligament (ligamentum patellase) and of the Achilles tendon (tendo calcaneous) in man. Bull Assoc Anat (Nancy) 1976;60:323–334. - PubMed
    1. Krackow KA, Thomas SC, Jones LC. Ligament-tendon fixation: analysis of a new stitch and comparison with standard techniques. Orthopedics. 1988;11:909–917. - PubMed
    1. Kartus J, Movin T, Karlsson J. Donor-site morbidity and anterior knee problems after anterior cruciate ligament reconstruction using autografts. Arthroscopy. 2001;17:971–980. - PubMed
    1. Butler DL, Juncosa-Melvin N, Boivin GP, Galloway MT, Shearn JT, Gooch C, et al. Functional tissue engineering for tendon repair: A multidisciplinary strategy using mesenchymal stem cells, bioscaffolds, and mechanical stimulation. J Orthop Res. 2008;26:1–9. - PubMed

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