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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 28, 2001 as doi:10.1096/fj.01-0656fje. |
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Department of Chemical and Biological Engineering, Bioengineering Center, Tufts University, Medford, Massachusetts 02155, USA;
* Department of Surgery, Research Division, University Hospital Basel, 4031 Basel, Switzerland;
Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA;
New England Medical Center, Boston, Massachusetts 02111, USA; and
Harvard Medical School, Center for Technology and Engineering, Boston, Massachusetts, USA
2Correspondence: Department of Chemical and Biological Engineering, Tufts University, 4 Colby St., Medford, MA 02155, USA. E-mail: david.kaplan{at}tufts.edu
SPECIFIC AIM
To demonstrate that mechanical stimulation in vitro, without ligament-specific exogenous growth and differentiation factors, induces the selective differentiation of mesenchymal progenitor cells from the bone marrow into a ligament cell lineage in preference to alternative paths (i.e., bone or cartilage cell lineages).
PRINCIPAL FINDINGS
1. Helically organized collagen fibers formed in the direction of the applied load at the periphery of the mechanically stimulated ligaments, a feature absent in the controls.
2. Characteristic collagen banding patterns were clearly visible in the longitudinal transmission electron microscopy sections of mechanically stimulated gels, a pattern either absent or markedly less abundant in static controls.
3. Collagen type I fiber bundles (20 µm diam.) formed in the mechanically stimulated gels.
4. Mechanical stimulation of ligaments based on human or bovine bone marrow-derived cells induced elongated ligament-like cell morphology and cell alignment in the direction of loading, in contrast to the round and randomly distributed cells in static controls.
5. Cell alignment in the direction of mechanical loading was 2.5-fold higher (significant, P<0.001, paired Student’s t test between mechanically stimulated and static ligaments).
6. Compared with static controls, mechanically stimulated gels contracted by a significantly higher percentage (
58% vs. 45%, respectively, for mechanical vs. controls), and had significantly higher cross-sectional cell density after 21 days of culture (P<0.001, paired Student’s t test).
7. Collagen types I and III and fibronectin were all expressed in mechanically stimulated ligaments but not in controls, as evidenced by immunostaining (Fig. 1
). Collagen types I and III could be detected in the form of fiber bundles oriented in the direction of loading.
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8. The mRNA expression of collagen types I and III and tenascin-C, typical markers of ligament cells, was higher in the mechanically stimulated than in the control ligaments, as assessed by real-time quantitative RT-PCR (Fig. 2
).
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9. After 14 days of culture, differences became statistically significant (P<0.05) and mRNA levels in the mechanically stimulated ligaments approached those quantified in native ligaments. These results are consistent with previous reports on the up-regulation of collagen types I and III and tenascin-C by fibroblasts isolated from anterior cruciate ligaments when exposed to mechanical stimulation.
10. mRNA expression of bone sialoprotein and collagen type II, typical markers of respectively bone and cartilage cells, was not detected in the engineered ligaments.
11. The mRNA expression of osteocalcin and osteopontin that characterizes the progression of mesenchymal progenitor cells into the osteogenic lineage was not up-regulated by mechanical stimulation, and was comparable in engineered and native ligaments (Fig. 3
).
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CONCLUSIONS AND SIGNIFICANCE
The role of mechanical stimulation in adult stem cell differentiation is clearly delineated in this paper. Furthermore, the effect of mechanics can be demonstrated in the absence of ligament-specific exogenous growth factors usually incorporated into media to foster differentiation. Mechanical stimulation enhanced cell differentiation for reasons that are not entirely clear. The influence of mechanical forces may be transduced via cell binding sites, leading to enhanced rates or extent of differentiation. It is reasonable to speculate that mechanical signals may trigger cell-surface stretch receptors and adhesion sites, resulting in cascades that involve activation of genes responsible for the synthesis and secretion of key ligament extracellular matrix components. Changes in rates of mass transfer of nutrients, metabolites, and oxygen may also be affected by the mechanical forces. Combinations of mechanical signaling via cell membrane receptors and changes in transport likely play a role in the results observed in the present study.
Several groups have recently developed apparatus for the application of uniaxial strain of cell-seeded constructs. In other studies, cyclic mechanical strain promoted smooth muscle cell proliferation, organization and extracellular matrix synthesis, and collagen type I synthesis by anterior cruciate ligament fibroblasts. Compression improved the structure ofcultured cartilage explants. The successful demonstration of bone marrow stromal cell differentiation into ligament-like cells with multidimensional strain in 3D matrices suggests that mechanical forces can play an important role in the processes of cellular differentiation and not just in promoting specific tissue-types from differentiated cells. More detailed studies of cell receptors involved in transducing the mechanical signals and the associated cascades as well as nutrient transport issues are needed in order to refine the sequence of events responsible for the enhanced differentiation and tissue formation.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0656fje; to cite this article, use FASEB J. (December 28, 2001) 10.1096/fj.01-0656fje 
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