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In addition, there appears to be only few studies that utilize uniaxial cyclic
stretching as a modality of mechanical stimulation, even though this stimulus is
physiologically relevant to the development of a functional musculoskeletal system
(Mackey et al. 2008). Nevertheless, these few studies do suggest that the duration
(Kuo and Tuan 2008; Park et al. 2004), magnitude (Chen et al. 2008; Qi et al. 2008),
and frequency (Tirkkonen et al. 2011) of the uniaxial cyclic stretching plays an
important role in determining the fate of hMSCs. Unfortunately such studies tend to
utilize a limited range of strain levels and rates, and seldom investigate the effects of
different frequencies of dynamic mechanical stimulation. In addition, what happens to
cells when these different load parameters are combined remains unclear.
Because cells formed strong anchorage on the flexible membranes (as shown
by the stress fiber staining in Fig. 3), the different types of mechanical magnitude on
the membranes at the bottom of the cells can be transmitted through cell adhesion
receptors (e.g. integrins) to the interconnected cytoskeleton structures. This in turn
induces either relatively uniform or polarized strain distribution within these cells.
The different strain distribution may have a role in the regulation the conformation
and/or localization of signaling molecules, and either turn on different signaling
pathways or have the opposite effects on the same pathway. This then leads to the
different responses observed when changes are made to the loading characteristics
(Park et al. 2004).
To confirm that changes in the collagen content is reflective to that of
tenogenic expression levels, our study examined the tenogenic related genes
expressed using various strain rates and amounts. There were several studies that have
been reported using selected genes but none had demonstrated the expression of all 6
established tenogenic genes in one experiment, such as that reported in our present
paper. Tendon extracellular matrix is composed of mainly type-I and III collagen
supported by more specific glycoproteins such as decorin, tenascin and tenomodulin.
These proteins have specific functions, for example, decorin is thought to facilitate
fibrillar slippage during mechanical deformation (Zhang et al. 2006). Tenascin-C,
although found in abundance in tendon, it is not specific to this tissue since it can also
be found in ligament (Canseco et al. 2012) and bone (Webb et al. 1997), and it is upregulated in tissue remodeling processes like embryogenesis, wound healing and
tumorigenesis (Mackie and Tucker 1999). Several genes that have been deemed
specific to tendon cells which includes TNMD and SCX, cannot be identified through
its cellular protein expressed product hence are used as direct markers of tenogenic
differentiation. TNMD is a good phenotypic marker for tenocytes and, plays a role in
tendon development and vascularization (Docheva et al. 2005). SCX on the other
hand, is a transcription factor that is present in tendon during the condensation stage
and persists into adulthood, also essential for matrix organization (Schweitzer et al.
2001). Additional evidences suggested a positive role of SCX in adult tendon
homeostasis, taking a role as a co-activator of other tendon correlated genes such as
COL1 (Léjard et al. 2007) and TNMD (Shukunami et al. 2006).
The clinical implications of this study are two-fold: Firstly, the use of uniaxial
stretching of hMSCs in vitro may result in the production of tendon specific cells
which can provide superior regeneration of damaged tendon, especially that involving
degenerated tendons. In rotator cuff tendinopathy or which is the result of a
degenerative disease process, the restoration of the tendon to a level that is able to
provide the appropriate mechanical loading for the tendon cells is seen as a major
clinical problem. In such cases, the use of pre-stretched hMSCs will be beneficial
since it will overcome the issue of the repair outcome being dependent of the intrinsic
tendon cells which are exposed to poor mechanical loading. Secondly, the loading
regime being observed in the present study suggests that similar regime may be
beneficial to prevent further tissue degeneration when used in patients and that such
protocols would be useful in rehabilitation or at least prevention of tendinopathy.
However, it must be made clear that in both instances, these are merely suggestions
since more robust and in-depth studies involving in vivo or even clinical subjects
would be needed to confirm its practical applications. It is therefore important that the
findings of this study will need to be interpreted with care. Nevertheless, the results
are strongly suggestive of a positive effect between stretching and tenogenic potential
of multipotent cells, and do provide a good insight to the potential of using
mechanically loaded cultured hMSCs for the possible use as a therapeutic alternative
or even a rehabilitation regime protocol that will be beneficial for patients with
specific tendon diseases.
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