In Addition, There Appears to Be Only Few Studies That Utilize Uniaxial Cyclic Stretching

Supplementary

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 musculoskeletalsystem (Mackeyet 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), andfrequency (Tirkkonenet al. 2011) of the uniaxial cyclic stretchingplays 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 loadparameters 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 (Cansecoet al.2012) and bone (Webb et al.1997), and it is up-regulated 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 (Dochevaet 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 (Léjardet al.2007) and TNMD (Shukunamiet al.2006).

The clinical implications of this study are two-fold: Firstly, the use of uniaxial stretching of hMSCsin 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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