previous study, we demonstrated that the three-dimensional

(3D) uniaxial loading protocol could provide a better environment

for the cell–matrix interactions, which closely mimic the mechanical

microenvironment of TDSCs, and preferentially generate a teno-

genic response [7]. In this chapter, we provided 2 options for 3D

uniaxial stretching to TDSCs, including the scaffold-based method

and scaffold-free method. The scaffold-based method was using

CelGro^® scaffold, a unique collagen scaffold providing a 3D space

for cell attachment [8]. The scaffold-free method was modified by

our previous protocol and designed to stimulate TDSCs to gener-

ate extracellular matrix and then was formed to a tendon organoid

[9, 10]. Our data showed both of these two options can induce

tenogenesis of TDSCs. These two methods have their own advan-

tages. The scaffold-free method may be time consuming but mimic

natural process. On the other hand, the scaffold-based method is

time effective and enables potential development of surgical

implantable biological devices.

Other types of mechanosensitive cells, such as cardiac myo-

cytes, endothelial cells, and osteocytes, can be investigated by this

method as well [11–13]. However, some cells without producing

extracellular matrix ability can only be seeded on scaffolds. More-

over, investigators should use specific loading regime and detect

specific outcome events on other types of cells. In the present

chapter, we use tenogenesis markers to prove our loading regime

can enhance tenogenesis for TDSCs than static culture (see Fig. 1).

Fig. 1 Tenogenesis marker in (a) scaffold-free model and (b) scaffold-based model. RNA was extracted from

1 cm from the middle of samples. Individual gene-expression levels were normalized against the internal

control, 36B4, and then normalized to gene-expression levels from static cultures

136

Ziming Chen et al.