An In Vitro System for Applying Tensile Force to Skeletal Stem Cells Using a Cyclic Cell Stress Tension System

Summary

Mouse periosteal skeletal stem cells (SSCs) were isolated by FACS and cultured in vitro. A controllable experimental system was established to apply cyclic tensile mechanical stimulation to SSCs, resulting in decreased expression of the cellular senescence-associated genes, specifically p16 and p21.

Abstract

Bone is a major mechanosensitive organ that continuously responds to physical cues in the body. Although mechanical stimulation plays important roles in skeletal development, homeostasis, and injury repair, the specific responses of skeletal stem cells (SSCs) to mechanical stress remain incompletely understood. To address this question, we established an in vitro model to isolate mouse periosteal SSCs and apply mechanical stimulation. Mouse periosteal cells were first obtained by enzymatic digestion, and SSCs were subsequently isolated by fluorescence-activated cell sorting (FACS) based on established surface markers. After attachment to culture, SSCs were subjected to tensile mechanical stimulation using a cyclic cell stress-tension system. This system enables the reproducible application of tensile force to cultured cells and provides a platform for analyzing SSC responses to mechanical input. Using this approach, we found that the expression of the cellular senescence-associated genes p16 and p21 was markedly reduced in SSCs following mechanical stimulation. These findings suggest that tensile stimulation may influence senescence-related changes in SSCs under in vitro conditions. Overall, this protocol provides a useful and suitable platform for studying SSC behavior under mechanical stimulation and for investigating how mechanical cues affect SSC function.

Introduction

As the largest and most important supporting organ in the human body, the skeletal system not only provides attachment sites and protection for other organs, but also serves as the foundation for motor function. Therefore, the skeletal system is constantly exposed to various mechanical stimuli from both internal and external environments. Mechanical stimulation signals are equally crucial for bone development and functional maintenance. As early as 1893, it was proposed that there is a certain positive correlation between bone density and the mechanical force that bones bear, which is Wolff’s law¹. In recent years, some studies have demon....

Protocol

The mouse experiments in this protocol have all been approved by the Animal Care and Use Committee of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (SIBCB-S350-2312-41).

1. Isolation of periosteal cells in mice

1. Place 4- to 6-week-old mice in a CO₂ euthanasia box and expose them to CO₂ continuously for 3 min until breathing stops.
2. Disinfect the surface of the mice's bodies with 75% alcohol. Use ophthalmic scissors to separate the hind limbs. Then use pointed forceps and ophthalmic scissors to remove the skeletal muscles from the hind limbs, ensuring tha....

Results

Following the implementation of this protocol, tensile force was successfully applied to periosteal SSCs of mice. Firstly, SSCs were obtained from the periosteum of mice by flow cytometry. The flow cytometry results showed that the proportion of SSCs at the periosteum accounted for nearly 30% of Lin^- (CD45^-, CD31^-, Ter119^-) CD90.2^- 6C3^- (Figure 1), which was much higher than that in the bone marrow. Given the signif.......

Discussion

In recent years, skeletal stem cell marker genes from different parts of bones have been continuously discovered^11,12,17. However, research on the direct regulation of SSCs function still needs to be deepened, especially in the aspect of mechanical force stimulation regulation. So far, research on the mechanical regulatory effects of skeletal stem cells/precursor cells has mainly been based on the construction of Piezo1.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
α-MEM	Corning	10-022-CV	periosteal cells Isolation
APC anti-mouse CD200	BioLegend	123809	SSCs sorting
BioFlex 6-Well Culture Plate	Flexcell	BF-3001A	Cell culture
Brilliant Violet 605 anti-mouse CD90.2	BioLegend	140317	SSCs sorting
Collagenase	Sigma	C0130	periosteal cells Isolation
Dispase II	Sigma	D4693	periosteal cells Isolation
DNase I	Sigma	DN25	periosteal cells Isolation
Fetal Bovine Serum	Gibco	A5356701	SSCs sorting
FITC anti-mouse 6C3/Ly-51	BioLegend	108305	SSCs sorting
Flexcell Tension System	Flexcell	Flexercell FX-6000	Cell tension
Flow Cytometry Sorter	BD	BD FACSAria Fusion	SSCs sorting
MesenCult Expansion Kit (Mouse)	Stem cell	05513	Cell culture
PE/Cy7 anti-mouse CD105	BioLegend	120409	SSCs sorting
PerCP/Cy5.5 anti-CD31	BioLegend	102522	SSCs sorting
PerCP/Cy5.5 anti-CD45	BioLegend	103132	SSCs sorting
PerCP/Cy5.5 anti-mouse TER-119	BioLegend	116228	SSCs sorting
PrimeScript RT Reagent Kit	TaKaRa	RR037A	RT-qPCR
Rat tail type I collagen	Corning	354236	Cell culture
real-time PCR system	Thermo Fisher Scientific	QuantStudio 1 Plus	RT-qPCR
red blood cell lysis buffer	Beyotime	C3702	periosteal cells Isolation
TRIzol	Sigma	T9424	RT-qPCR
Phosphatase Inhibitor Cocktail II	MCE	HY-K0022	WesterBlot
RIPA Lysis Buffer I	Sangon Biotech	NO. C500005	WesterBlot
Protease Inhibitor Cocktail	MCE	HY-K0010	WesterBlot
p44/42 MAPK (Erk1/2) Antibody	Cell Signaling Technology	9102	WesterBlot
Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) Rabbit Monoclonal Antibody	Cell Signaling Technology	4370	WesterBlot