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 demonstrated the significance of the skeletal system bearing mechanical forces for the normal development of bones^2,3,4,5. The response of the skeletal system to mechanical force partly depends on the cellular components in the bone⁶. Osteocytes account for 85% to 90% of the cellular components in the skeletal system. Due to their wide distribution and unique dendritic cell morphology, osteocytes are currently the most thoroughly studied type of skeletal cell in terms of mechanical response^7,8. Besides the widely recognized ability of osteocytes to respond to mechanical force, skeletal precursor cells also play an indispensable role in it. It was found that the response of periosteal precursor cells to mechanical signals regulated their fate transformation, thereby influencing tissue damage repair⁹.

Skeletal stem cells (SSCs) are a type of tissue-specific stem cells with self-renewal ability, which can differentiate into mature bone cell types required for bone growth, maintenance and repair. In 2015, surface marker genes for mouse SSCs were identified¹⁰. Subsequently, multiple studies have also demonstrated that SSCs play a very important role in various aspects such as bone development, homeostasis maintenance, and bone injury repair^11,12. Due to the characteristic of the skeletal system responding to mechanical forces, we can consider mechanical force signals as an indispensable factor in regulating the function of skeletal stem cells. To study the behavioral changes of SSCs under mechanical stimulation, we need to establish a suitable experimental system for applying mechanical force in vitro.

The main methods of in vitro mechanical stimulation for cells include fluid shear stress, compressive stress, tensile force, hydrostatic pressure, matrix stiffness and matrix topology^6,13. Based on the surface marker genes of mouse SSCs, we obtained mouse periosteal SSCs by flow cytometry sorting, since the SSCs involved in fracture repair mainly originate from the periosteum¹⁴. Subsequently, an appropriate tensile force was applied to the periosteal SSCs through a cyclic cell stress tension system. After tensile force stimulation, cell samples can be obtained, and multi-omics analyses can be conducted, including transcriptome sequencing, proteome sequencing, and ATAC-seq. Therefore, establishing this protocol provides a solid foundation for studying SSC responses to mechanical stimulation.

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 that the joint regions of the femur and tibia remain intact.
3. Preserve the isolated bones in chilled PBS, and then coat both ends of the bones with low-melting agarose (5-10% in TAE buffer).
4. Digest the femurs and tibias in minimum essential medium alpha (α-MEM) containing 1 mg/mL collagenase, 2 mg/mL dispase II, and 0.1 mg/mL DNase I at 37°C and 0.3 × g for 30 min twice. Collect the digested cells into the culture medium (α-MEM + 10% fetal bovine serum (FBS)).
5. Filter the cell suspension through a 40 µm cell strainer and then centrifuge at 500 × g for 5 min to remove the supernatant.
6. Resuspend the cell precipitate with red blood cell lysis buffer for 2 min and terminate the reaction with flow cytometry buffer (PBS + 2% FBS).
7. Centrifuge the cells at 500 × g for 5 min and discard the supernatant. Resuspend the cells with flow cytometry buffer at 1 × 10⁷ cells/mL for cell sorting.

2. Fluorescence-activated cell sorting

1. Stain the cell suspension with the following antibodies as follows: PerCP/Cy5.5-conjugated anti-mouse CD45, PerCP/Cy5.5-conjugated anti-mouse CD31, PerCP/Cy5.5-conjugated anti-mouse Ter119, FITC-conjugated anti-mouse 6C3/Ly-51, Brilliant Violet 605-conjugated anti-mouse CD90.2, PE/Cy7-conjugated anti-mouse CD105, APC-conjugated anti-mouse CD200.
2. Dilute the above antibodies at a ratio of 1:500 and incubate them on ice for 30 min, protected from light.
3. Wash the cells with flow cytometry buffer, and perform cell sorting with a fluorescence-activated cell sorter to obtain CD45^- CD31^- Ter119^- CD90.2^- 6C3^- CD105^- CD200⁺ SSCs. Collect the sorted SSCs with the culture medium. Analyze the acquired data with FlowJo v10.8.1 software.
4. Expand the sorted SSCs in culture dishes with expansion medium in a 5% CO₂ cell incubator. One passage could be performed before mechanical stimulation.
   NOTE: Approximately 1 × 10⁵ periosteal SSCs can be obtained from each pair of 6-week-old mice.

3. Mechanical stimulation of SSCs

1. Dilute the rat tail type I collagen in sterilized 0.02 M acetic acid to a final concentration of 0.15 mg/mL. This solution can be reused 3 times and should be kept at 4 °C.
2. Add 1.5 mL of diluted Rat tail type I collagen to each well of the 6-well flexible-bottomed culture plate. After coating the plates for 1 h at room temperature, aspirate the excess collagen and wash the plates once with PBS for later use.
3. NOTE: For immediate use, the plate should be rinsed with PBS to remove any residual acid. Before storage at 4 °C, the plate should be completely dried with the lid removed.
4. Digest the cultured mouse periosteal SSCs, centrifuge at 500 × g for 5 min, resuspend the cell precipitate in the culture medium to achieve a cell density of 1 × 10⁵ cells/mL, add 2 mL of cell suspension to each well of the 6-well flexible-bottomed culture plate, and place it in the cell incubator.
5. 24 h after seeding the cells, place the flexible-bottomed culture plate in the stretched base plate, and retain another cell flexible-bottomed culture plate as a control group without mechanical force stimulation.
6. Set the parameters in the FX-6000 Version 1.0 software as follows: Stretch strength 8%, frequency 0.1 Hz, waveform as a square wave, waveform duty cycle 80%, and stretch duration for 12 h.
   ​NOTE: There are four positions on the stretched base plate for placing a 6-well flexible-bottomed culture plate. If the cells required for the experiment are fewer than 4, the empty positions should be filled with new 6-well flexible-bottomed culture plates. After applying appropriate mechanical stimulation, SSCs can undergo functional tests, such as cell differentiation or proliferation, or samples can be extracted for multi-omics sequencing.

4. Real-time quantitative PCR (RT-qPCR)

1. After the mechanical stimulation of the cells was completed, discard the culture medium, wash the cells once with PBS, extract total RNA with TRIzol Reagent, and perform RNA reverse transcription.
   NOTE: After RNA extraction is completed, it can be stored at -80 °C. The cDNA obtained from reverse transcription can also be stored at -20 °C for subsequent testing.
2. Perform the RT-qPCR with a real-time PCR system. The primer sets used were as follows:
   Hprt: forward 5’-GTTAAGCAGTACAGCCCCAAA-3’,
   Hprt: reverse 5’-AGGGCATATCCAACAACAAACTT-3’,
   p16: forward 5’-GGTCACACGACTGGGCGATT-3’,
   p16: reverse 5’-GCACCGTAGTTGAGCAGAAGAG-3’,
   p21: forward 5’-GCCTGGTTCCTTGCCACTTCTT-3’,
   ​p21: reverse 5’-ATTACGGTTGAGTCCTAACTGCCATC-3’.

5. The effects of short-term treatment of mechanical stress

1. Set the parameters in the FX-6000 Version 1.0 software as follows: Stretch strength 8%, frequency 0.1 Hz, waveform as a square wave, waveform duty cycle 80%. The cells are lysed with RIPA lysis buffer I (with phosphatase inhibitor cocktail and protease inhibitor cocktail) at 10 min and 60 min after mechanical tension stimulation.
2. Perform the Western blot with Erk and p-Erk antibody.

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 significant role of periosteal SSCs in bone development and fracture repair^14,15. It is necessary to fully analyze the changes in periosteal SSCs after responding to mechanical stimulation.

To detect the changes that occurred in periosteal SSCs after mechanical force stimulation, the expression of cellular senescence marker genes was detected, as primary cells are prone to cell senescence during culture passage¹⁶. The results showed that after mechanical stimulation, the expression of cellular senescence marker genes p16 and p21 in periosteal cells was found to be significantly downregulated (Figure 2A–B). Short-term treatment can increase the phosphorylation level of Erk in skeletal stem cells; however, longer treatment duration does not further increase the phosphorylation level of Erk (Figure 2C).

Figure 1: Fluorescence-activated cell sorting of periosteal SSCs. (A) Singlet gating on SSC-A vs FSC-A and FSC-W vs FSC-H. (B) Lin^- cell gating within the singlet population using CD31, CD45, and Ter119 antibodies. (C) SSC gating within the Lin⁻ population was performed using 6C3, CD90.2, CD105, and CD200 antibodies. The proportions of SSCs, pre-BCSPs, and BCSPs in the Lin⁻ 6C3⁻ CD90.2⁻ population were 28.3%, 52.7%, and 16.1%, respectively. Abbreviations: BCSP = bone cartilage stromal progenitor. Please click here to view a larger version of this figure.

Figure 2: Detection of cell senescence marker gene expression in periosteal SSCs. (A, B) After mechanical force stimulation, the mRNA expression of senescence marker genes p16 (A) and p21 (B) in mouse periosteal SSCs decreased significantly. Data are presented as the means ± SD. n = 4 refers to biological replicates. Unpaired t-test. (C) The protein levels of p-ERK and ERK were detected in cells at 10 min and 60 min after the application of mechanical tension stimulation. Please click here to view a larger version of this figure.

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 knockout mouse models^9,18, and there is still a lack of direct evidence to reveal what changes have occurred in SSCs under mechanical stimulation. The cyclic stretch system can provide precise, controllable, repeatable, static, or periodic stress changes for cells cultured in vitro, making it a very suitable experimental platform for applying mechanical force to cells. Many studies have utilized the cyclic stretch system to investigate the cellular responses of osteoblasts, osteocytes, chondrocytes, tenocytes, ligament cells, and myoblasts under mechanical stimulation¹⁹. Here, mouse periosteal SSCs were collected by flow cytometry sorting, and then mechanical stimulation was applied with the cyclic stretch system, providing a reliable experimental system for further research on the regulation of SSCs' function by mechanical stimulation. The advantage of this protocol is that it can apply specific and controllable mechanical stimulation to SSCs, eliminating the interference of other bone cell types in animal models, such as exercise models and models that directly apply mechanical loading to bones^20,21.

The first key point of this protocol is to obtain a cell suspension with sufficient viability for cell sorting. During bone digestion on a shaker, the rotation speed should not exceed 0.5 × g. This parameter can be adjusted based on experimental outcomes. In some cases, the speed may be reduced to 0.1 × g, with a corresponding increase in the digestion time. Second, due to differences in equipment and experimental systems among laboratories, researchers may need to further optimize the parameters used for mechanical stimulation based on our protocol in order to achieve the desired results.

One limitation of this protocol is that it requires a relatively large number of SSCs (2 × 10⁵ cells per well), whereas SSCs are present in bone tissue at low abundance. Although this limitation can be partially addressed by increasing the number of experimental mice, such a strategy may restrict its broader applicability. For future clinical translation of mechanically stimulated SSCs, the development of an appropriate in vitro expansion system that preserves SSC properties in a stable manner will be essential, analogous to approaches used for the in vitro expansion of hematopoietic stem cells²².

In conclusion, a protocol for the direct application of mechanical stimulation to mouse periosteal SSCs is presented, and mechanical stimulation was found to reduce the expression of p16 and p21 in SSCs. It is worth noting that short-term treatment enhances Erk phosphorylation in skeletal stem cells, whereas prolonged stimulation does not further elevate this phosphorylation level. Therefore, observing distinct biological effects requires trying different durations or magnitudes of mechanical stimulation. The implementation of this protocol may promote further investigation into the regulation of SSC function by mechanical force and improve understanding of the mechanistic network through which the skeletal system responds to mechanical signals.

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