The Enhancing Effect of Mechanical Stimulation on the Chondrogenic Function of Infrapatellar Fat Pad Stem Cells

Summary

This study focuses on developing a multimodal technology combining cyclic tensile mechanical stimulation (10% strain, 1 Hz) with infrapatellar fat pad-derived stem cells (IPFP-SCs) therapy, investigating their synergistic effects on promoting chondrogenic differentiation of IPFP-SCs, and establishing a more efficient regenerative strategy for cartilage tissue engineering.

Abstract

The infrapatellar fat pad (IPFP), a specialized fibrofatty structure within the anterior compartment of the knee joint, is characterized by a unique microarchitecture featuring interspersed collagen bundles and adipose lobules, which collectively establish its viscoelastic biomechanical properties. Emerging evidence highlights the distinctive transcriptional profile of IPFP-SCs, specifically their association with cartilage degradation mediators during osteoarthritis progression. Recent research demonstrates that dynamic compression and hydrostatic pressure effectively enhance chondrogenic differentiation of both bone marrow-derived and IPFP-derived mesenchymal stem cells while inhibiting calcification deposition. This study utilized a cell stretching system to simulate the cyclic mechanical stress environment of the knee joint, demonstrating that dynamic tensile stimulation (10% strain, 1 Hz) significantly enhances the chondrogenic differentiation capacity of IPFP-SCs. This enhancement was manifested by upregulated expression of chondrogenic markers, including SOX9 and COMP, confirming that the joint-specific mechanical microenvironment plays a critical regulatory role in the terminal differentiation of mesenchymal stem cells. These results provide crucial experimental evidence for cartilage tissue regeneration strategies, emphasizing the importance of biomechanical modulation in regenerative therapies for cartilage repair.

Introduction

The regeneration and repair of cartilage tissue represent a central challenge in osteoarthritis treatment, as conventional cell therapies often fail to reconstruct functional cartilage matrices due to inadequate microenvironmental regulation. In recent years, multimodal strategies integrating physical mechanical stimulation with cell therapy have emerged as a research hotspot¹, aiming to enhance the chondrogenic differentiation potential of stem cells by simulating in vivo biomechanical microenvironments. This study focuses on developing a multimodal technology combining cyclic tensile mechanical stimulation (10% strain, 1 Hz) with IPFP-SCs therapy, investigating their synergistic effects on promoting chondrogenic differentiation of IPFP-SCs, and establishing a more efficient regenerative strategy for cartilage tissue engineering.

The core objective of this study is to enhance the chondrogenic capacity of IPFP-SCs through dynamic tensile stimulation. Previous studies have demonstrated that mechanical signals can drive chondrogenic differentiation of mesenchymal stem cells (MSCs) by regulating cytoskeletal reorganization, ion channel activation (e.g., Piezo1), and downstream signaling pathways (e.g., the YAP-SOX9 axis)². Existing literature provides critical insights into the synergistic effects of multimodal stimulation. Buckley and Kelly et al. confirmed that periodic hydrostatic pressure stabilizes chondrogenic phenotypes by enhancing sGAG and type II collagen deposition³. Guo et al. reported that dynamic culture combined with appropriate mechanical stimulation facilitates the efficient expansion of progenitor cells, enabling the generation of clinically relevant articular chondrocytes for cartilage defect repair. However, shear stress alone resulted in inferior chondrogenic differentiation of hMSCs compared to static conditions, while additional compressive loading upregulated chondrogenic markers such as Sox9, aggrecan, and type II collagen⁴. Notably, shear stress alone fails to induce significant cartilage-specific gene expression⁵. Zhang et al. further demonstrated that combining mechanical stimulation with exogenous factors (e.g., SOX-9) significantly improves differentiation efficiency⁶. Studies reveal that dynamic compression induces chondrogenic differentiation, whereas inhibition of the ERK1/2 pathway abolishes this response. Conversely, ERK1/2 inhibition under dynamic compression enhances osteogenic differentiation, marked by increased expression of alkaline phosphatase (ALP), type I collagen (COLI), and osteocalcin (OCN)⁷. Building on these findings, this study adopts tensile stimulation as a core intervention, exploring its synergistic role with endogenous signaling pathways in IPFP-SCs (e.g., Piezo1-mediated calcineurin activation)². This approach aligns more closely with the multimodal mechanical environment of joint motion. Additionally, recent research highlighting the spatiotemporal specificity of mechanical stimulation⁸ informs the design of a staged loading protocol in this study.

Compared to conventional static culture, this technology innovates in two key aspects. First, a delayed stimulation protocol is implemented to avoid early inhibitory effects of mechanical loading on differentiation. For instance, Luo et al. demonstrated that dynamic compression applied 21 days post-cell encapsulation significantly enhances chondrogenesis in BMSCs⁹, a principle integrated into the current loading protocol. Second, a precision mechanical stimulation system enables accurate control of tensile stress parameters (intensity, frequency, and duration), replicating physiologically relevant biomechanical conditions. Precise parameter control is critical to obtaining reliable results, as excessive mechanical loading may induce cell damage¹⁰, while oversimplified mechanical environments may yield counterproductive outcomes¹¹. Inspired by advanced multi-mechanical coupling systems⁵, the developed apparatus ensures stable and reproducible stimulation, with high scalability for translational applications. Furthermore, leveraging the unique advantages of IPFP-SCs, including their developmental homology with articular cartilage and high chondrogenic potential³, enhances the technology's clinical feasibility.

This study advances mechanistic insights into IPFP-SCs chondrogenesis and supports the development of clinical cartilage repair strategies. By integrating multimodal mechanical stimulation with cell therapy, this technology addresses limitations of traditional methods and provides insights for postoperative biomechanical rehabilitation protocols. In summary, through the innovative integration of tensile stimulation and IPFP-SCs therapy, this study aims to establish an efficient and controllable cartilage regeneration strategy. Its design draws extensively on prior mechanobiological research while opening new avenues for the clinical translation of tissue engineering technologies.

Protocol

The study was performed in compliance with the Institutional guidelines and was approved by the Ethics Committee of Inner Mongolia Autonomous Region People's Hospital(SC-07/01KT2024008). The reagents and the equipment used are listed in the Table of Materials.

1. Thawing and expansion of IPFP-SCs

1. Thaw IPFP-SCs.
   1. Rapidly thaw cryopreserved IPFP-SCs (P2-P5) in a 37 °C water bath.
   2. Transfer the cells into a 15 mL centrifuge tube containing pre-warmed complete growth medium (α-DMEM + 10% FBS + 1% penicillin/streptomycin).
   3. Centrifuge at 300 × g for 5 min at room temperature. Discard the supernatant and resuspend the cell pellet in fresh growth medium.
2. Expand the cells.
   1. Seed the cells into two 10 cm culture dishes at a density of 5,000-8,000 cells/cm². Incubate the cells at 37 °C with 5% CO₂.
   2. Replace the medium every 2-3 days until cells reach 80-90% confluence.
   3. Passage the cells using 0.25% trypsin-EDTA and maintain in culture for subsequent steps.

2. Seeding cells for chondrogenic differentiation

1. Prepare cell suspension.
   1. Digest the cells with 0.25% trypsin-EDTA at 37 °C for 5 min when they reach 80%-90% confluence.
   2. Add complete medium at a 1:1 ratio to terminate digestion.
   3. Centrifuge at 300 × g for 5 min at room temperature. Discard the supernatant and resuspend the cell pellet in fresh growth medium.
2. Seed the cells.
   1. Count the cells using a hemocytometer or automated cell counter.
   2. Dilute the resuspended cells to a density of 4 × 10⁵ cells per chamber of a specialized culture dish.
   3. Place the chambers in a 37 °C, 5% CO₂ incubator for 12-16 h to allow complete cell attachment.

3. Chondrogenic pre-culture

1. Initiate pre-differentiation.
   1. Remove the growth medium from the chambers.
   2. Rinse the cells twice with 1× DPBS (pre-warmed to 37 °C) to remove residual serum and debris.
   3. Wash the cells twice with pre-mixed chondrogenic induction medium (without growth factors).
   4. Replace the wash medium with complete chondrogenic induction medium.
   5. Maintain the cultures at 37 °C with 5% CO₂ for 3 days.
2. Monitor the cell morphology.
   1. Confirm cell adherence and early aggregation using microscopy.

4. Cyclic tensile mechanical stimulation (Day 7)

1. Prepare the mechanical stimulation device.
   1. Sterilize the device and culture chambers with 75% ethanol, followed by UV irradiation.
   2. Calibrate the uniaxial cell stretching system to deliver 10% strain at a frequency of 1 Hz.
2. Initiate stimulation.
   1. Apply cyclic tensile stress (10% strain, 1 Hz) for 1 h per day over 7 consecutive days.
   2. Maintain environmental conditions at 37 °C with 5% CO₂ throughout the stimulation period.
   3. Replace the chondrogenic induction medium every 72 h.
3. Control setup
   1. Include parallel static control chambers without mechanical stimulation under identical culture conditions.

5. Immunofluorescence staining

1. Sample preparation and cutting
   1. Cut the mechanically stimulated cell-seeded membrane with sterile surgical scissors or a scalpel to match the size and shape of the confocal dish.
   2. Ensure the trimmed sample fully covers the imaging area of the dish.
2. Fix the cells.
   1. Wash the cells twice with PBS.
   2. Fix with 4% paraformaldehyde (PFA) for 15 min at room temperature (RT).
   3. Treat with 0.1% Triton X-100 in PBS for 10 min at RT.
   4. Wash twice with PBS.
   5. Block with 2% BSA in PBS for 30 min at RT.
3. Stain for markers.
   1. Incubate the sample overnight at 4 °C with primary antibodies diluted in blocking solution: anti-SOX9 (1:200) and anti-COMP (1:200).
   2. Wash the sample 3 times with PBS.
   3. Dilute fluorescent-conjugated secondary antibodies in PBS at a 1:1000 ratio.
   4. Add 100 µL of diluted secondary antibody solution to cover the sample.
   5. Incubate at RT for 1 h on an orbital shaker protected from light.
   6. Wash the sample 3 times with PBS.
4. Image acquisition
   1. Visualize the sample using a fluorescence microscope.
   2. Capture images at consistent exposure settings.

6. Quantitative PCR (qPCR) analysis

1. Extract RNA
   1. Lyse the cells in TRIzol reagent.
   2. Isolate total RNA following the manufacturer's protocol.
   3. Measure RNA concentration and purity using a spectrophotometer.
2. Synthesize cDNA.
   1. Reverse-transcribe 1 µg of RNA into cDNA using a high-capacity cDNA synthesis kit.
3. Perform qPCR.
   1. Prepare qPCR reactions using SYBR Green Master Mix and specific primers for chondrogenic markers (SOX9, COMP) and housekeeping genes.
   2. Run the assays in triplicate on a real-time PCR system. Include no-template controls.

7. Reproducibility

1. Sterility
   1. Perform all steps under a laminar flow hood to prevent contamination.
2. Parameter consistency
   1. Verify mechanical stimulation settings (strain, frequency, and duration) daily.
3. Replicates
   1. Include at least three biological replicates per condition.
4. Data normalization
   1. Normalize qPCR data to housekeeping genes and static controls.

Results

The representative findings of this study demonstrate that cyclic tensile mechanical stimulation enhances the chondrogenic differentiation of IPFP-SCs under chondrogenic induction. Specifically, IPFP-SCs subjected to 7-day tensile loading exhibited significant upregulation of chondrogenic markers at both transcriptional levels, as evidenced by increased SOX9 expression and COMP production compared to static control groups. These coordinated molecular and structural responses confirm that biomechanical s...

Discussion

The protocol outlined integrates cyclic tensile mechanical stimulation (10% strain, 1 Hz) with IPFP-SCs to enhance chondrogenic differentiation, addressing critical challenges in cartilage regeneration. Key steps include precise cell expansion, staged chondrogenic pre-culture (3 days), delayed mechanical stimulation (initiated at Day 3 for 7 days), and post-stimulation analysis via immunofluorescence (SOX9, COMP) and qPCR. The mechanical stimulation of protocol, informed by prior mechanobiological studi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4% Paraformaldehyde	biosharp	BL539A
Anti-Fluorescence Quenching Blocking Solution (containing DAPI)	Beyotime	P0131
Blocking Solution	Beyotime	P0260
Cell Culture Chamber	AIRTECH	AZ2020062530
Cell Tank	CELL &FORCE
Col II Primary Antibody	Abcam	AB34712
E.Z.N.A. HP Total RNA Kit	OMEGA	R6812-00S
F-actin Primary Antibody	Proteintech	PF00001
Fetal Bovine Serum	CellMax	SA101.02
Goat to Rb IgG	Abcam	AB50077
Hieff qPCR SYBR Green Master Mix(Low Rox Plus)	YEASEN	11202ES50
Human Mesenchymal Stem Cell Chondrogenic Induction Kit	Fuyuanbio	FY200008
Leica DMi8 inverted biomicroscope	LEICA	DMi8
LightCycler 96 Instrument	Roche	16056
MEM-α	Gibco	C12571500BT
PBS	Servicebio	G4202
Penicillin-Streptomycin-Amphotericin B	NCM Biotech	C100C8
Piezo1 Primary Antibody	Proteintech	15939-1-AP
PrimeScript RT reagent Kit (Perfect Real Time)	Takara	RR037A
SOX9 Primary Antibody	Abcam	AB185230
TritonX-100	SCIGE	SG6193
Veriti Dx 96-well Thermal Cycler	Thermo Fisher	EN61326