Combining Collagenase Injections with Exercise-Induced Mechanical Overload: A Mouse Model Simulating Overload Stress Injury Knee Osteoarthritis

Summary

This protocol describes the establishment of a mouse model of overload stress-induced knee osteoarthritis by combining intra-articular collagenase injections with exercise-induced overexertion, providing a minimally invasive platform for investigating degenerative pathogenesis.

Abstract

Knee osteoarthritis (KOA) is a multifactorial degenerative joint disease driven by the complex interplay of biological tissue degradation and sustained mechanical overload. conventional surgical animal models often induce trigger post-traumatic joint destruction, which fails to accurately replicate the gradual and cumulative pathogenesis of human degenerative KOA. To bridge this translational gap, the present study establishes a novel and reproducible mouse model simulating overload stress-induced KOA by synergizing mild enzymatic cartilage degradation with sustained mechanical loading. Specifically, standardized intra-articular injections of type II collagenase were paired with daily mechanical overexertion via a strictly calibrated rotator-type fatigue apparatus over a four-week period. The efficacy of this dual-factor intervention was validated using comprehensive behavioral, histological, and molecular analyses. Automated gait analysis revealed severe locomotor deficits in the combined group. These were characterized by significant reductions in stride length, stride width, and peak stance paw area. Histomorphological evaluations using safranin O-fast green staining confirmed these functional deficits, a showing progressive cartilage structural damage, surface defects, and significantly elevated Osteoarthritis Research Society International (OARSI) scores that faithfully mirror the pathological hallmarks of early-to-mid stage human KOA. Furthermore, immunohistochemical evaluations demonstrated marked upregulation of the mechanosensitive channel Transient Receptor Potential Vanilloid 4 (TRPV4) and concurrent reduction in the crucial matrix protein Collagen Type II Alpha 1 (COL2A1) in articular chondrocytes. By integrating localized joint instability with controlled mechanical stress, this minimally invasive strategy avoids the acute trauma associated with surgical models. This protocol establishes a clinically relevant experimental platform for tracking dynamic molecular mechanisms, evaluating chronic pain phenotypes, and testing long-term therapeutic interventions for early-stage osteoarthritis.

Introduction

Osteoarthritis (OA) is a multifactorial degenerative condition characterized by pain, swelling, stiffness, and impaired function, arising from factors such as obesity, mechanical strain, and trauma¹. Knee osteoarthritis (KOA) is highly prevalent and increasingly common with aging, however clinical trials are often limited in scope due to diverse clinical manifestations. Therefore, the development of effective treatments necessitates appropriate animal models². Given the intrinsic heterogeneity of OA, no universally accepted "gold standard" model exists, requiring researchers to identify specific models that align with their scientific inquiry.

KOA is currently characterized in modern medicine as a condition that involves the interplay of mechanical and biological factors on the articular cartilage and perichondral tissue. The consequence of this intricate interplay is primarily the progressive deterioration of the articular cartilage and its surrounding matrix³. Mechanical stress serves as a crucial transduction signal governing various physiological and pathological processes within joint cells and tissues⁴. Maintaining articular cartilage in a healthy state necessitated appropriate mechanical loading within the physiological range. Conversely, excessive loading overload often results in the degeneration of cartilage and the subsequent onset of KOA⁵. Spontaneous models are hindered by substantial time and economic investments⁶, while surgically-induced models utilize a destructive approach that primarily represents mid- to late-stage KOA, deviating from natural degenerative progression^7,8. Conversely, chemically-induced models target the collagen matrix but fail to capture the comprehensive changes of human degenerative OA when used without mechanical stress⁹. Combining enzymatic degradation with mechanical overexertion addresses these gaps by more faithfully recapitulating the gradual pathogenesis of overload stress injury.The goal of this study is to establish and validate a novel mouse model of KOA by combining intra-articular type II collagenase injections with overexertion using a rotating wheel fatigue apparatus.

Protocol

All animal experiments were conducted in line with protocols approved by the Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine. Experiments were performed under a project license (NO.: PZSHUTCM201016022) granted by Experimental Animal Center Of Shanghai University Of Traditional Chinese Medicine Ethics, in compliance with National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals.

1. Animal Model Production Protocol

1. Randomly assign 32 10-week-old C57BL/6 mice to four groups: control, collagenase, excessive exercise, and collagenase + excessive exercise.
   1. Acclimate all mice for one week under standard feeding and housing conditions before initiating the modeling procedures.
2. Induce the animal models over a 4-week period according to the assigned experimental groups.
   1. Administer type II collagenase via intra-articular injection to the mice in the collagenase group.
   2. Subject the mice in the excessive exercise group to exercise training using a rotating wheel apparatus.
   3. Apply both the intra-articular collagenase injection and the rotating wheel exercise training to the mice in the collagenase + excessive exercise group.
   4. Maintain the exact same training frequency and duration across all relevant exercise groups.

2. Type II Collagenase Intra-Articular Injection Protocol

1. Anesthesia
   1. Induce anesthesia using isoflurane delivered through a calibrated gas anesthesia machine. Perform rapid induction at a concentration of 2%–3% isoflurane.
   2. Maintain anesthesia at 1.5%–2% isoflurane. Ensure the proper operation of the anesthetic scavenging system to prevent occupational exposure.
   3. Monitor the respiratory rate and reflexes of the mouse continuously throughout the procedure.
2. Skin Preparation
   1. Apply hair removal cream to the area surrounding the left knee joint. Remove the hair completely using a scraper.
   2. Clean the exposed skin thoroughly. Maintain aseptic conditions throughout the preparation.
3. Type II Collagenase Preparation
   1. Equilibrate the type II collagenase powder to room temperature for 30 min in a sterile biosafety cabinet. Use sterile phosphate-buffered saline (PBS, pH 7.2–7.4) as the exclusive solvent and buffer for the dissolution process.
   2. Precisely weigh the required mass of collagenase powder using an analytical balance. Slowly add the sterile PBS to the powder to reach a final working concentration of 0.04 U/µL.
   3. Mix the solution gently by pipetting the mixture up and down until it is clear and free of visible particles. Avoid violent vortexing during this step to prevent the loss of enzymatic activity.
   4. Centrifuge the prepared 0.04 U/µL solution at 1,000 rpm for 5 min at 4 °C. This removes undissolved micro-particles and prevents needle blockage during the subsequent injection.
   5. Aspirate the supernatant into sterile tubes and store them in a 4 °C ice bath for immediate use. Prepare the solution freshly for each session to ensure maximum enzymatic activity.
4. Intra-Articular Injection
   1. Grasp the dorsal skin of the mouse using the thumb and index finger of the non-dominant hand. Secure both lower limbs with the ring and little fingers to extend the knee joints naturally and fully expose the injection area.
   2. Hold a 10 µL microsyringe equipped with a 30 G needle. Insert the needle parallel to the skin surface into the superficial skin layer for approximately 0.2 cm at the entry point.
   3. Adjust the needle direction slowly to a perpendicular orientation relative to the skin when tissue resistance decreases.
   4. Continue advancing the needle for approximately 0.3–0.5 cm until a loss of resistance indicates entry into the joint cavity. Withdraw the syringe plunger slowly to confirm the absence of blood reflux.
   5. Inject 5 µL of the type II collagenase solution (concentration: 0.04 U/µL) into the joint cavity at a constant speed¹⁰.
   6. Maintain the needle position immediately after the injection. Gently press the skin surrounding the injection site for 10–15 s.
   7. Withdraw the needle slowly. Press the entry point lightly with a sterile gauze for 30 s to prevent drug leakage.
   8. Place the mouse into a warm and dry home cage. Allow the animal to recover naturally from anesthesia, which typically takes 5–10 min.
   9. Resume standard housing and feeding conditions after the mouse has fully recovered.
   10. Perform these intra-articular injections twice per week with a 3–4 day interval between the injections, exclusively during the 4-week modeling period (following the initial 1-week acclimatization).
   11. Dispose of all sharps immediately in approved biohazard containers.
       NOTE: Ensure trained personnel perform all injections to minimize joint injury and experimental variability.

3. Rotating Wheel–Induced Overexertion Protocol

1. Apparatus Preparation and Safety Check
   1. Set the rotator-type fatigue apparatus speed to 12 rpm (equivalent to approximately 0.063 m/s) and the duration to 2 h per session.
   2. Turn on the electrical stimulation module. Confirm the current output is within the preset safe range of 0.1–0.3 mA. Do not increase the current intensity manually.
      ​CAUTION: Use the electrical stimulation module strictly to guide the mice back to the wheel, not as a punitive stimulus. Check the apparatus wiring for damage and ensure good electrode contact before operation to prevent short circuits and electrical shock injuries.
2. Acclimatization Training
   1. Ensure the rotating wheel chambers of the fatigue apparatus are clean, dry, and free of debris. Deactivate the electrical stimulation electrodes at the wheel entrance to eliminate any potential stimulation-related stress.
   2. Handle each mouse gently by the base of the tail or scoop them using sterile soft tissue. Place mice individually into the central region of the rotating wheel’s running surface.
   3. Position the mouse in a natural quadrupedal standing posture on the horizontal running surface. Orient the body parallel to the wheel’s central rotation axis with the front paws forward and hind paws slightly back.
   4. Use a temporary, non-restrictive soft barrier to prevent the mouse from entering the narrow electrode zone during the first 2 min. Remove the barrier after this period to allow the mouse to explore the entire chamber freely.
   5. Leave the mice in the stationary apparatus for 10 min daily for the first 3 days. Do not start the wheel rotation or initiate any electrical stimulation during this period.
   6. Monitor the mouse for signs of severe stress such as persistent freezing or excessive rearing. Reposition the mouse gently to the central wheel area if it exhibits abnormal behavior to restore a natural posture.
   7. Start the wheel at a speed of 5 rpm for a 30 min duration on day 4. This step acclimates the mice to the movement rhythm of the apparatus without the use of electrical cues.
   8. Guide the mice manually back to the center of the wheel if they fall or stop during the rhythmic adaptation phase. Ensure the electrical stimulation remains deactivated throughout the entire 4-day acclimatization period.
      ​CAUTION: Avoid using electrical stimulation during the acclimatization phase to prevent fear-induced stress responses and ensure future training compliance.
3. Formal Exercise Execution
   1. Conduct the formal training at a fixed time each day (e.g., 9:00 AM–11:00 AM). Wear insulated gloves to place the mice into the rotator-type fatigue apparatus.
   2. Close the apparatus door. Start the preset parameters of 12 rpm (~0.063 m/s) for a maximum duration of 2 h per session (See Figure 1).
      ​CAUTION: Monitor the mice continuously during the training. Pause the training for 5 min if a mouse falls continuously (more than 3 times within 1 min) to prevent severe exhaustion and stress-induced injury.
4. Fatigue Endpoint Determination and Stimulation
   1. Monitor the mice continuously to evaluate their fatigue state. Terminate the training session early, without forcing the full 2 h duration, if a mouse meets at least two of the four specific fatigue criteria to avoid over-stress.
   2. Define Criterion 1 as the inability to remain on the running wheel for more than 5 s before falling. Define Criterion 2 as a significantly increased falling frequency of 3 or more times within a 1 min period.
   3. Define Criterion 3 as a lack of voluntary intention to return to the wheel, where the mouse remains immobile or moves only briefly despite electrical stimulation.
   4. Define Criterion 4 as the presentation of abnormal locomotor postures. Look for obvious signs such as dragging limbs or a staggering gait.
   5. Allow the apparatus to automatically trigger a mild electrical stimulation when a mouse falls to the spinner entrance. Ensure the electrical stimulation stops immediately once the mouse returns to the wheel¹¹.
      ​CAUTION: Terminate the training immediately if the mouse exhibits abnormal conditions such as convulsions or dyspnea. Remove the mouse from the apparatus and provide symptomatic treatment if necessary.
5. Post-Training Processing and Frequency
   1. Turn off the apparatus power after the training ends. Wear insulated gloves to remove the mice and return them to their home cages.
   2. Provide the mice with adequate drinking water. Observe the animals for any abnormal activity, mental status, or limb issues (e.g., claudication or muscle tremors).
   3. Record any abnormalities and provide timely treatment.
   4. Maintain this training frequency of once daily, 2 h per session, for 4 consecutive weeks. Keep the training parameters and safety monitoring consistent throughout the period.
      NOTE: Verify that the fatigue apparatus and electrical stimulation module are functioning properly before each daily training session to ensure consistent stimulation intensity.

4. Gait Analysis

1. System Overview
   1. Perform the gait acquisition and analysis using an automated gait imaging system.
   2. Conduct all gait procedures on 32 10-week-old C57BL/6 mice following the 4-week modeling period.
   3. Position the high-speed camera beneath the transparent treadmill belt to capture ventral images during locomotion.
2. Acclimatization and Setup
   1. Acclimate all mice to the treadmill apparatus prior to formal data collection to minimize stress.
   2. Set the treadmill speed to 20 cm/s to match the physiological walking pace of the mice.
   3. Record three independent valid trials per mouse with a 2-min rest interval between trials.
3. Trial Recording and Stride Selection
   1. Allow the mouse to walk continuously for a 60-second acquisition period during each trial.
   2. Perform a replacement trial if a recording is invalid due to "freezing," jumping, or turning.
   3. Initiate data collection only after the mouse achieves stable, consistent locomotion for at least 10 s.
   4. Select a minimum of 4 consecutive full strides from each 60-second trial for analysis.
   5. Ensure two independent blinded observers perform the stride selection to eliminate subjective bias.
   6. Confirm the mouse maintains a natural quadrupedal posture without skipping or sudden speed fluctuations.
   7. Verify the stride includes both a weight-bearing stance phase and a forward-propulsion swing phase.
   8. Check for clear paw-belt contact images without tracking loss, blurring, or contamination.
4. Image Processing and Parameter Extraction
   1. Use the system software's built-in algorithms to digitize captured images and detect paw-belt contact points.
   2. Calculate the Swing/stance ratio as the ratio of swing phase duration to stance phase duration.
   3. Measure Stride length (cm) as the linear distance traveled during a single full stride.
   4. Measure Stride width (cm) as the horizontal distance between the medial edges of contralateral hind paws at peak stance.
   5. Determine the Paw area at peak stance (cm²) representing the total contact area at maximum weight-bearing.
   6. Calculate Paw area variability (cm²) as the coefficient of variation of the contact area across consecutive strides.
   7. Calculate the average value of the 3 valid trials for each individual mouse and export the final datasets.
      NOTE: Exclude trials with irregular stopping, jumping, or turning behaviors from the analysis.

5. Histology

1. Tissue Collection and Fixation
   1. Euthanize all mice humanely by cervical dislocation following the 4-week induction period. Dissect the left knee joint carefully and remove the surrounding excess soft tissue.
   2. Immerse the specimen immediately in pre-cooled 4% paraformaldehyde (PFA) fixative (w/v). Ensure the fixative volume is 10–15 times the specimen volume to submerge the tissue completely.
   3. Fix the tissue at room temperature for 48 h. Agitate the fixation container gently every 8 h to ensure adequate fixative penetration.
2. Tissue Decalcification
   1. Rinse the fixed specimen with slowly running water for 10 min. Repeat this rinsing process three times to remove the residual PFA.
   2. Transfer the specimen into a 0.5 mol/L ethylenediaminetetraacetic acid (EDTA) decalcification solution (pH 7.2–7.4). Maintain the decalcification solution volume at 20 times the specimen volume and store it at 4 °C.
   3. Decalcify the tissue for 14–21 days. Replace the old fluid with fresh EDTA decalcification solution every 3 days.
   4. Test the decalcification endpoint by gently piercing the subchondral bone with a sterile needle. Conclude the decalcification process when the needle penetrates smoothly without resistance.
   5. Wash the completely decalcified specimen under running water for 2 h to remove the residual EDTA. Wash the specimen subsequently with phosphate-buffered saline (PBS) three times for 5 min each.
3. Embedding and Sectioning
   1. Dehydrate the decalcified specimen sequentially using a graded ethanol series (70%, 80%, and 90% for 2 h each; 95% for 1 h; absolute ethanol twice for 1 h each).
   2. Clear the dehydrated tissue in xylene twice for 10 min each. Embed the cleared tissue in paraffin.
   3. Cut the paraffin-embedded tissue into 5 µm thick sagittal continuous sections using a microtome. Mount the sections onto poly-L-lysine-coated glass slides and bake them in a slide drying hotplate at 60 °C for 2 h.
4. Histomorphological Evaluation
   1. Stain the sections with safranin O-fast green to evaluate the articular cartilage histomorphology.
   2. Assess the cartilage degeneration using the Osteoarthritis Research Society International (OARSI) scoring system under double-blind conditions¹².
5. Immunohistochemistry (IHC) Core Workflow
   1. Deparaffinize the sections in xylene twice for 10 min each. Rehydrate the sections sequentially through a graded ethanol series to deionized water.
   2. Immerse the sections in 0.01 mol/L citrate buffer (pH 6.0) for microwave-induced heat epitope retrieval. Heat the buffer to a boil on high power, then maintain a gentle simmer on medium power for 15 min.
   3. Cool the sections naturally to room temperature. Wash the slides with PBS three times for 5 min each
   4. Incubate the sections in a 3% hydrogen peroxide (H2O2) methanol solution for 20 min at room temperature to block endogenous peroxidase activity. Wash the slides with PBS three times for 5 min each.
   5. Apply a 5% goat serum blocking solution to the sections. Incubate for 60 min at room temperature to block non-specific binding sites.
   6. Discard the blocking solution without washing. Apply the specific primary antibody working solution (Transient Receptor Potential Vanilloid 4 (TRPV4) at 1:100, Caspase-3 at 1:200, or Collagen Type II Alpha 1 (COL2A1) at 1:150 dilution).
   7. Incubate the sections with the primary antibody in a humidified chamber at 4 °C overnight for 12–16 h.
   8. Equilibrate the sections to room temperature for 30 min the following day, then wash with PBS three times for 5 min each. Apply a horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG secondary antibody (1:500 dilution).
   9. Incubate the sections with the secondary antibody for 60 min at room temperature. Wash the slides with PBS three times for 5 min each.
   10. Apply a 3,3'-diaminobenzidine (DAB) chromogen solution and incubate in the dark for 3–5 min at room temperature. Stop the reaction immediately with deionized water when a clear brown signal appears under microscopic observation.
   11. Counterstain the sections with a hematoxylin solution for 5 min. Rinse under running water for 10 min to allow the stain to turn blue.
   12. Differentiate the sections using a 1% acid alcohol solution for 30 s. Rinse the slides under running water for 5 min.
   13. Dehydrate the sections rapidly through a graded ethanol series and clear the tissue in xylene twice for 10 min each.
   14. Mount the slides using neutral balsam and cover them with optical resin coverslips. Allow the mounted slides to dry at room temperature.
6. IHC Controls and Image Analysis
   1. Establish a negative control by replacing the primary antibody with PBS during the staining procedure. Include a known positive cartilage tissue section as a positive control to verify antibody efficacy.
   2. Acquire the stained section images using a whole slide scanner at 40× and 100× magnifications.
      NOTE: Perform all histological evaluations independently by at least two blinded observers to reduce subjective bias.

6. Statistical Analysis

1. Data Handling and Overall Testing
   1. Conduct all data collection and analysis under blinded conditions to minimize experimental bias.
   2. Analyze the continuous variables among the four experimental groups using a one-way analysis of variance (ANOVA) to test for overall differences.
   3. Apply the Kruskal-Wallis H test to any variables that do not meet the necessary parametric assumptions.
2. Post Hoc Testing and Significance Thresholds
   1. Perform post hoc pairwise comparisons using the Tukey test to identify specific group differences if the initial ANOVA indicates statistical significance.
   2. Rely solely on the Tukey test to control for the family-wise error rate. Do not combine this with any additional multiple-comparison correction methods.
   3. Define statistical significance uniformly as p < 0.05

Results

Gait Analysis
Gait cycle parameters: The swing/stance ratio—defined as the ratio of the duration of the non-weight-bearing swing phase to the weight-bearing stance phase of a single stride—was evaluated to reflect the balance of weight-bearing and propulsion. Compared to the control group (2.10 ± 0.06), mice that received an intra-articular injection of collagenase (1.45 ± 0.05) or underwent excessive exercise (1.68 ± 0.05) showed significantly lower swing/stance ratios. It is worth noting that this ...

Discussion

In this study, we developed a novel mouse model of KOA that mimics the effects of excessive stress on the joint by combining intra-articular injection of collagenase with wheel-based fatigue-inducing exercise training. These findings once again substantiate the notion that KOA arises from the interplay of biological and mechanical factors. Understanding and identifying these factors hold promise for devising individualized treatment plans and preventive strategies to alleviate symptoms and improve the quality of life for...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4% Paraformaldehyde fixative	Beyotime	P0099
Acetic acid	Sinopharm	631-61-8
DigiGait imaging system for mice	RWD Life Science	MSI-DIG-MS
EDTA solution	Procell	PB180320
Embedding cassette	Leica	IP C
Ethanol	Sinopharm	64-17-5
Fast green stain	Leyan	1085995
Hair removal cream	Veet	G20161330
Hematoxylin	Adamas	61753A
High-speed refrigerated centrifuge	Hermle	Z366K
Incubator	TAITEC	M-210FN
Inhalation anesthesia machine	Ranger	RZ-Viking
Isoflurane	Webio	w100
Microtome	Ted Pella	10180-220
Mouse rotator-type fatigue apparatus	Beijing Zhishu Duobao Biological Technology	YLS-10B
Neutral balsam	Sinopharm	96949-21-2
Safranin O stain	Bioss	S0062
Semi-automatic tissue embedding center	Thermo Fisher	HistoStar
Slide drying hotplate	Electron Microscopy Sciences	XH-2002
Tissue flotation water bath	BIOBASE	BT-I
Type II collagenase	Sigma-Aldrich	C4-22-1G
Whole slide scanner	Leica	Aperio VERSA
Xylene	Sinopharm	1330-20-7