A Surgical Method to Establish a Murine Model of Knee Osteoarthritis using a Modified Hulth Procedure

Summary

This protocol outlines the refined surgical steps of the modified Hulth model, providing a standardized, reproducible method for inducing experimental osteoarthritis.

Abstract

Knee osteoarthritis (KOA) is a chronic degenerative joint disease characterized by articular cartilage degeneration, subchondral bone remodeling, osteophyte formation, and synovitis, which is the most common type of osteoarthritis.

This protocol presents a surgical method to establish a murine model of knee osteoarthritis using a modified Hulth procedure. Compared to injection-induced or conventional surgical models such as destabilization of the medial meniscus (DMM) and anterior cruciate ligament transection (ACLT), the modified Hulth model induces an earlier disease onset and more severe joint inflammation. By transecting the medial collateral ligament, medial meniscus, and anterior cruciate ligament, this method disrupts the mechanical alignment of the knee joint, thereby promoting OA progression. This model is particularly suitable for studying acute OA pathology and evaluating potential therapeutic interventions. To evaluate arthritis severity, we used small-animal ultrasound combined with three-dimensional (3D) reconstruction of the joint cavity to quantify its volume and assess synovial fluid accumulation. In addition, safranin-O/fast green staining of histological sections was performed for Osteoarthritis Research Society International (OARSI) scoring to evaluate cartilage destruction.

Introduction

Knee osteoarthritis (OA) is a common, chronic, degenerative joint disease characterized clinically by pain, stiffness, limited range of motion, and muscle weakness around the knee. Its prevalence is higher in women than in men. If inadequately controlled, OA can lead to progressive loss of physical capacity, sleep disturbance, chronic fatigue, and eventual disability. As global populations age, the incidence of knee OA continues to rise, posing a substantial challenge to health-care systems and economies worldwide^1,2.

Pathophysiologically, cartilage loss represents the central feature of OA, accompanied by structural and functional impairment of the entire joint and peri-articular tissues. To elucidate disease mechanisms and evaluate therapeutic interventions, a variety of animal models have been developed that recapitulate key aspects of human OA. Among these, surgically induced models are most frequently employed. Such as destabilization of the medial meniscus (DMM)³ and anterior cruciate ligament transection (ACLT)⁴, disrupt normal joint structure, which results in cartilage degeneration and OA-like changes. Alternatively, chemical models rely on intra-articular injection of agents that degrade the extracellular matrix (e.g., collagenase or monosodium iodoacetate), leading to rapid chondrocyte death and cartilage erosion^5,6. Whereas injection models typically achieve endpoint pathology within weeks, surgical models generally require a longer time course.

Among the available surgical techniques, we selected and further refined the Hulth model because of its accelerated onset of OA⁷. By combining transection of the anterior cruciate and posterior cruciate ligaments with medial collateral ligament resection and total medial meniscectomy, this model reliably induces severe, rapidly progressive cartilage damage and subchondral bone alterations. The short time frame to structural joint failure makes the modified Hulth model particularly suitable for acute or subacute OA studies, and we propose that our optimized protocol offers a practical, reproducible platform for future preclinical investigations.

Protocol

All experimental procedures were approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine. Male C57BL/6 mice aged 6-8 weeks and weighing approximately 20 g were purchased from commercial vendors (see Table of Materials) (Ethics approval number: PZSHUTCM2312210005).

1. Preoperative care

1. Allow mice to acclimate for at least 1 week before surgery.
2. Sterilize all surgical instruments before surgery and disinfect the surgical workspace.
3. Turn on the gas anesthesia machine, and place the mouse in an induction chamber for 5 min using 2-3% isoflurane to initiate general anesthesia.
   NOTE: Ensure that the oxygen flow rate is maintained between 0.5 NL/min and 1 NL/min.
4. Once anesthetized, transfer the mouse to the surgical platform and maintain anesthesia using 1-1.5% isoflurane via an anesthesia mask until completion of the surgery (1-1.5% isoflurane).
5. Position the mouse in a supine position with the knee joints facing upward, ensuring stability and proper access to the surgical site.
6. Confirm the depth of anesthesia by checking for the absence of reflexes, then apply depilatory cream to completely remove the hair around the knee joint.
7. Remove the residual cream carefully using gauze or cotton swabs, then disinfect the exposed skin first with 75% ethanol, followed by iodine tincture to ensure aseptic conditions.

2. Surgical procedure (Figure 1)

1. Extend the mouse's hind limb and apply traction to tighten the skin. Make a 0.5-1 cm longitudinal incision along the medial aspect of the right knee joint using a sterile surgical blade.
   NOTE: All surgical procedures described below were performed under a surgical microscope.
2. Retract the skin and perform blunt dissection of the underlying fascia to fully expose the joint capsule, and visualize the patellar ligament.
3. Probe and incise the fascia overlying the medial collateral ligament (MCL), using a fine injection needle.
4. Transect the MCL using microscissors under magnification.
5. Displace the patellar ligament using a needle to expose the intra-articular space and identify the medial meniscus.
6. Transect the medial meniscotibial ligament (MMTL) using micro-scissors after cutting, and gently probe the tissue with a needle to confirm complete detachment and mobility of the meniscus.
7. Identify and transect the anterior cruciate ligament (ACL), taking care to avoid damage to the posterior cruciate ligament (PCL) or lateral meniscus. This will typically result in a positive anterior drawer sign post-operatively, confirming joint instability.
   NOTE: Drawer test: Prior to wound closure, the femur was stabilized with one hand while the ankle was grasped with the other hand, maintaining the tibia and femur in a perpendicular orientation. Relative anteroposterior displacement between the tibia and femur was manually applied; observable translational movement indicated a positive drawer sign, confirming cruciate ligament disruption.
8. To induce more severe osteoarthritis, alternatively, perform simultaneous transection of both the anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) combined with medial meniscectomy (MMT) (as in the classic Hulth model).
   NOTE: This step is optional and may be performed as required; if selected, this constitutes the modeling approach of the classic Hulth model.

3. Postoperative care

1. Close the incision with 4-0 nylon sutures and disinfect the surgical site with iodine tincture.
2. Place the mouse on a temperature-controlled heating pad until it regains consciousness and exhibits normal breathing and motor activity. Once fully recovered and showing no signs of distress, return it to its home cage.
3. Monitor suture integrity continuously for 3-5 days following surgery. If suture breakage is observed, re-anesthetize the mouse and perform re-suturing
4. Administer intramuscular antibiotics for up to 3 days post-surgery to prevent postoperative infection.
   NOTE: Antibiotic administration is employed for prophylaxis against postoperative infection. Postoperative analgesia is determined by experimental objectives; in osteoarthritis research, analgesics are typically withheld during the acute phase to avoid confounding therapeutic efficacy assessment.
5. Monitor the surgical site daily for signs of swelling, discharge, or local inflammation.
6. Euthanize the mouse at the study endpoint, following approved protocols, and dissect the knee joint for histological or imaging analysis.
   NOTE: At this step, we adopted the protocol of euthanizing the experimental animals by overdose anesthesia. This was achieved by intraperitoneal injection of pentobarbital sodium solution at a dosage of >100 mg/kg in mice.

4. Evaluation of OA development

1. Prior to euthanasia, assess joint swelling and perform ultrasonography to evaluate structural changes in the knee joint. Evaluate motor performance in the mouse by performing the rotarod test.
   NOTE: Isoflurane anesthesia is required for ultrasound examination in mice.
2. Following euthanasia, dissect the knee joint and carefully remove the surrounding muscle and fascia while preserving the tibia, fibula, and femur.
   ​Fix the joints and decalcify them in 10% ethylenediaminetetraacetic acid (EDTA) solution for at least 30 days, replacing the solution twice per week to ensure complete decalcification.
3. Embed the decalcified knee joints in paraffin and prepare sagittal sections at a thickness of 4 µm⁸.
4. Begin collecting sections once the meniscus is clearly visible. Stain the sections with safranin O and fast green to visualize cartilage and subchondral bone morphology. Evaluate cartilage degradation using the OARSI⁹ scoring system.
5. Safranin O-Fast Green staining procedure (Commercial kit used, see Table of Materials)
   1. Deparaffinization and rehydration
      1. Deparaffinize sections in xylene for 3 min, repeat 3 times.
      2. Rehydrate through a graded ethanol series (100%, 90%, 80%) to remove residual xylene, 2 min each.
         NOTE: Ethanol series must be used in descending order of concentration.
      3. Rinse in distilled water for 1 min and drain briefly.
   2. Weigert's Iron Hematoxylin staining
      1. Apply Weigert's working solution to sections, stain for 5 min.
      2. Rinse in distilled water for 1 min and drain briefly.
   3. Acid differentiation
      1. Apply the acid differentiating solution to sections for 15 s.
      2. Wash in water for 10 min and drain excess water.
         NOTE: Differentiation time is critical -- prolonged differentiation prevents fast green staining, whereas insufficient differentiation results in deep fast green staining with poor safranin uptake
   4. Fast Green staining
      1. Apply fast green solution to sections for 5 min, drain briefly.
      2. Apply a weak acid solution to remove residual fast green stain for 15 s, and drain briefly.
   5. Safranin O staining
      1. Apply safranin O solution to sections for 4.5 min.
      2. Rapidly transfer sections to 90% ethanol to remove excess safranin O, 2-3 s.
         NOTE: This step may be extended appropriately depending on the intensity of safranin staining.
   6. Dehydration and clearing
      1. Dehydrate sections in 100% ethanol for 1 min.
      2. Clear sections in xylene for complete dehydration, 2 min, repeat 3 times.
         NOTE: The xylene used in this step must not be mixed with that used in Section 4.5.1; cross-contamination may compromise section quality.
   7. Mounting with Neutral Balsam
      1. Place sections in a fume hood to allow excess xylene to evaporate.
      2. Remove sections, apply an appropriate amount of neutral balsam to the specimen, and carefully cover with a coverslip.
      3. Examine sections under a light microscope for air bubbles and carefully eliminate any present.
      4. Store sections in a slide box and place in a 60 °C oven for 24 h to ensure complete curing of the neutral balsam.

Results

Longitudinal scans of the knee joint were performed using a micro-ultrasound⁸ system with a scan step of 4 mm and an axial resolution of 0.04 mm. On postoperative day 7, 3D reconstruction revealed a marked increase in joint-cavity volume in the model group compared with the sham group (Figure 2A,B). Rotarod fatigue testing^10,11 corroborated this finding, demonstrating a significant reduction i...

Discussion

Comparison with other surgery modelling methods, like the destabilization of the medial meniscus (DMM) and anterior cruciate ligament transection (ACLT) models, the modified Hulth model induces osteoarthritis through combined ligamentous and meniscal injury. The specific structural disruption determines alterations in joint biomechanics across different planes. ACLT⁴ compromises rotational stability (internal and external rotation) and reduces anteroposterior stability, resulting in positive drawe...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL injection syringe	/	/	Specifications: 30 G × 0.13 mm
3D Ultrasound Vevo 3100 Imaging System	Optic	Vevo 2100	A high-frequency ultrasound imaging system integrated with Vevo LAB software for data analysis.
4-0 nylon suture	/	/
75% ethanol	Shanghai Titan Scientific Co., Ltd.	G73537O
C57BL/6J male mice (6-8 weeks old)	Shanghai Jiesijie Laboratory Animal Co., Ltd.	/
Depilatory cream	Veet	Lot: C021123001
Iodine tincture	/	/
Isoflurane	RWD	R650-IE
Micro forceps	RWD	F12013-10
Micro scissors	RWD	S11035-08
Modified Safranin O and Fast Green Stain Kit	Solarbio	G1371
Needle holder	RWD	F21001-12
Surgical blades (10#)	RWD	S31010-01
Surgical microscope	Olympus	SZ61