Establishment and Evaluation of a Sheep Model of Full-thickness Osteochondral Defect

Summary

Here, we present an integrated protocol for generating controlled osteochondral lesions in sheep knees to establish an experimental model suitable for bone and cartilage tissue regeneration studies. The protocol outlines a method for inducing a full-thickness osteochondral defect in the femoral condyle, utilizing techniques that minimize animal welfare impacts and variability in results.

Abstract

Aging, trauma, genetic predisposition, and lifestyle impact the human body´s cartilage degradation. Once injured, continuous insults resulting from daily activities can trigger pathological conditions such as osteoarthritis, the primary cause of disability and socioeconomic loss worldwide. The main goal of all orthopedic surgeons treating joint cartilage injuries has been to reduce the extent of the lesion anatomically, repair the cartilage surface, and reestablish joint stability. Animal models are essential for the development of therapeutic drugs, but current models for cartilage defects are unsatisfactory. Osteochondral lesions in sheep are a valuable model for testing new therapies and biomaterials that can aid in the recovery of cartilage and bone in human joint environments. This study established an efficient protocol for inducing acute osteochondral defects in large animals. A standardized lesion was created in both medial femoral condyles. One knee was randomly assigned to receive gelatin-methacryloyl treatment, while the contralateral knee served as a control. Six months after the surgical procedure, the femoral condyle area was removed, the dissected knee joints were decalcified, embedded in paraffin, and cut into sections, which were stained with hematoxylin and eosin. Scores were used to evaluate the lesion. This methodology allows immediate macroscopic observation after injury induction. Additionally, this model effectively replicates clinical cartilage defects, providing a valuable model for studying their pathology and developing innovative therapeutic approaches.

Introduction

Cartilage is essential for preserving the mechanical competence of the skeletal system by providing a frictionless surface between bones in diarthrodial joints, thereby preventing articular surface erosion¹. The ability of cartilage to perform these demanding mechanical functions relies heavily on the unique composition and organization of its extracellular matrix-particularly its exceptionally high-water content. Water plays a crucial role in the structure and function of articular cartilage, representing approximately 70-80% of its composition. Water is the primary element responsible for load bearing during compression. The tissue's ability to resist compressive forces depends on how water is structured and restricted within the matrix. This organization is possible through the interaction of water with the main macromolecules of cartilage, such as collagen and proteoglycans, especially hyaluronic acid-bound aggregates¹. These act as containment structures that order and limit water movement. This interaction provides the tissue not only with stiffness but also with resilience and the ability to recover after deformation. The interaction between free water, bound water, and the macromolecular matrix constitutes the core of cartilage biomechanical behavior².

Despite its remarkable ability to support and distribute physical and mechanical loads that reach the joints, once injured, articular cartilage (AC) has limited potential for spontaneous repair due to low chondrocyte mitotic activity and poor tissue vascularization². Joint damage is usually associated with the progressive loss of extracellular matrix (ECM) molecules and proteins, which constitute 95% of the tissue, and confer biomechanical features to AC³. An imbalance between synthesis and degradation of ECM components, shifting toward catabolic processes, can result in osteoarthritis (OA), the most prevalent degenerative joint disease worldwide. Osteoarthritis affects approximately 10-12% of the adult population, causing pain, limiting mobility, and markedly reducing patients' autonomy⁴. Osteoarthritis is more prevalent in aging populations due to cumulative insults on the joint over a lifespan; however, assorted risk factors have been associated with the clinical condition onset, such as gender, genetic predisposition, obesity, and daily habits⁴. Until now, therapeutic approaches to treat OA have consisted of clinical symptom management and total joint replacement in the most severe cases⁵. Without a robust, reproducible, and reliable therapeutic strategy to effectively treat OA, innovative approaches to understanding and addressing the problems associated with articular cartilage injury are being studied.

To repair, regenerate, and enhance joint movement in arthritic joints, progress in regenerative medicine has emerged as a new hope for restoring damaged AC^6,7. The use of autologous chondrocytes combined with ECM implantation and intra-articular injection of stem cells represents the current state-of-the-art in this area^8,9,10. The main challenge with these approaches is ensuring appropriate differentiation and functional ECM synthesis by implanted cells. Autologous chondrocytes tend to form fibrocartilage or progress to a hypertrophic state, creating new dysfunctional tissue that does not recapitulate the physiological features of AC, leading to incomplete recovery and often late deterioration¹¹. Biomaterials can be used to guide cell behavior during tissue regeneration, providing a temporary scaffold for cells to adhere and synthesize new, natural, and functional extracellular matrix^12,13,14. The ideal biomaterial for cartilage engineering must ensure strong integration with surrounding tissues, especially the subchondral bone, as failure at this interface compromises long-term repair. However, weak bonding between the cartilage and bone layers of osteochondral scaffolds and poor integration with host tissue remain major challenges¹⁵. Although scaffolds attempt to mimic the native osteochondral architecture, reproducing the complex interface microstructure is still difficult. This reinforces the importance of research and development of scaffolds with biological properties that can simultaneously support cartilage and bone regeneration while maintaining interface stability¹⁶. Gelatin, derived from collagen-a key component of cartilage tissue-possesses properties such as cell-binding motifs and biodegradability, suggesting its potential as a supportive biomaterial when combined with regenerative factors for the treatment of osteochondral lesions. When chemically modified with methacrylic anhydride to form gelatin methacryloyl (GelMA), it becomes photo-crosslinkable, enabling injection and in situ photopolymerization, which enhances mechanical stability for applications in regenerative medicine¹⁷.

Osteochondral lesions have limited regenerative capacity, which represents a clinical challenge. Preclinical investigations that employ large-animal models of knee joint injury are essential for translational research because they allow systematic evaluation of variables affecting disease mechanisms and therapeutic outcomes. Commonly used species include pigs, goats, dogs, horses, and sheep, each presenting specific advantages and limitations that must be considered in experimental design. Moreover, all these species exhibit limited endogenous repair of chondral and osteochondral defects, as observed in humans¹⁸. To ensure that these models are predictive of human outcomes, they must reproduce relevant parameters such as anatomy, physiology, and biomechanical properties¹⁸. For comparison, the thickness of human articular cartilage in the femoral condyles ranges from 1.8 to 2 mm¹⁹, and the diameter of cartilage lesions typically requiring treatment is 10 mm or greater²⁰.

The porcine model offers advantages for cartilage and osteochondral regeneration studies, as joint size, cartilage thickness, and weight-bearing requirements closely resemble those observed in humans¹⁸. Some miniature pig breeds, such as the Göttingen minipig, are easy to handle and reach skeletal maturity between 18 and 22 months of age²¹. Their cartilage thickness ranges from 1 to 2 mm, and defects of 6 to 8 mm can be created¹⁸. Bone characteristics, including trabecular thickness, bone apposition rate, and collagen fiber organization, are comparable to those of humans²². However, the porcine model also has limitations: even in miniature breeds, the knee (stifle) joint is smaller than in humans, limiting the creation of large experimental defects. Furthermore, most studies use skeletally immature animals due to the associated maintenance costs, which may overestimate the repair potential²³.

Goats exhibit joint anatomy, biomechanics, and cartilage thickness like those of humans²⁴. They do not require specialized facilities, are easy to handle, and are relatively inexpensive²⁵. Goat cartilage thickness ranges from 0.8 to 2.0 mm, and the most reported critical defect size is 6 mm²⁶. However, there is significant variation in cartilage thickness across breeds, sizes, and sexes, which can lead to inconsistent results²⁷. The cartilage-to-subchondral bone ratio and trabecular bone structure are also comparable to those of humans²⁸. Skeletal maturity occurs relatively late, between 24 and 36 months of age²⁹. From a biomechanical standpoint, the goat knee experiences joint pressures and cyclic loads like those of humans²⁵, although knee flexion during gait is greater (50°-70° in goats vs. <30° in humans), resulting in distinct contact areas³⁰. This difference, combined with higher peak pressures, may contribute to less efficient cartilage repair³⁰.

Among large animal models, dogs stand out for their ability to tolerate rehabilitation regimens, joint immobilization, and training for treadmill walking, swimming, and controlled load-bearing exercises¹⁸. Articular cartilage thickness ranges from 0.9 to 1.3 mm, and the critical defect size is approximately 4 mm, limiting direct comparison to the human model²⁵. Skeletal maturity occurs between 8.5 and 13.7 months of age³¹. Anatomically, the canine knee differs from the human knee, and variations in biomechanics, loading patterns, and maturity across breeds complicate the extrapolation of results³². Despite these anatomical differences and the small defect size, the canine model remains valuable for studies including rehabilitation protocols or spontaneous cartilage pathologies similar to those in humans. However, its experimental use is restricted by ethical considerations²⁹.

The equine model is the largest available animal model for cartilage repair research and naturally develops age- or trauma-related chondral lesions and osteoarthritis similar to those in humans³³. Horses exhibit anatomical and biomechanical features comparable to humans, such as an upright knee joint, large joint size, cartilage thickness between 2 and 3 mm-closer to human cartilage-full knee extension during gait, and comparable bone mineral density³⁴. Skeletal maturity occurs between 24 and 48 months, and defect sizes commonly studied range from 6 to 20 mm^32,33. Limitations include the high vertical loading on the equine stifle joint during locomotion, which exposes implants to greater forces than in humans and hinders long-term healing³⁵. Moreover, acquisition and maintenance costs are high, and specialized facilities are required³⁶.

Sheep are widely used as a model in cartilage repair studies due to their availability, docile temperament, good tolerance to joint surgery, and ease of handling³⁷. Skeletal maturity in sheep is typically reached at approximately 2-3 years of age²⁵. In addition, the ovine knee demonstrates anatomical and biomechanical similarities to the human knee joint³⁸. Cartilage thickness in the medial femoral condyle ranges from 0.7 to 1.7 mm, and bone mineral density and contact pressures are also comparable, although bone volume fraction is higher in sheep³⁹. Due to reduced cartilage thickness, induced defects often extend into the subchondral bone, making this model more suitable for osteochondral defect studies³². Lesions of 6 to 8 mm in diameter are typically created in locations such as the femoral condyles and trochlea³². Furthermore, the ovine model offers advantages for bilateral studies, allowing comparison of treated and control sites within the same animal, thereby reducing host-related variability, improving consistency and reproducibility, and minimizing the number of animals required⁴⁰. Each model presents different degrees of similarity to human physiology and biomechanics. Therefore, model selection should balance biological relevance, practical feasibility, and ethical considerations. In the present study, nulliparous female sheep aged 12 months, evaluated during the seasonal anestrus period, were included.

This study sought to design an effective protocol for generating acute osteochondral defects in large animals. The protocol involves creating a full-thickness osteochondral lesion to establish a model suitable for regenerative medicine studies. Additionally, the application of GelMA hydrogel is explored as a guide for tissue healing and as a prospective method for delivering regenerative molecules and cells. The protocol outlines the surgical procedure for creating osteochondral lesions in the sheep's knee articular cartilage. It is adaptable to various tissue-engineering applications, including the induction of similar injuries in other large-animal models for regenerative medicine.

Protocol

The study received approval from the Ethics Committee in Research and was certified with a Certificate of Ethical Presentation numbered 6092250823/2023. In the present study, nine White Suffolk female sheep aged 12 months were selected. The defect was created in both medial femoral condyles of each animal. Empty defects were used as controls. Each animal had one knee randomized to treatment with GelMA and the other as a control. This strategy allowed us to assess the natural regenerative capacity and reduce the impact of individual variations, such as genetic factors and skeletal maturity. The animals were followed for 6 months to evaluate hyaline cartilage formation.

1. Anesthetic procedure

1. Deprive the animals of food and water for approximately 24 h before the surgical procedure.
2. Premedicate the animals with intramuscular meperidine 4 mg/kg combined with midazolam 0.3 mg/kg.
3. Fill the anesthetic vaporizer one day before use, with the room empty, and wear protective gloves to minimize the risk of occupational exposure.
4. Check the anesthetic circuit for leaks before each procedure, and perform all manipulations in a well-ventilated area, following institutional biosafety guidelines.
5. After 15 min, cannulate the animals with an 18 G catheter and administer Lactated Ringer's solution at a rate of 10 mL∙kg^-1∙h^-1.
6. Administer intravenous anesthesia using 1% propofol at a dose of 4 mg/kg.
7. Intubate the animals with a 7.5-8.0 mm cuffed endotracheal tube and maintain them under spontaneous ventilation using a semi-closed circular breathing system that delivers oxygen at an FiO₂ of 1.0 and a flow rate of 50 mL/kg.
8. Use the partial intravenous anesthesia (PIVA) technique, maintain propofol as a continuous infusion at 0.1 mg∙kg^-1 min^-1, and only adjust the isoflurane concentration to achieve an adequate surgical plane of anesthesia. Monitor anesthetic depth throughout the procedure.
9. Perform perineural blocks of the femoral and sciatic nerves, guided by a neuro localizer. Administer 0.2 mL/kg of 2% lidocaine for each blockade.
   NOTE: The surgery should only begin once the animal is confirmed to be under surgical anesthesia (no response to a firm hoof pinch and no jaw tone).

2. Surgical procedure in vivo

1. Disinfect the shaved knee using topical antiseptic chlorhexidine gluconate and place the animal in the dorsal decubitus position in the surgery room.
2. Locate the articular cavity by touching the animal's knee to determine the precise site of incision. Make a double-curved skin incision (starting distal to the lateral femoral epicondyle and ending medially at the level of the tibial tuberosity).
3. Identify the patellar tendon, patella, patellar ligament, and the medial portion of the joint capsule. Incise the capsule to expose the medial femoral condyle. Hyperflex the knee to visualize the most distal region of the condyle's articular cartilage.
4. Make a surgical incision of approximately 3-5 cm in the region of the animal´s condyle. Remove the tissue layers underlying the skin to access the condyle and keep the incision area clean to prevent contamination.
5. Collect synovial fluid by using a syringe and needle. Transfer part of the sample to an EDTA tube for analysis and use the remaining material to prepare smear slides.
6. Position the retractors to expose the condylar surface fully. The more exposed the condyle is, the easier it will be to induce injury in the next step.
   NOTE: At this time, be aware of the animal's paw. Flexion or extension of the knee can help expose the condyle.
7. Drill an 8 mm diameter and 5 mm deep defect in the center of the medial femoral condyle using an orthopedic drill fitted with an 8 mm trephine bit, ensuring proper alignment to avoid unintended dimensions or shapes, and maintain a 0° angle relative to the most distal region of the articular cartilage.
   NOTE: Perform the trepanation at the lowest possible speed, gently operating the drill control and using only enough rotation to cut the cartilage and subchondral bone, also performing local cooling with 0.9% NaCl at room temperature, even at low speed.
8. Perform the surgical extraction of the osteochondral plug using the orthopedic hammer and osteotome.
9. Clean the wound extension with sterile gauze, ensuring the inside of the lesion is free of blood and tissue debris.
10. Apply 200 µL of the liquid 20% (w/v) GelMA formulation, containing 0.25% (w/v) lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as the photoinitiator, to the inside of the injury until it reaches the surface.
11. Photocrosslink the GelMA hydrogel under a 395-405 nm LED (9 W total power) positioned approximately 5 cm from the sample for 180 s. Under these conditions, the estimated irradiance at the surface is ~10 mW/cm², resulting in a total exposure energy of approximately 1.8 J/cm².
    NOTE: Use protective eyewear when using ultraviolet light.
12. Remove the retractors carefully.
13. Close the lesion with independent sutures in the different tissue layers to prevent seroma formation. The number of sutures required will depend on the extent of the surgical incision.
14. Apply a sterile bandage to the sutures to prevent infections.

3. Postoperative care

1. Monitor the animals during anesthetic recovery, including vital signs such as heart rate (70-90 beats/min), respiratory rate (12-20 breaths/min), body temperature (39 °C to 40.5 °C), and normal mucous membranes with a capillary refill time of 1-2 s. Palpate the facial and femoral arteries regularly to check if the pulse is easily perceptible.
   NOTE: Anesthetic recovery should occur in a quiet, clean, warm, padded environment, with minimal lighting and low noise levels.
2. Perform surgical site dressings using topical penicillin (benzathine penicillin G: 1,250,000 IU; procaine penicillin G: 1,250,000 IU; dihydrostreptomycin sulfate: 1.25 g; urea: 2.50 g), once a day for 10 days.
3. Ensure that the animals remain in a quadrupedal position immediately after waking from anesthesia, put weight on the operated limb, and show no signs of discomfort.
4. Postoperative analgesia consisted of meloxicam (0.4 mg/kg every 24 h) and tramadol (2 mg/kg every 12 h) for 5 days. An additional rescue dose of tramadol was administered when pain-related facial expressions were identified using the Sheep Grimace Scale⁴¹, a validated tool based on specific facial action units (including orbital tightening, flehmen response, ear position, and head position) for assessing postoperative pain and distress in sheep.

4. Histology analysis

1. Six months after the surgical procedure, perform euthanasia. To achieve deep sedation before administering the euthanasia agent, premedicate the animals intravenously with ketamine (10 mg/kg) and xylazine (1 mg/kg), using doses intentionally higher than those typically recommended for clinical procedures to ensure rapid onset of profound sedation, as supported by accepted euthanasia protocols. Subsequently, administer propofol to induce an anesthetic coma. Then, administer potassium chloride at 2 mEq/kg IV until respiratory and cardiovascular arrest occur and all protective reflexes are absent.
2. Section the distal femoral epiphysis to remove the area of the condyles. Fix the dissected knee joints in 10% neutral-buffered formalin and decalcify in 5% EDTA (ethylenediaminetetraacetic acid) at 34 °C, changing the EDTA solution every 3 days.
3. Embed the material in paraffin and cut into 4 µm sections. Remove paraffin from the slides by soaking them in xylene at 65 °C for 3 x 5 min. Hydrate with 10-20x immersions in ethanol (99%, 95%, 70%) followed by deionized water for 5 min⁴².
4. For hematoxylin and eosin (H&E) staining, stain the sections with Harris hematoxylin and counterstain with eosin, scan at 20× magnification using an automated slide scanning microscope, and analyze using the linked software.
5. Evaluate the lesion according to the Osteoarthritis Research Society International (OARSI) histopathology initiative⁴³: a score of 0 indicates normal cartilage; a score of 1 reflects superficial fibrillation without cartilage loss; a score of 2 corresponds to vertical clefts that reach the layer immediately beneath the superficial zone, accompanied by partial loss of the surface lamina; a score of 3 indicates vertical clefts or erosion that extend into the calcified cartilage and affect less than 25% of the articular surface; scores of 4, 5, and 6 represent vertical clefts or erosion that reach the calcified cartilage and involve 25-50%, 50-75%, and more than 75% of the articular surface, respectively.
   NOTE: For this study, lesions with vertical clefts or erosions extending into the calcified cartilage and involving more than 25% of the articular surface were assigned a score of 4.

Results

Osteochondral defect induction

This study aimed to develop an efficient protocol for inducing acute osteochondral defects in large animals. The injuries were created mechanically during a surgical procedure, resulting in a lesion measuring approximately 8 mm in diameter and 5 mm-deep (Figure 1). Considering the scores established by the International Cartilage Regeneration & Joint Preservation Society, this type of injury represents a grade 4 ...

Discussion

To overcome challenges in cartilage therapy, such as the inadequacy of chondrogenic differentiation and ECM maturation, non-clinical studies remain essential for evaluating therapeutic strategies. Methods for non-clinical models of injured cartilage tissue have been reported previously using various techniques⁴⁵. However, the literature still lacks detailed reports and standardized procedures for inducing acute osteochondral injury in the knee of large animals, resulting in considerable variation ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
25 poliglecaprone suture thread, no. 2-0	Ethicon		Suture thread for general soft tissue closure, was used for suturing the joint capsule and subcutaneous cellular tissue.
Antibody anti-β-Tubulin	Invitrogen	PA516863	Antibody against β-tubulin, two subunits (α/β) that make up the microtubules of the cytoskeleton
Anesthesia device	HB hospitalar		Inhalation anesthesia equipment
Axio Scan Z1	Zeiss, Munich, Germany		Microscope
Benzathine penicillin G	Ourofino		Antibiotics
Catheter 18 G	Solidor		Intravenous catheter
Cephalexin	Virbac		Antimicrobial therapy
Chlorhexidine Digluconate Aqueous Solution	RIOQUÍMICA		Antiseptic used for skin antisepsis
Cuffed endotracheal tube 7.5–8.0 mm	Solidor		Cuffed endotracheal tube
DAPI	Sigma	D9542	Fluorescent dye
Dulbecco's Modified Eagle Medium	Gibco	12100-046	Cell culture medium
Dihydrostreptomycin sulfate	Ourofino		Antibiotics
EDTA tube	Becton Dickinson	362788	Collection tube
Eosin	Neon	1795	A dye used to visualize cell morphology, staining cytoplasm.
Ethylenediaminetetraacetic acid	Sigma	E5134	Promotes the gradual demineralization of bone tissue
Fetal Bovine Serum	Gibco	12657-029	Supplement for cell culture with growth factors
Formalin	Sigma-Aldrich	47608	Preserve tissue morphology
GelMA Lyophilized	TissueLabs	GLMA-LYO-1	Photocrosslinkable hydrogel
Harris hematoxylin	Laborclin	620503	A dye used to visualize cell morphology, staining nuclei.
Isoflurano	Cristália		anesthetic maintenance
Ketamine 10%	VETNIL		Euthanasia procedure
Lactated Ringer's solution	Fresenius Kabi		Fluid therapy
L-Glutamine 200 mM	Gibco	25030-081	Essential amino acid for cell culture
Lidocaine hydrochloride	Cristália		Local anesthesia
LIVE/DEAD viability/citotoxity Kit	Invitrogen	L3224	Fluorescent assay kit to evaluate cell viability
Meloxicam	Ouro fino		Anti-inflammatory and analgesic for the postoperative period
Midazolam hydrochloride	Cristália		Preanesthetic medication
Mononylon suture thread, no. 3-0	Ethicon		Suture used for skin closure
NaCl 0.9%	Cristalia		Saline solution
Neuro localizer	Neurolocalizador DL 250 DeltaLife		Local anesthesia guided by a neurolocalizer and peripheral nerve electrostimulator.
Orthopedic Drill	Cannulated Mini Orthopedic Drill (Delicate) - Chromed Mandrel - Makita Brand		Used for orthopedic surgeries.
Orthopedic hammer	Edlo	Hammer Neufield (10-3155)	General characteristics: 20cm in length and 560gr in weight; material: high-quality stainless steel; use: indicated for traumatology and orthopedics procedures requiring controlled impact. Certifications: the products have quality certifications such as ISO ((International Organization for Standardization): 13485:2016, with European compliance and Good Manufacturing Practices standards.
Lambotte osteotome	Edlo	Lambotte osteotome (10-0749)	The Lambotte osteotome (6 mm x 17 cm / 6 3/4") is a high-precision surgical instrument used primarily in orthopedic surgeries and neurosurgical procedures involving bone manipulation. With a 6 mm wide tip, it is ideal for delicate cuts, reshaping, and removal of compact cartilage and subchondral bone.
Penicillin Streptomycin	Gibco	15140-122	Broad spectrum antibiotic widely used in cell culture
Pethidine hydrochloride	Cristália		Preanesthetic medication
Potassium chloride 19.1%	Samtec		Euthanasia procedure
Procaine penicillin G	Ourofino		Antibiotics
Propofol	Cristália		Anesthetic induction and maintenance
Ringer lactate	Cristália		Fluid therapy
Semi-closed circular breathing system	HB hospitalar		Respiratory circuit for anesthesia
Sterile crepe bandage	Cremer		Sterile crepe bandage, used to cover the distal ends of the limbs being operated on.
Stimuplex D, 22 Ga. x 2 in. (50 mm) Insulated Needle with Extension Set, 30° Bevel (STIMD2250/30)	BBRAUN		Perineural local anesthesia
Tramadol hydrochloride	Cristália		Postoperative analgesia
Trephine Drill	Bone Trephine Drill, mm diameter – model: contra-angle, brand: surgical WF		Bone Collecting Trephine Drill 8.0 mm; produced in surgical steel; used in bone graft surgeries; serves to remove the bone block for histological analysis or autografting.
Xylazine 10%	VETNIL		Euthanasia procedure