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 abil....

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.....

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