A total of 24 rabbits were obtained from the Multidisciplinary Laboratory for Experimental Animals, University Faculty of Medicine. The experimental animals were male, white New Zealand rabbits weighing between 3100 and 4300 g, skeletally mature (56 weeks). Before inclusion in the study, the animals were subjected to a 1-week quarantine period. During this period, they were housed under standard laboratory conditions (50%-60% humidity, ambient temperature of 20-22°C, with a 12-hour light/dark cycle). The 24 rabbits were randomly assigned into 4 groups, with interventions applied to their left hips. To establish a healthy control group, the right femoral heads of 6 randomly selected rabbits killed humanely at the time of assessment were excised and evaluated, without any surgical intervention ( Table 1 ).

Before surgery, all experimental animals were administered general anesthesia via intramuscular injection of 5 mg/kg xylazine (Alfazyne, Ege Vet Hayvancılık San. ve Tic) and 35 mg/kg ketamine (Ketalar, Eczacıbaşı İlaç San. ve Tic). An anterolateral approach to the left hip was used, with a vertical skin incision approximately 6 to 8 cm in length. After dissection of the subcutaneous soft tissue, the joint capsule was visualized. A 2-cm transverse incision was made in the joint capsule over the femoral head. The left thigh of the subjects was externally rotated and abducted to bring the femoral head into a subluxated position ( Fig 1 ). A 3-mm-deep osteochondral defect was created on the femoral head, corresponding approximately to zone 6 according to the geographic zone classification by Ilizaliturri et al., using a 5-mm diameter flattened drill bit and a rotary tool. The defect was considered contained, as it was surrounded by intact cartilage borders. A depth gauge marked at 3 mm was used on the drill bit to ensure consistency in the depth of the defect ( Fig 2 ). The microfracture procedure was performed using a 0.9-mm diameter and 3-mm deep awl prepared in advance. In the randomized groups, to reduce the joint’s “suction seal (SS)” effect, both a 1-cm labral excision (between the 1-o’clock and 3-o’clock positions on the anterior quadrant of the left acetabulum) and a 2 × 6-mm capsular excision were performed in 12 animals from group 2 (microfracture [MF]) and Group 4 (Control), with the capsule left open and unrepaired. In contrast, in groups 1 (SS + MF) and 3 (SS), the labrum remained intact, and capsular repair was carried out to preserve the joint’s negative pressure effect. Capsular closure, including the joint capsule and aponeurosis, was performed using absorbable polyglycolic acid sutures (VICRYL 2-0; Ethicon). After thorough sterile irrigation and hemostasis, the tissue layers were closed appropriately. The animals were monitored during the postoperative period. The rabbits were fed ad libitum with standard feed and water. Daily wound care and clinical observations were performed regularly. All animals were allowed unrestricted movement within their cages. No antibiotic or analgesic treatment was administered postoperatively.

Surgical procedure steps. Under sterile conditions, a longitudinal anterolateral skin and subcutaneous incision of the left hip was performed to expose the joint capsule. A 2-cm transverse incision was then made on the capsule, partially exposing the femoral head (from left to right).

The 5-mm diameter, flat drill bit used to create the osteochondral defect and the 0.9-mm diameter instrument (awl) used for the microfracture procedure (left). The defect diameter is measured using a gauge to verify its size (right).

The left femoral heads of the subjects were macroscopically evaluated. The International Cartilage Regeneration & Joint Preservation Society (ICRS) scoring system was used for the grading. The materials were examined and assessed by 2 orthopaedic surgeons and 2 histologists. The average of the evaluation scores from all 4 individuals was calculated. In addition, the reliability of the scores given by both observers was measured using the intraclass correlation coefficient (ICC). Rulers were placed alongside the femoral head specimens, and photographs were taken using a camera. The extent of the repair tissue covering the defect area was quantitatively evaluated in a digital environment using software (Image J version 1.54g) on the basis of the photographs taken ( Fig 3 ).

Quantitative calculation of the extent of the defect area in the rabbit femoral heads filled with repair tissue using digital software (Image J version 1.46; National Institutes of Health).

All tissue samples were preserved in formaldehyde. Bone tissues were decalcified using an ethylenediaminetetraacetic acid solution and subsequently embedded in paraffin. The paraffin blocks were sectioned into 5-μm slices using a rotary microtome (RM 2255; Leica). The sections were then stained with hematoxylin-eosin, Masson trichrome, and Safranin O for histomorphologic analysis under a light microscope. The images were examined using a fluorescence microscope (BX-51; Olympus).

To assess the similarity between the newly formed tissue and hyaline cartilage, as well as the structural composition of matrix proteoglycans, histologic analysis was performed using immunohistochemical staining. Collagen deposition was evaluated through immunohistochemical labeling with anti-type I and anti-type II collagen, whereas proteoglycan deposition in cartilage tissue was identified using anti-aggrecan.

The modified O’Driscoll scoring system was used to evaluate cartilage tissue healing, the amount of repair tissue, and cell quality. Each preparation was scored between 0 and 27 points on the basis of criteria such as surface regularity, cartilage morphology, integration of the regenerative structure, its relationship with surrounding tissue, and its distance from healthy cartilage. The connection of the newly formed cartilage with neighboring tissues was separately scored from 0 to 2 as a subparameter of the scoring system. The healing tissue in sections stained with anti-type I collagen, anti-type II collagen, and antiaggrecan antibodies was examined using an Olympus BX51 fluorescence microscope. Collagen I, II, and aggrecan immunoreactivities in the regenerative area were scored on a scale from 0 (none) to 3 (strong).

Data were analyzed using IBM SPSS Statistics V25 (IBM Corp.). Descriptive statistics were presented as number of units (N), percentage (%), mean, standard deviation, and standard error. The normality of numerical data was assessed using the Shapiro-Wilk test and Q-Q plots. For variables with normal distribution, between-group comparisons were performed using one-way analysis of variance; when a significant difference was found, multiple comparisons were performed using the Tukey HSD test. The ICC was calculated to evaluate interobserver agreement. P <.05 was considered statistically significant. The data underlying this article will be shared on reasonable request to the corresponding author.