All animals survived the duration of the study. In this animal study, 15 mixed breed (with varying genetic lines from Malin, Dorset, Siamese Long Tail, and Barbados Blackbelly) male sheep were used. All the sheep were adults aged between 12 and 36 months of age (average age was 21.7 months) and weighed between 36 and 52 kg (average weight was 44.0 kg) before surgery. Each animal was housed individually in a barn to prevent fighting and reduce the risk of injury. They were fed twice daily with freshly cut grass and commercial pellets.

Ethics approval was obtained from the Institutional Animal Care and Use Committee, Universiti Putra Malaysia (reference number: AUP-R014/2022). All procedures were conducted in accordance with the Animal Welfare Act 2015, Institutional Animal Care and Use Committee Policy and Code of Practice for the Care and Use of Animals for Scientific Purposes.

Fifteen sheep were divided into 3 groups comprising 5 sheep in each group, namely, group A (control), group B (HA), and group C (PBSC + HA). All the animals underwent surgery. As control, no injection was administered to group A. PBSC harvesting was only performed in group C. Surgical, harvesting, and injection procedures are described in the following sections. Six months after surgery, all sheep were killed humanely. Left stifle joints were collected and examined macroscopically and histologically.

All animals were clinically healthy at baseline. Sheep were included on the basis of availability, with only minimum weight and age requirements applied. Because of sourcing constraints, no preselection was done. Randomization was not applied in this study. The initial batch of sheep acquired were assigned to group C for apheresis in order to allow adequate recovery time before surgery. This was necessary due to the additional procedure required for stem cell mobilization and collection, which needed to be completed before the surgical intervention. All animals were healthy and maintained under similar housing and handling conditions throughout the study period, and the allocation method was not expected to introduce systematic bias between the groups.

In group C sheep, the collection of PBSC was conducted 1 month before surgery. An injection dose (5 mg/kg) of G-CSF (Neupogen; Amgen, Thousand Oaks, CA) was given to each animal subcutaneously once a day for three consecutive days prior to apheresis. On day 4, autologous PBSC were collected by an automated cell separator (apheresis) via central venous access. A catheter with 11-F dual lumen was flushed with heparinized saline before insertion into the jugular vein using sterile technique. Apheresis was performed using Spectra Optia Apheresis Machine (Caridian BCT, Denver, CO). All collected PBSC were cryopreserved in 10% dimethyl sulfoxide and divided into 2-mL cryovials for storage in liquid nitrogen at–196°C. Hemocytometer was used to obtain viable cell counts and viability, whereby CD34 ⁺ CD45 ⁺ cell population was analyzed from flow cytometric analysis. Further details with results and analysis of the cells are available from our recent publication. The mean viable cell counts, viability and CD34 ⁺ CD45 ⁺ cell population were found to be 51.69 ± 29.21 × 10 ⁶ cells per mL, 99.11% ± 0.42 and 5.77% ± 2.72, respectively.

All surgeries were performed under general anesthesia at Universiti Putra Malaysia Veterinary Hospital. Sedation was induced in the sheep using ketamine-diazepam, who were intubated and maintained on isoflurane in oxygen. The surgeries were carried out by 2 surgeons (K-Y.S. and N.H.K.) with extensive clinical backgrounds. One is a veterinary specialist with a doctoral degree in equine surgery and fellowship training in orthopaedics. The other is a consultant orthopaedic surgeon with multiple international fellowships in joint and trauma surgery and experience in regenerative therapy with stem cells.

The left stifle joint was used for the study. Surgery involved a lateral parapatellar approach to dislocate the patella medially to expose the entire trochlear of the stifle joint. An 8-mm diameter full-thickness chondral defect was created in the central trochlear by using a punch and curettage to produce a standard articular defect ( Fig 1 ). Nine drill holes of 6 mm depth were performed for each defect using a specially made 2-mm drill (patent number: US 10,702,289 B2). The wounds were closed in layers and the sheep were awoken from general anesthesia and allowed to mobilize immediately.

(A) View after creating an 8-mm diameter full-thickness articular cartilage defect in the central trochlear to the left stifle joint of the sheep, equivalent to an area size of 50.2 mm ². (B) A specially made drill for subchondral drilling with a diameter of 2 mm, drilling to a depth of 6 mm. (C) Each articular cartilage defect accommodated 9 drill holes.

The choice of the central trochlear region for defect creation was based on anatomical and functional similarities between the ovine and human knee. Although the stifle joint in sheep bears a greater proportion of weight due to the animal’s flexed posture, its accessibility and surface area make it a suitable site for evaluating chondral repair. The central trochlear surface, as used in this study, also mirrors a common site of injury in human knees.

In group A (control), no injections were given. For the sheep in group B (HA), intra-articular injections of HA (Ostenil; TRB Chemedica AG, Germany) were performed at day of surgery, then 7 and 14 days postoperatively. Two milliliters of HA was injected at each interval. For those in group C (PBSC+HA), intra-articular injections of 2 mL PBSC mixed with 2 mL of HA were given with the same schedule as group B: at day of surgery, then 7 and 14 days postoperatively. In total, 3 intra-articular injections were administered to the sheep in groups B and C. For the 2nd and 3rd postoperative injections, the sheep were intravenously sedated with ketamine-diazepam (at 2 and 0.2 mg/kg respectively) while they remained in their animal cages.

All animals were killed humanely 6 months after the surgery. The left stifle joints were harvested. The harvested stifle joint was exposed, evaluated macroscopically, and photographed. A block was then cut out from the previously drilled area. For the histologic evaluation, 5 samples of normal cartilage were harvested from joint of the sheep in group A (Control) to serve as a normal point of reference for comparison with the treatment groups. The specimens were preserved in 10% formalin, decalcified, and embedded in paraffin for tissue blocking. Sectioning of tissues at 4 μm was done. Samples were taken from the middle section of the chondral defect and these were stained with hematoxylin and eosin and 0.5% safranin-O. Positive safranin-O staining was detected as a reddish color reaction.

Immunochemistry was performed to assess the presence of type I collagen (rabbit polyclonal antibody; Gene Tex, San Antonio, TX) and type II collagen (mouse monoclonal antibody; Thermo Fisher Scientific, Fremont, CA). Antigen retrievals were done by overnight heating in the oven at 80°C of the tissue slides in target retrieval solution at pH9. Three percent hydrogen peroxide was used for blocking endogenous peroxidase for 15 minutes. Then, incubation of each slide with primary antibody type I collagen was done in 30 minutes. This was followed by incubation with a secondary antibody (Anti Rabbit, HRP conjugate [Abcam]) for 30 minutes. For type II collagen stain, the aforementioned procedures were repeated with replacement of the antibody of type I collagen to type II collagen. Application of 3,3′-diaminobenzidine gave the brownish color reaction for both type I and type II antibodies.

The tissue samples were then evaluated by 2 independent-blinded histopathologists. The International Cartilage Regeneration & Joint Preservation Society (ICRS) II grading systems was used in this study. The total score for each sample was calculated by adding up the individual scores of these 14 components. The maximum possible total score is 1,400, and this is only achievable if each of the 14 components has a perfect score of 100.

Histologic scoring was carried out by 2 independent histopathologists who were blinded to the treatment groups. Blinding was not applied during the surgical procedures or treatment administration because of the practical constraints of performing stem cell mobilization and apheresis.

Sample size was calculated using G∗Power 3.1.9.4 for 1-way analysis of variance, based on the method and data from published animal studies, in which the total ICRS II score difference was calculated to be at least 400 with average standard deviation of 124.6. The analysis showed that for a 5% type-I error rate (α level 0.05) and 20% type II error rate (80% power) with an ability to detect a difference of 400 scores among the 3 groups (Cohen f = 1.513; standard deviation = 124.6, groups of equal size), a minimum of 3 sheep per group were needed in order to have a study of adequate statistical power, but 5 were included per group instead to accommodate potential withdrawal due to illness or mortality.

Statistical analysis of ICRS II scores was performed using SPSS, version 26 (IBM, Armonk, NY). Outliers were identified using box plots. Normality of data was evaluated with the Shapiro-Wilk test, and the equality of variances was tested using the Levene test. As the result of unequal variances, the nonparametric Kruskal-Wallis test was used to compare the ICRS II scores among the 3 treatment groups and the normal cartilage samples included for reference. Subsequent post-hoc comparisons were analyzed using the Mann-Whitney U test. Comparative analysis was 2-tailed, and the level of statistical significance was P <.05. Inter-rater reliability between the 2 histopathologists was assessed using the intraclass correlation coefficient (ICC), applying a 2-way mixed-effects model for absolute agreement, denoted as ICC(3,2).

Two outliers (A2 & NC3) were identified via box plot analysis and excluded from further analysis. Inter-rater reliability yielded an ICC of 0.97 (95% confidence interval 0.92-0.99, P <.001), indicating excellent agreement between the 2 histopathologists in their ICRS II scoring. A detailed breakdown of scores is presented in Table 1 . The Kruskal-Wallis test on the ICRS II scores showed a significant difference among the 3 treatment groups and the normal cartilage reference samples, with a test statistic of H = 13.982 and 3 degrees of freedom ( P =.003), indicating that at least one group differed significantly. Post-hoc comparisons using the Mann-Whitney U test showed that group C (PBSC+HA) had significantly greater ICRS II scores (902 ± 229; mean ± standard deviation) compared with group A (536 ± 81, P =.014) and group B (563 ± 83, P =.016). However, when compared with normal cartilage (1266 ± 35), group C ICRS II scores were significantly lower ( P =.014), which is expected, given that native cartilage represents the ideal benchmark for histologic evaluation of cartilage repair. Achieving full restoration comparable with healthy cartilage remains a challenge in this sheep model. Nevertheless, group C ICRS II scores were notably greater than those of groups A and B. This suggests that group C produced repair tissue that more closely resembles hyaline cartilage compared to groups A and B.

Table 1

Breakdown of ICRS II Scores for All Animals

Category	A1	A2	A3	A4	A5	B1	B2	B3	B4	B5	C1	C2	C3	C4	C5	NC1	NC2	NC3	NC4	NC5
Tissue morphology	40	80	60	50	50	15	30	30	65	40	50	80	40	20	65	95	90	70	90	100
Matrix staining	0	45	0	0	20	0	0	0	0	0	5	93	0	20	0	55	60	60	60	60
Cell morphology	10	85	0	60	10	0	30	10	30	20	25	75	50	65	18	100	100	80	90	100
Chondrocyte clustering	55	85	90	80	70	45	65	65	65	50	100	90	50	10	53	5	80	70	70	80
Surface architecture	60	40	40	40	60	25	75	60	65	60	70	100	60	95	70	100	100	90	100	100
Basal integration	90	30	60	40	50	40	60	50	70	70	90	95	75	100	80	100	90	80	90	90
Tidemark formation	0	10	0	0	0	5	20	0	10	10	10	68	15	80	33	85	90	80	80	90
Subchondral bone abnormalities/ marrow fibrosis	85	10	10	35	45	80	30	50	60	90	0	100	75	100	95	95	100	90	100	100
Inflammation	100	95	93	100	100	95	100	100	100	90	100	100	100	100	95	100	100	90	100	100
Abnormal calcification/ ossification	50	40	40	50	50	95	50	50	50	85	70	100	40	100	75	100	90	90	90	90
Vascularization (within repaired tissue)	45	90	10	45	20	55	30	20	45	10	45	75	65	95	50	100	100	80	100	100
Surface assessment	50	70	20	50	30	10	30	20	60	25	50	95	60	90	90	100	90	80	90	100
Mid-/deep zone assessment	0	40	0	10	0	15	10	10	20	45	30	80	55	100	75	100	100	90	90	100
Overall assessment	20	50	8	35	10	20	10	10	25	40	15	55	50	93	45	100	90	80	90	100
Total	605	770	430	595	515	500	540	475	665	635	660	1,205	735	1,068	843	1,235	1,280	1,130	1,240	1,310
Mean per group	536 ± 81 (A2 excluded)					563 ± 83					902 ± 229					1266 ± 35 (NC3 excluded)

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