Regenerative Effect of Mesenchymal Stem Cell on Cartilage Damage in a Porcine Model

Osteoarthritis (OA) is a major public and animal health challenge with significant economic consequences. Cartilage degradation plays a critical role in the initiation and progression of degenerative joint diseases, such as OA. Mesenchymal stem cells (MSCs) have become increasingly popular in the field of cartilage regeneration due to their promising results. The objective of this preclinical study was to evaluate the regenerative effects of mesenchymal stem cells (MSCs) in the repair of knee cartilage defects using a porcine model. Seven healthy LYD breed white pigs, aged 9–10 weeks and weighing approximately 20 ± 3 kg, were used in the experimental protocol. Full-thickness defects measuring 8 mm in diameter and 5 mm in depth were induced in the lateral femoral condyle of the posterior limbs in both knee joints using a sterile puncture technique while the knee was maximally flexed. Following a 1-week induction phase, the pig treatment groups received a 0.3 million/kg MSC transplant into the damaged knee region, while the placebo group received a control solution as a treatment. Magnetic resonance imaging (MRI), computerized tomography (CT), visual macroscopic examination, histological analysis, and cytokine concentration analysis were used to assess cartilage regeneration. The findings revealed that human adipose-derived mesenchymal stem cells (hADSCs) were more effective in repairing cartilage than pig umbilical cord-derived mesenchymal stem cells (pUCMSCs). These results suggest that MSC-based treatments hold promise as a treatment option for cartilage repair, which aid in the treatment of OA. However, further studies with larger sample sizes and longer follow-up periods are required to fully demonstrate the safety and efficacy of these therapies in both animals and humans.


Introduction
Knee osteoarthritis (OA) is a degenerative joint disease that affects both humans and animals, resulting in significant pain and disability with economic loss [1][2][3]. The progressive degradation of articular cartilage is a significant process for the progression of OA. The loss of cartilage can result from a range of factors, including age, injury, and obesity, which can all contribute to the degenerative process. The reduction in cartilage thickness and quality leads to increased friction and pressure on the underlying bone, leading to further damage and a vicious cycle of cartilage loss and joint deterioration. Knee osteoarthritis can be categorized into primary or secondary forms based on its

Materials and Methods
The experiment titled "Regenerative effect of mesenchymal stem cell on knee cartilage damage in a porcine model" was conducted from 2022 to 2023 at the National Pingtung University of Science and Technology (NPUST), Taiwan (22 • 38 16.19 N and 120 • 35 26.39 E). A schematic illustration of the experimental design is presented (Figure 1), with the objective of making it more accessible to readers from diverse fields, thereby ensuring easier comprehension and understanding. The methodology for this study was as follows.
Biomedicines 2023, 11, x FOR PEER REVIEW 3 of 21 tables, graphs, and microscopy images, and a discussion section that explains the theoretical basis of our results.

Materials and Methods
The experiment titled "Regenerative effect of mesenchymal stem cell on knee cartilage damage in a porcine model" was conducted from 2022 to 2023 at the National Pingtung University of Science and Technology (NPUST), Taiwan (22°38′16.19″ N and 120°35′26.39″ E). A schematic illustration of the experimental design is presented ( Figure  1), with the objective of making it more accessible to readers from diverse fields, thereby ensuring easier comprehension and understanding. The methodology for this study was as follows.

Preparation of MSCs
MSCs were funded by Bionet company, (Taipei City, Taiwan). They were unable to reveal the specific culture medium chosen for the study due to its unique element, which was a valuable trade secret for the company. With minor modifications to the protocol [27], human adipose-derived mesenchymal stem cells were isolated and collected after receiving informed consent. Briefly, lipoaspirates were rinsed in phosphate-buffered saline (PBS) and digested with an enzyme, such as collagenase, to break down the tissue. The stromal vascular fraction (SVF) is subsequently separated from the other cells by centrifugation. ASCs were then grown in a suitable condition from the SVF. When there were sufficient number of ASCs, they were collected for use. The method presented here with minor modification was used to isolate pig umbilical cord mesenchymal stem cells [28]. At first, the umbilical cords were disinfected with 75% ethanol, followed by a PBS wash to remove any blood contamination. Following that, the blood vessels were excised to avoid endothelial cell contamination, and the cord tissue was divided into small 0.5-1 mm 3 fragments. These fragments were placed on 10 cm culture dishes and cultured in alpha-MEM (GIBCO, Grand Island, NY, USA) supplemented with 5% UltraGROTM (AventaCell, Atlanta, GA, USA) and antibiotics (PSA, GIBCO). The cultures were incubated at 37 degrees Celsius in a humidified atmosphere with 5% CO2. Every 3-4 days, new media was introduced. When the cultures reached confluence, they were washed with DPBS, harvested with 0.05% TrypLE (GIBCO, USA), and transferred to fresh 10 cm culture dishes at a

Preparation of MSCs
MSCs were funded by Bionet company, (Taipei City, Taiwan). They were unable to reveal the specific culture medium chosen for the study due to its unique element, which was a valuable trade secret for the company. With minor modifications to the protocol [27], human adipose-derived mesenchymal stem cells were isolated and collected after receiving informed consent. Briefly, lipoaspirates were rinsed in phosphate-buffered saline (PBS) and digested with an enzyme, such as collagenase, to break down the tissue. The stromal vascular fraction (SVF) is subsequently separated from the other cells by centrifugation. ASCs were then grown in a suitable condition from the SVF. When there were sufficient number of ASCs, they were collected for use. The method presented here with minor modification was used to isolate pig umbilical cord mesenchymal stem cells [28]. At first, the umbilical cords were disinfected with 75% ethanol, followed by a PBS wash to remove any blood contamination. Following that, the blood vessels were excised to avoid endothelial cell contamination, and the cord tissue was divided into small 0.5-1 mm 3 fragments. These fragments were placed on 10 cm culture dishes and cultured in alpha-MEM (GIBCO, Grand Island, NY, USA) supplemented with 5% UltraGROTM (AventaCell, Atlanta, GA, USA) and antibiotics (PSA, GIBCO). The cultures were incubated at 37 degrees Celsius in a humidified atmosphere with 5% CO 2 . Every 3-4 days, new media was introduced. When the cultures reached confluence, they were washed with DPBS, harvested with 0.05% TrypLE (GIBCO, USA), and transferred to fresh 10 cm culture dishes at a plating density of 3-6 × 10 3 cells per cm 2 for continued growth. Finally, the MSCs were cryopreserved in culture media containing 10% DMSO in a vapor phase liquid nitrogen tank at −190 • C using a control rate freezer (Icecube, Sylab, Purkersdorf, Austria). Both hADSC and pUCMSC cells from passage three were used for the experiment. The MSCs were cultured in the BIONET Lab (No. 28, Lane 36, Xinhuyi Rd, Neihu District, Taipei City, 114065) under Good Manufacturing Practice conditions. They reported that the expanded cells were then evaluated for number, viability, purity, and identity. Before injection, MSCs were counted using a hemocytometer and loaded into a syringe.

Experimental Animals
The research was performed in the animal research laboratory of National Pingtung University of Science and Technology (NPUST). White LYD (the crossbred Landrace, Yorkshire, and Duroc) pigs (n = 7) body weight of 20 to 25 kg with age group 9-10 weeks were used. Pigs were purchased from a commercial farm near NPUST and placed at the NPUST positive control animal facility room. When performing this study at National Pingtung University of Science and Technology, International Animal Care and Use Committee (IACUC) protocols were followed. The International Animal Care and Use Committee (IACUC) approved the animal research and issued an IACUC permit number (NPUST-110-079). Because the ethical care of animals is of the highest concern, all pre-operative, surgical, and post-operative procedures were carried out in compliance with IACUC standards. The pigs were allowed to become acclimated to their surroundings prior to the commencement of the experiment. The pigs had unrestricted access to food and water and were reared in a controlled environment with a 12-h/12-h light/dark cycle at 23 ± 2 • C with relative humidity of 65 ± 5%. All pigs were physically evaluated to ensure their safety and wellbeing, and were immediately ear tagged with a specific number for identification purposes during this study (Table 1).

Experimental Design
In this study, we analyzed the effectiveness of the healing of cartilage using straightforward experimental designs. Twelve knees of seven pigs were randomly assigned to four groups [ Table 1] (Figure 2). In the treatment group, both human adipose derived mesenchymal stem cells and pig umbilical cord derived mesenchymal stem cells were used. Each pig in the study involved a singular defect on both knees in the posterior limbs (It is important to note that no defects were created in the front limbs), which were specifically induced using a sterile puncture technique. The induced defects were carefully created in the lateral femoral condyle, with dimensions measuring 8 mm in diameter and 5 mm in depth. The knee joint was maximally flexed during the puncture procedure, as illustrated in Figure 3. For the administration of the stem cell treatment, an intra-articular injection method was employed ( Figure 4). The stem cells were delivered locally and precisely to the injured area of the knee joint using this distribution method. By employing this technique, the researchers sought to increase the therapeutic efficiency of the stem cells in healing the knee abnormalities of the experimental animals.
This study was carried out in three stages, employing a consistent methodology to effectively manage workload and ensure data validity. Moreover, to address the limitations of using MRI and radiography, and to manage a large number of animals, the investigation was divided into three stages, as illustrated in Table 1. Transportation of animals to the hospital for MRI and CT examinations added to the time required for conducting the study. This approach facilitated improved planning and resource allocation, while also ensuring the welfare of the animals. medium only. In the third and final stage of the experiment, two pigs were selected. One pig received an average of 30 × 10 4 /kg of pig umbilical mesenchymal stem cells on both knees one week after induction surgery, while the other pig received an average of 36 × 10 4 /kg of adipose-derived mesenchymal stem cells on both knees one week after induction surgery. Through this experimental design, our objective was to evaluate the impact of human and pig stem cells on knee joint healing through cartilage regeneration, while also observing the effect of the negative control group.   pig received an average of 30 × 10 4 /kg of pig umbilical mesenchymal stem cells on both knees one week after induction surgery, while the other pig received an average of 36 × 10 4 /kg of adipose-derived mesenchymal stem cells on both knees one week after induction surgery. Through this experimental design, our objective was to evaluate the impact of human and pig stem cells on knee joint healing through cartilage regeneration, while also observing the effect of the negative control group.

Surgical Procedure
Zoletil ® 100 (virbac) along with xylazine 10%(w/v) (Health-Tech Pharmaceutical Co., Ltd., Taoyun, Taiwan) was used as anesthesia for minimally invasive surgery ( Figure 2). The dosage was 5-10 mg/kg. Zoletil ® 100 was administered intravenously (IV). Isoflurane was used to maintain anesthesia in operation by the anesthesia machine. Atropine (Tai Yu Chemical& Pharmaceutical Co., Ltd., Hsinchu, Taiwan) pre anesthesia medicine was used with a dosage of 0.05 mg/kg administered subcutaneously. The antibiotic cephazolin (Sintong Taiwan Biotech Co., Taoyuan, Taiwan) was used for infection control. The dosage was 25 mg/kg twice a day, which was administered intramuscularly. Carprofen was pro- In the first stage of the experiment, two pigs were selected, with one serving as the treatment group, receiving 28 × 10 4 /kg of human adipose derived mesenchymal stem cells on both knees one week after induction surgery. The other pig acted as the placebo group and received only the medium. However, in the first round, one of the pigs received the medium solution only in the right leg due to a technical error during anesthesia [ Table 1].
In the second stage, three pigs were employed, with one serving as the treatment group, receiving an average of 26 × 10 4 /kg of pig stem cells derived from umbilical cords of pigs on both knees one week after surgery. Another pig acted as the control group and received no treatment, while the remaining pig served as the placebo group, receiving 1 mL of medium only. In the third and final stage of the experiment, two pigs were selected. One pig received an average of 30 × 10 4 /kg of pig umbilical mesenchymal stem cells on both knees one week after induction surgery, while the other pig received an average of 36 × 10 4 /kg of adipose-derived mesenchymal stem cells on both knees one week after induction surgery. Through this experimental design, our objective was to evaluate the impact of human and pig stem cells on knee joint healing through cartilage regeneration, while also observing the effect of the negative control group.

Surgical Procedure
Zoletil ® 100 (virbac) along with xylazine 10%(w/v) (Health-Tech Pharmaceutical Co., Ltd., Taoyun, Taiwan) was used as anesthesia for minimally invasive surgery. The dosage was 5-10 mg/kg. Zoletil ® 100 was administered intravenously (IV). Isoflurane was used to maintain anesthesia in operation by the anesthesia machine. Atropine (Tai Yu Chemical& Pharmaceutical Co., Ltd., Hsinchu, Taiwan) pre anesthesia medicine was used with a dosage of 0.05 mg/kg administered subcutaneously. The antibiotic cephazolin (Sintong Taiwan Biotech Co., Taoyuan, Taiwan) was used for infection control. The dosage was 25 mg/kg twice a day, which was administered intramuscularly. Carprofen was provided to pigs after surgery as a pain relief method for knee pain. The drug's recommended dose was 2.2 mg/kg, which was administered intramuscularly once or twice a day, depending on the severity of the wound and pain condition.
Before skin incision, betadine (Taipei, Taiwan) was applied near surgical parts. Following the skin incision, an articular cartilage defect was formed in the lateral femoral condyle of both knees. An 8 × 5 mm sterilized biopsy punch (Kai industries Co., Ltd., Seki, Japan) was used to create an incision. The wounds were sutured with 4-0 Vicryl and surgical staples (Weck Visistat ® 35 W) after the procedure (See Supplementary Video S1 for surgical staples) ( Figure 5).

MRI and CT Examinations
Pre-anesthesia medication was administered to the animals; 15 min before the anesthetic dosage, atropine (0.05 mg/kg) was administered intramuscularly. Animals were in-

MRI and CT Examinations
Pre-anesthesia medication was administered to the animals; 15 min before the anesthetic dosage, atropine (0.05 mg/kg) was administered intramuscularly. Animals were induced by full dosage of mixture of Zoletil 100 (0.04 mg/kg) with xylazine (0.003/kg). Animals were taken to mercy animal hospital (Kaohsiung, Taiwan) with special care for MRI and CT examinations on every 4th and 6th weeks of stem cell implantation. Baseline was taken as 0 week before stem cell therapy ( Figure 6). MRI and CT images were recorded to evaluate the articular cartilage by an experienced professor. The study used T2 mapping with frequency selective fat suppression to evaluate the condition of the cartilage. The frequency encoding direction was oriented in the anterior-posterior direction, and each measurement was repeated three times.

MRI and CT Examinations
Pre-anesthesia medication was administered to the animals; 15 min before the anesthetic dosage, atropine (0.05 mg/kg) was administered intramuscularly. Animals were induced by full dosage of mixture of Zoletil 100 (0.04 mg/kg) with xylazine (0.003/kg). Animals were taken to mercy animal hospital (Kaohsiung, Taiwan) with special care for MRI and CT examinations on every 4th and 6th weeks of stem cell implantation. Baseline was taken as 0 week before stem cell therapy ( Figure 6). MRI and CT images were recorded to evaluate the articular cartilage by an experienced professor. The study used T2 mapping with frequency selective fat suppression to evaluate the condition of the cartilage. The frequency encoding direction was oriented in the anterior-posterior direction, and each measurement was repeated three times.

Macroscopic Images
After eight weeks of stem cell treatments, the animals were euthanized in accordance with the protocol established by the Institutional Animal Care and Use Committee (IACUC). The animals were first administered a full dosage of a mixture of Zoletil 100 (0.04 mg/kg) and Xylazine (0.003/kg) to induce unconsciousness. Then, the knee joints were collected, pictures were taken, and evaluated using ICRS scoring [29] (Table 2). Afterwards, the knee joints were preserved in 4% paraformaldehyde for further histological examination.

Macroscopic Images
After eight weeks of stem cell treatments, the animals were euthanized in accordance with the protocol established by the Institutional Animal Care and Use Committee (IACUC). The animals were first administered a full dosage of a mixture of Zoletil 100 (0.04 mg/kg) and Xylazine (0.003/kg) to induce unconsciousness. Then, the knee joints were collected, pictures were taken, and evaluated using ICRS scoring [29] (Table 2). Afterwards, the knee joints were preserved in 4% paraformaldehyde for further histological examination.

Microscopic Evaluation
For three days, MCs from joint cartilages were preserved in 4% buffered paraformaldehyde. After two weeks of decalcification in 0.5 M ethylene diamine tetra-acetate (EDTA) solution at four degrees Celsius, the samples were desiccated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). An optical microscope (CKX41 Olympus Corporation, Tokyo, Japan) and a digital camera (EOS 80D, Canon Inc., Tokyo, Japan) were used to evaluate the histologic sections. Five independent evaluators independently analyzed the severity of articular cartilage sections and scored them using the International Cartilage Repair Society (ICRS) histology grading method [30] (Table 3). A higher score demonstrated superior quality of the repaired tissue.

Enzyme-Linked Immunosorbent Assay for Cytokine Concentrations
For the cytokine concentrations, blood from each group were collected; blood samples were centrifuged at 1500 g for 10 min and serum was preserved at −80 • C until use. The levels of various cytokines (interleukin-10, transforming growth factor-beta, interleukin-4, tumor necrosis factor alpha, and interleukin-1 beta) were measured using the ELISA (enzyme-linked immunosorbent assay) technique with Quantikine ® ELISA kits (R&D Systems, Minneapolis, MN, USA) as per the manufacturer's instructions. The ELISA kits for all five cytokines were purchased from R&D Systems and used as per the manufacturer's instructions. After the samples were diluted as appropriate, cytokine levels were evaluated using a four-parameter logistic (4-PL) curve fit. The results were presented as mean standard error (SEM).

Statistical Analysis
Descriptive statistics such as mean and standard errors (S.E.) were used to summarize the data. The ANOVA was used to determine if there were significant differences among treatment groups in terms of cytokine concentration, gross grading score, and visual histological findings score. The one-way ANOVA in IBM SPSS version 26 software was used for analysis. Multiple comparisons were performed to compare treatment means using the Duncan and Tukey post hoc tests at a statistical significance level of 5%. The graphs were created with Microsoft Excel 2022 and the most recent version of R software which is R package version 1.3-5 (Agricolae 10).

MRI and CT Observations
This study used magnetic resonance imaging (MRI) and computerized tomography (CT) as imaging modalities to accurately assess and monitor alterations in the knee joint. Specifically, MRI and CT scans were used at the fourth and sixth weeks of the study to validate the expected localization of damage resulting from treatments. The identification of any extra cases of joint infections or injuries in addition to arthritis was also made possible by these imaging techniques. At weeks 0, 4 and 6, MRI and CT scanning were carried out ( Figure 6). Week 0 was used as the baseline time for MRI and CT imaging observations in order to evaluate the defect's initial condition prior to any intervention ( Figure 7). The results obtained from magnetic resonance imaging and computerized tomography scans revealed that the group treated with human adipose derived mesenchymal stem cells demonstrated superior outcomes with respect to cartilage growth and wound healing. At week 6, it was observed that the volume of articular cartilage was lower in the defect control group, while the filling consisted of homogeneous tissue with an abnormal signal intensity of the repair tissue. Figure 8 demonstrates that the human adipose-derived mesenchymal stem cells (hADSC) group exhibits superior outcomes in terms of filling defect margins and promoting cartilage regeneration compared to the other groups, such as the pig umbilical cord-derived mesenchymal stem cells (pUCMSCs), control, and placebo group. pig umbilical cord-derived mesenchymal stem cells (pUCMSCs), control, and placebo group. MRI results have been categorized according to various conditions. Sagittal views were chosen to compare each group. The T2 condition is an excellent method for demonstrating the difference between the damaged and repaired parts. The results of the MRI and CT scans showed notable improvements in the human stem cell-treated group, especially in terms of improved cartilage formation and wound healing. The repair stages of hADSC groups seemed better than those of pUCMSC groups. Based on the results of this study, it appears that MSCs can be used as a new way of regenerating cartilage. A viable approach to repairing articular cartilage and addressing the associated risks of OA could be achieved through this approach. Representative MRI and CT images before cell implantation (week 0). Red circles and red box indicate the defect area. It was taken as the baseline time. MRI images taken after the induction of the knee defect but before the administration of stem cell therapy (a). The purpose of these images is to assess the extent of the knee defect and provide a visual representation of the initial condition before treatment. CT images taken after both knee defect before stem cell therapy (b). CT images provide additional information about the structural aspects of the defected knee before cell implantation.

Figure 7.
Representative MRI and CT images before cell implantation (week 0). Red circles and red box indicate the defect area. It was taken as the baseline time. MRI images taken after the induction of the knee defect but before the administration of stem cell therapy (a). The purpose of these images is to assess the extent of the knee defect and provide a visual representation of the initial condition before treatment. CT images taken after both knee defect before stem cell therapy (b). CT images provide additional information about the structural aspects of the defected knee before cell implantation.
MRI results have been categorized according to various conditions. Sagittal views were chosen to compare each group. The T2 condition is an excellent method for demonstrating the difference between the damaged and repaired parts. The results of the MRI and CT scans showed notable improvements in the treatment group treated human adipose derived mesenchymal stem cells, especially in terms of improved cartilage formation and wound healing (Figures 8 and 9). Figure 9 demonstrated that treatment group especially hADSC exhibited reduced defect margins compared to other groups. The repair stages of hADSC groups seemed better than those of pUCMSC groups. Based on the results of this study, it appears that MSCs can be used as a new way of regenerating cartilage. A viable approach to repairing articular cartilage and addressing the associated risks of OA could be achieved through this approach.

Gross Observations
Eight weeks after surgery, the animals were euthanized with overdose of zoletil and xyzaline and the gross appearances of the defects were taken and studied ( Figure 9). The control group s left and right knee defects had large fissures and penetration cracks. In the placebo group, smaller fissures and penetration cracks compared to control group were observed. In the treatment group (pUCMSC), the defect was covered by a thin layer of repair tissue, but the defected region was not completely covered with tissue. Surprisingly, healing was almost complete in the other treatment group (hADSC), and the reparative tissue was effectively integrated into the repair site, with flush and smooth surfaces on the restored cartilage ( Figure 10). Using the International Cartilage Repair Society (ICRS) score, five independent evaluations evaluated the regeneration cartilages for coverage, neocartilage color, defect margin, and surface roughness [29]. Figure 11 demonstrates the distribution of the mean gross scores among different groups.

Gross Observations
Eight weeks after surgery, the animals were euthanized with overdose of zoletil and xyzaline and the gross appearances of the defects were taken and studied ( Figure 10). The control group's left and right knee defects had large fissures and penetration cracks. In the placebo group, smaller fissures and penetration cracks compared to control group were observed. In the treatment group (pUCMSC), the defect was covered by a thin layer of repair tissue, but the defected region was not completely covered with tissue. Surprisingly, healing was almost complete in the other treatment group (hADSC), and the reparative tissue was effectively integrated into the repair site, with flush and smooth surfaces on the restored cartilage ( Figure 10). Using the International Cartilage Repair Society (ICRS) score, five independent evaluations evaluated the regeneration cartilages for coverage, neocartilage color, defect margin, and surface roughness [29]. Figure 11 demonstrates the distribution of the mean gross scores among different groups.

Histological Observations
Hematoxylin and eosin staining (H&E) was used to demonstrate the partial histological changes of regenerated cartilage in sample slides from all experimental groups ( Figure  12). The microscopic examination of reparative tissue in the treatment group, particularly

Histological Observations
Hematoxylin and eosin staining (H&E) was used to demonstrate the partial histological changes of regenerated cartilage in sample slides from all experimental groups ( Figure 12). The microscopic examination of reparative tissue in the treatment group, particularly hADSC, revealed comparatively smooth restored hyaline-like cartilage with the columnar formations of chondrocytes. The findings of histological evaluations and pathological images from each experimental group indicated that cell distribution, cell population, and cartilage mineralization are better in those who were treated with human stem cells. The mean histopathology value in hADSC seems to be higher than in pUCMSC, placebo, and control groups. Several indicators, such as subchondral bone, matrix, and surface, are significantly higher in the treated group when compared to the control group ( Figure 13). Figure 11. Macroscopic examination of regenerative cartilages based on overall scoring values. Error bars represent mean ± SE (n = 5). Here, n represents technical replications. The same lowercase letters are not significantly different among the experimental treatments (p < 0.05).

Histological Observations
Hematoxylin and eosin staining (H&E) was used to demonstrate the partial histological changes of regenerated cartilage in sample slides from all experimental groups ( Figure  12). The microscopic examination of reparative tissue in the treatment group, particularly hADSC, revealed comparatively smooth restored hyaline-like cartilage with the columnar formations of chondrocytes. The findings of histological evaluations and pathological images from each experimental group indicated that cell distribution, cell population, and cartilage mineralization are better in those who were treated with human stem cells. The mean histopathology value in hADSC seems to be higher than in pUCMSC, placebo, and control groups. Several indicators, such as subchondral bone, matrix, and surface, are significantly higher in the treated group when compared to the control group ( Figure 13).  The ICRS histological scoring demonstrated that hADSC had significantly higher scores (p < 0.05) than the control group for most parameters measured eight weeks after surgery. The placebo group had a higher mean overall score at eight weeks than the control group for surface and cartilage mineralization; however the difference was not statistically significant. Treatment groups, both hADSC and pUCMSC, had higher mean scores in some parameters, such as cell population, cell distribution, and subchondral bone. The mean score for cell population of hADSC was 1.875 ± 0.256, which was followed by pUCMSC with 1.687 ± 0.198. The mean score for the subchondral bone parameter of hADSC was 2.187 ± 0.208, which was also followed by pUCMSC with a mean score of 2.062 ± 0.192. There was a high level of agreement among the valuators. The mean histology score is shown in Figure 13. The ICRS histological scoring demonstrated that hADSC had significantly high scores (p < 0.05) than the control group for most parameters measured eight weeks aft surgery. The placebo group had a higher mean overall score at eight weeks than the co trol group for surface and cartilage mineralization; however the difference was not stati tically significant. Treatment groups, both hADSC and pUCMSC, had higher mean scor in some parameters, such as cell population, cell distribution, and subchondral bone. Th mean score for cell population of hADSC was 1.875 ± 0.256, which was followed b pUCMSC with 1.687 ± 0.198. The mean score for the subchondral bone parameter hADSC was 2.187 ± 0.208, which was also followed by pUCMSC with a mean score 2.062 ± 0.192. There was a high level of agreement among the valuators. The mean histo ogy score is shown in Figure 13.

Cytokine Observations
The study observed different cytokines and it revealed a significant difference in th levels of anti-inflammatory and pro-inflammatory cytokines. The results indicated th the administration of stem cells resulted in an increase in the concentration of anti-inflam matory cytokines, such as IL-4, IL-10, and TGF-beta, compared to the control group an placebo group (Figures 14-18). This suggests that stem cell therapy may have a positiv impact on reducing inflammation and promoting a healthy immune response. Cytokin concentrations were measured every two weeks following stem cell treatment an throughout the study (Figure 14a).
(a) (b) Figure 13. International Cartilage Repair Society (ICRS) histology scores were used to evaluate repair cartilages. The statistical error bars display the mean ± SE (n = 5). All the same lowercase letters are not significantly different between experimental treatments (p < 0.05).

Cytokine Observations
The study observed different cytokines and it revealed a significant difference in the levels of anti-inflammatory and pro-inflammatory cytokines. The results indicated that the administration of stem cells resulted in an increase in the concentration of antiinflammatory cytokines, such as IL-4, IL-10, and TGF-beta, compared to the control group and placebo group (Figures 14-18). This suggests that stem cell therapy may have a positive impact on reducing inflammation and promoting a healthy immune response. Cytokine concentrations were measured every two weeks following stem cell treatment and throughout the study (Figure 14a). The ICRS histological scoring demonstrated that hADSC had significantly higher scores (p < 0.05) than the control group for most parameters measured eight weeks after surgery. The placebo group had a higher mean overall score at eight weeks than the control group for surface and cartilage mineralization; however the difference was not statistically significant. Treatment groups, both hADSC and pUCMSC, had higher mean scores in some parameters, such as cell population, cell distribution, and subchondral bone. The mean score for cell population of hADSC was 1.875 ± 0.256, which was followed by pUCMSC with 1.687 ± 0.198. The mean score for the subchondral bone parameter of hADSC was 2.187 ± 0.208, which was also followed by pUCMSC with a mean score of 2.062 ± 0.192. There was a high level of agreement among the valuators. The mean histology score is shown in Figure 13.

Cytokine Observations
The study observed different cytokines and it revealed a significant difference in the levels of anti-inflammatory and pro-inflammatory cytokines. The results indicated that the administration of stem cells resulted in an increase in the concentration of anti-inflammatory cytokines, such as IL-4, IL-10, and TGF-beta, compared to the control group and placebo group (Figures 14-18). This suggests that stem cell therapy may have a positive impact on reducing inflammation and promoting a healthy immune response. Cytokine concentrations were measured every two weeks following stem cell treatment and throughout the study (Figure 14a).  The different time points are denoted by the x-axis, with "Wo" and "W1" signifying the week before surgery and cell implantation, respectively. The following time points, "W2", "W4", "W6", and "W8", represent two, four, six, and eight weeks after treatment in each group, respectively. The y-axis illustrates the total cytokine concentrations measured throughout the study for each group.
(b) Total cytokines concentrations during the study in each group, where the x-axis represents different treatment groups and the y-axis represents the total cytokine concentration in picograms per milliliters (pg/mL). Cytokine concentration was measured by commercial ELISA. Error bars represent mean ± SE (n = 3). The same lowercase letters are not significantly different among the experimental treatments (p < 0.05).
tively. The y-axis illustrates the total cytokine concentrations measured throughout the study f each group. (b) Total cytokines concentrations during the study in each group, where the x-ax represents different treatment groups and the y-axis represents the total cytokine concentration picograms per milliliters (pg/mL). Cytokine concentration was measured by commercial ELISA. E ror bars represent mean ± SE (n = 3). The same lowercase letters are not significantly different amon the experimental treatments (p < 0.05).
(a) (b) Figure 15. Anti-inflammatory cytokine concentration. (a) Determination of IL-10 cytokine conce tration over time. The different time points are denoted by the x-axis, with "Wo" and "W1" signif ing the week before surgery and cell implantation, respectively. The following time points, "W2 "W4", "W6", and "W8", represent two, four, six, and eight weeks after treatment in each grou respectively. The y-axis illustrates the total cytokine concentrations measured throughout the stud for each group. (b) Total cytokine concentrations during the study in each group, where the x-ax represents different treatment groups and the y-axis represents total cytokine concentration picograms per milliliters (pg/mL). Cytokine concentration was measured by commercial ELISA. E ror bars represent mean ± SE (n = 3). The same lowercase letters are not significantly different amon the experimental treatments (p < 0.05).
(a) (b) Figure 16. Anti-inflammatory cytokine concentration. (a) Determination of TGF-beta cytokine co centration over time. The different time points are denoted by the x-axis, with "Wo" and "W1" si nifying the week before surgery and cell implantation, respectively. The following time poin "W2", "W4", "W6", and "W8", represent two, four, six, and eight weeks after treatment in eac group, respectively. The y-axis illustrates the total cytokine concentrations measured througho the study for each group. (b) Total cytokine concentrations during the study in each group, whe the x-axis represents different treatment groups and the y-axis represents the total cytokine conce tration in picograms per milliliters (pg/mL). Cytokines concentration was measured by commerci ELISA. Error bars represent mean ± SE (n = 3). The same lowercase letters are not significantly d ferent among the experimental treatments (p < 0.05). The different time points are denoted by the x-axis, with "Wo" and "W1" signifying the week before surgery and cell implantation, respectively. The following time points, "W2", "W4", "W6", and "W8", represent two, four, six, and eight weeks after treatment in each group, respectively. The y-axis illustrates the total cytokine concentrations measured throughout the study for each group.
(b) Total cytokine concentrations during the study in each group, where the x-axis represents different treatment groups and the y-axis represents total cytokine concentration in picograms per milliliters (pg/mL). Cytokine concentration was measured by commercial ELISA. Error bars represent mean ± SE (n = 3). The same lowercase letters are not significantly different among the experimental treatments (p < 0.05).
represents different treatment groups and the y-axis represents the total cytokine concentration in picograms per milliliters (pg/mL). Cytokine concentration was measured by commercial ELISA. Error bars represent mean ± SE (n = 3). The same lowercase letters are not significantly different among the experimental treatments (p < 0.05).
(a) (b) Figure 15. Anti-inflammatory cytokine concentration. (a) Determination of IL-10 cytokine concentration over time. The different time points are denoted by the x-axis, with "Wo" and "W1" signifying the week before surgery and cell implantation, respectively. The following time points, "W2", "W4", "W6", and "W8", represent two, four, six, and eight weeks after treatment in each group, respectively. The y-axis illustrates the total cytokine concentrations measured throughout the study for each group. (b) Total cytokine concentrations during the study in each group, where the x-axis represents different treatment groups and the y-axis represents total cytokine concentration in picograms per milliliters (pg/mL). Cytokine concentration was measured by commercial ELISA. Error bars represent mean ± SE (n = 3). The same lowercase letters are not significantly different among the experimental treatments (p < 0.05).
(a) (b) Figure 16. Anti-inflammatory cytokine concentration. (a) Determination of TGF-beta cytokine concentration over time. The different time points are denoted by the x-axis, with "Wo" and "W1" signifying the week before surgery and cell implantation, respectively. The following time points, "W2", "W4", "W6", and "W8", represent two, four, six, and eight weeks after treatment in each group, respectively. The y-axis illustrates the total cytokine concentrations measured throughout the study for each group. (b) Total cytokine concentrations during the study in each group, where the x-axis represents different treatment groups and the y-axis represents the total cytokine concentration in picograms per milliliters (pg/mL). Cytokines concentration was measured by commercial ELISA. Error bars represent mean ± SE (n = 3). The same lowercase letters are not significantly different among the experimental treatments (p < 0.05). The different time points are denoted by the x-axis, with "Wo" and "W1" signifying the week before surgery and cell implantation, respectively. The following time points, "W2", "W4", "W6", and "W8", represent two, four, six, and eight weeks after treatment in each group, respectively. The y-axis illustrates the total cytokine concentrations measured throughout the study for each group. (b) Total cytokine concentrations during the study in each group, where the x-axis represents different treatment groups and the y-axis represents the total cytokine concentration in picograms per milliliters (pg/mL). Cytokines concentration was measured by commercial ELISA. Error bars represent mean ± SE (n = 3). The same lowercase letters are not significantly different among the experimental treatments (p < 0.05). The different time points are denoted by the x-axis, with "Wo" and "W1" signifying the week before surgery and cell implantation, respectively. The following time points, "W2", "W4", "W6", and "W8", represent two, four, six, and eight weeks after treatment in each group, respectively. The y-axis illustrates the total cytokine concentrations measured throughout the study for each group.
(b) Total cytokine concentrations during the study in each group, where the x-axis represents different treatment groups and the y-axis represents the total cytokine concentration in picograms per milliliters (pg/mL). Cytokine concentration was measured by commercial ELISA. Error bars represent mean ± SE (n = 3). The same lowercase letters are not significantly different among the experimental treatments (p < 0.05).
The different time points are denoted by the x-axis, with "Wo" and "W1" signifying the week before surgery and cell implantation, respectively. The following time points, "W2", "W4", "W6", and "W8", represent two, four, six, and eight weeks after treatment in each group, respectively. The yaxis illustrates the total cytokine concentrations measured throughout the study for each group. (b) Total cytokine concentrations during the study in each group, where the x-axis represents different treatment groups and the y-axis represents the total cytokine concentration in picograms per milliliters (pg/mL). Cytokine concentration was measured by commercial ELISA. Error bars represent mean ± SE (n = 3). The same lowercase letters are not significantly different among the experimental treatments (p < 0.05).

Discussion
Mesenchymal stem cells (MSCs) have garnered considerable attention as a potential source for cartilage regeneration in large animal models, including pigs [31][32][33]. In this study, we sought to evaluate the regenerative effects of two different MSC sources, namely human adipose-derived mesenchymal stem cells (hADSCs) and porcine umbilical cord derived mesenchymal stem cells (pUCMSCs), for their potential application in cartilage regeneration in pigs. The reason for selecting miniature porcine breeds as our large animal model in this study was because of their resemblance to humans in regard to joint size, loading mechanics, weight, and their inherent inability to regenerate cartilage, as well as their comparable collagen fiber arrangement, bone apposition rate, and trabecular The different time points are denoted by the x-axis, with "Wo" and "W1" signifying the week before surgery and cell implantation, respectively. The following time points, "W2", "W4", "W6", and "W8", represent two, four, six, and eight weeks after treatment in each group, respectively. The y-axis illustrates the total cytokine concentrations measured throughout the study for each group. (b) Total cytokine concentrations during the study in each group, where the x-axis represents different treatment groups and the y-axis represents the total cytokine concentration in picograms per milliliters (pg/mL). Cytokine concentration was measured by commercial ELISA. Error bars represent mean ± SE (n = 3). The same lowercase letters are not significantly different among the experimental treatments (p < 0.05). The different time points are denoted by the x-axis, with "Wo" and "W1" signifying the week before surgery and cell implantation, respectively. The following time points, "W2", "W4", "W6", and "W8", represent two, four, six, and eight weeks after treatment in each group, respectively. The y-axis illustrates the total cytokine concentrations measured throughout the study for each group.
(b) Total cytokine concentrations during the study in each group, where the x-axis represents different treatment groups and the y-axis represents the total cytokine concentration in picograms per milliliters (pg/mL). Cytokine concentration was measured by commercial ELISA. Error bars represent mean ± SE (n = 3). The same lowercase letters are not significantly different among the experimental treatments (p < 0.05).
The different time points are denoted by the x-axis, with "Wo" and "W1" signifying the week before surgery and cell implantation, respectively. The following time points, "W2", "W4", "W6", and "W8", represent two, four, six, and eight weeks after treatment in each group, respectively. The yaxis illustrates the total cytokine concentrations measured throughout the study for each group. (b) Total cytokine concentrations during the study in each group, where the x-axis represents different treatment groups and the y-axis represents the total cytokine concentration in picograms per milliliters (pg/mL). Cytokine concentration was measured by commercial ELISA. Error bars represent mean ± SE (n = 3). The same lowercase letters are not significantly different among the experimental treatments (p < 0.05).

Discussion
Mesenchymal stem cells (MSCs) have garnered considerable attention as a potential source for cartilage regeneration in large animal models, including pigs [31][32][33]. In this study, we sought to evaluate the regenerative effects of two different MSC sources, namely human adipose-derived mesenchymal stem cells (hADSCs) and porcine umbilical cord derived mesenchymal stem cells (pUCMSCs), for their potential application in cartilage regeneration in pigs. The reason for selecting miniature porcine breeds as our large animal model in this study was because of their resemblance to humans in regard to joint size, loading mechanics, weight, and their inherent inability to regenerate cartilage, as well as The different time points are denoted by the x-axis, with "Wo" and "W1" signifying the week before surgery and cell implantation, respectively. The following time points, "W2", "W4", "W6", and "W8", represent two, four, six, and eight weeks after treatment in each group, respectively. The y-axis illustrates the total cytokine concentrations measured throughout the study for each group. (b) Total cytokine concentrations during the study in each group, where the x-axis represents different treatment groups and the y-axis represents the total cytokine concentration in picograms per milliliters (pg/mL). Cytokine concentration was measured by commercial ELISA. Error bars represent mean ± SE (n = 3). The same lowercase letters are not significantly different among the experimental treatments (p < 0.05).

Discussion
Mesenchymal stem cells (MSCs) have garnered considerable attention as a potential source for cartilage regeneration in large animal models, including pigs [31][32][33]. In this study, we sought to evaluate the regenerative effects of two different MSC sources, namely human adipose-derived mesenchymal stem cells (hADSCs) and porcine umbilical cord derived mesenchymal stem cells (pUCMSCs), for their potential application in cartilage regeneration in pigs. The reason for selecting miniature porcine breeds as our large animal model in this study was because of their resemblance to humans in regard to joint size, loading mechanics, weight, and their inherent inability to regenerate cartilage, as well as their comparable collagen fiber arrangement, bone apposition rate, and trabecular thickness, as previously reported in previous studies [34,35]. The experimental data provided suggests that treatment with human stem cells may be a promising therapy for promoting cartilage growth and wound healing. The results obtained from MRI and CT scans demonstrated that the group treated with stem cells showed superior outcomes with respect to cartilage growth and repair. This is consistent with previous studies that have reported the potential of stem cells for regenerating cartilage tissue [36][37][38].
The concern about immune rejection was a crucial consideration in the experiment as the xenograft model was carried out. Upon conducting physical observations on the animal subjects during the study, no instances of immune rejection or tumor formation were observed in the pig's body during the study. Gross observations at eight weeks after surgery revealed that the stem cell treatment group, particularly hADSC, had almost completely healed defects, with smooth surfaces on the restored cartilage. This contrasts with the control group, which had large fissures and penetration cracks. These findings are also consistent with previous studies that have reported the potential of stem cells for promoting tissue repair [16,23,28]. Based on the results of this study, it demonstrated that human adipose-derived mesenchymal stem cells (hADSCs) showed better results in cartilage repair compared to other groups. The histological evaluations demonstrated that cell distribution, cell population, and cartilage mineralization were better in the stem cell treatment groups compared to the control group. Specifically, the microscopic examination of reparative tissue in the hADSC group revealed smooth restored hyaline-like cartilage and more chondrocytes compared to controls ( Figure 12). Better improvement in cartilage repair in the hADSC is believed to be the result of a better cell quality and cell doubling time for hADSCs compared to pUCMSCs. However, according to other findings, the basic biological characteristics of mesenchymal stem cells derived from adipose and umbilical cord tissues are comparable to one another, with both possessing significant selfrenewal capacity, anti-apoptotic capacity, and multi-differentiation potential. Furthermore, investigations have discovered that the different types of cytokines generated by ASCs and UC-MSCs are comparable, although there are variations in the levels of expression of cytokines. Furthermore, several research have indicated that ASCs reacted better to various neural induction techniques than UC-MSCs, indicating that hADSCs have the potential for cartilage regeneration [39].
The process by which stem cells contribute to tissue regeneration and wound healing is underpinned by two hypotheses, namely the differentiation theory and the paracrine theory [40,41]. While both hypotheses have merit, the latter is more widely accepted and involves the concept of cell homing. This mechanism is characterized by the migration of stem cells to areas of tissue damage and subsequent secretion of cytokines that influence the behavior of adjacent cells. By promoting tissue repair and reconstruction through this paracrine mechanism, this study is also aligned with this hypothesis [42]. Specifically, this study has found that the regenerative effect of human adipose-derived mesenchymal stem cells on chondrocyte growth in pigs is associated with immune modulation, without immune rejection or tumor formation. Although this study demonstrates promising results in terms of cartilage regeneration solely using MSCs, it has been suggested by other literature by Gugliandolo et al. [43] that the bone regenerative capacity of scaffolds enriched with MSCs can be influenced and enhanced through the addition of biomolecules, such as bone morphogenetic proteins (BMPs), or the modification of biomaterial characteristics, such as pore dimensions [43]. Administration of MSCs significantly decreased the proinflammatory factors (TNF-alpha, or IL-1 beta), whereas the anti-inflammatory factors (IL-4 or Il-10) were significantly increased in this study, which is also supported by previous studies [15,16,[34][35][36]. The dysregulation of proinflammatory and anti-inflammatory cytokines, with a predominance of proinflammatory cytokines, plays a pivotal role in driving the secretion of enzymes and other inflammatory mediators involved in the pathogenesis of osteoarthritis. This imbalance subsequently leads to detrimental morphological transformations within the joint, including cartilage degeneration, osteophyte formation, and various inflammatory alternations such as synovitis. Moreover, it seems necessary to investigate the epigenetic regulation of cytokine generation, as this could potentially lead to alternative treatment strategies for osteoarthritis [44]. This stud also investigated the impact of stem cell therapy on cytokine levels by examining three distinct anti-inflammatory cytokines (IL-4, IL-10, and TGF-beta) and two pro-inflammatory cytokines (TNF-alpha and IL-1Beta). The objective was to observe and analyze any alterations in the concentrations of these cytokines before and after the administration of stem cell therapy, thus providing insights into the therapeutic effects on cytokine profiles. The treatment group in this study was observed to have high levels of IL-10 and showed lower levels of pro-inflammatory cytokines after stem cell transplantation. This is due to IL-10 suppressing the secretion of cytokines, such as TNFα, IL-1, IL-6, IL-8, and IL-12, by dendritic cells and reducing the expression of MHC II molecules and the B7 co-stimulatory complex on their surfaces [45]. On the other hand, MSCs release many different types of cytokines, which regulate and reduce inflammatory responses, including transforming growth factor (TGF), hepatocyte growth factor (HGF), prostaglandin E2 (PGE2), soluble HLA-G5 protein, indolamine-2,3-dioxygenase (IDO), nitric oxide (NO), and interleukin-10 (IL-10) [46].
This study used a low-dose, single intra articular injection of MSCs that had no adverse effects on the experimental animals. The findings suggested a degree of safety and efficacy for a limited time as this xenograft model study is supported by the literature in terms of safety [16,47,48]. However, this study was unable to examine the results after eight weeks because the study was carried out for eight weeks after stem cell therapy. Therefore, a further study with a bigger sample number and longer duration is required to assess efficacy over a longer length of time. If the nation invests in regenerative research to develop alternative remedies for knee osteoarthritis, there is a good probability that patients' quality of life and the country's economy will improve.

Conclusions
The regenerative effect of human adipose MSCs on chondrocyte growth in a porcine model is associated with an immune modulation without immune rejection or tumor formation in this study. The study used a pig model to evaluate the effectiveness of mesenchymal stem cells for regenerating cartilage in the knee. According to the study's findings, hADSCs can aid in cartilage regeneration by promoting anti-inflammatory cytokines while reducing pro-inflammatory cytokines. This study opens the door to cartilage regeneration, potentially improving treatment techniques for OA in both humans and pets. However, further studies are needed to confirm these findings and establish it as a therapeutic option.