Background: Stem cells have been investigated as a measure of regenerative medicine for cartilage regeneration. While the transplanted stem cells were originally anticipated to survive and engraft to the recipient site, it was soon noted that these cells actually disappear from the transplantation site rapidly. To ensure durable effects and achieve structural improvement, long-term survival and engraftment of transplanted cells is necessary. Because cells can survive better with close interaction with adjacent cells, retention of transplanted stem cells and prevention of their dispersion into the joint cavity can be expected to promote cell survival and subsequent effective engraftment. Three-dimensional spheroids create cell-cell and cell-extracellular matrix (ECM) interactions while allowing diffusion of oxygen and waste products at the same time.

Objectives: We had demonstrated that ASC spheroids have better in vitro and in vivo survival and chondrogenic potential, and that when injected into osteoarthritic (OA) joints, they exert greater regenerative effects for articular cartilage and arrest the progression of surgically induced OA better than adipose stem cells (ASCs) in single-cell suspension. In the present study, we combined the concept of spheroid form and cell immobilization using bio-adhesive to promote the stem cell-based regeneration of articular cartilage in an animal OA model.

Methods: 1) Chondrogenic spheroids (chondrospheroids) were produced from human adipose stem cells (hASCs) using specially designed plastic culture plates. The plastic culture plate has 389 wells, each well having a diameter of 600µm. We produced chondrospheroids by seeding 2x10 ⁶ cells on this plastic culture plate, waiting for 10 minutes for the cells to sink into the well, and then washing to remove the remaining cells. Chondrospheroids were collected 3 days after production, evaluated for chondrogenic differentiation ability, stored in the refrigerator with a preservative solution, and used in animal experiments (Figure 1a). 2) In vivo efficacy of chondrospheroids in rabbits: To induce OA in rabbits, we removed the medial meniscus ligament and implanted Chondrospheroids 6 weeks later. The chondrospheroids were implanted and immobilized on the site using fibrin glue on the chondral defect created in the patellar groove of OA-induced rabbits. Rabbits were allocated to two different groups as follows: Fibrin only group; Fibrin with Chondrospheroid (Chondrospheroid+Fibrin: 3.2 x 10 ⁶ cells/700-750 chondrospheroids) group. All animals were sacrificed for analysis 3 months post-transplantation.

Results: We confirmed the chondrogenic potential of spheroids using Safranin-O staining and qRT-PCR for chondrogenic differentiation-related genes (SOX9, Aggrecan, Collagen type 2) 3 days after formation (Figure 1b). Twelve weeks after implantation of chondrospheroids, it was confirmed that cartilage regeneration was superior to that of the control group (Fibrin only group). ELISA analysis of TNF-α and IL-1β showed that the degree of inflammatory response within the joint was significantly lower in Chondrospheroid+Fibrin group, similar to that in normal rabbits (Figure 2A). Histological assessment also demonstrated better quality of repair with Chondrospheroid+Fibrin transplantation, defects filled with hyaline cartilage-like tissue (Figure 2B). The ICRS macroscopic score was significantly higher in the Chondrospheroid+Fibrin group than in the control group implanted with Fibrin only. Histological scores were also significantly better in the Chondrospheroid+Fibrin group than in the Fibrin only group (Figure 2C). Immunohistochemical staining using human nuclear antigen (HN) which was performed to confirm the mode of action (MoA) showed the presence of human cells in the regenerated articular cartilage of the rabbit, indicating that a portion of implanted hASCs in the chondrospheroids survived (Figure 2D).

Conclusion: Our results prove that the combined concept of using spheroid form of ASCs and in situ immobilization using bioadhesive fixation is an effect strategy to promote cartilage regeneration in OA by enhancing survival and in situ retention of implanted cells.

REFERENCES: [1] Ko JY, Park JW, Kim J, Im GI. Characterization of adipose-derived stromal/stem cell spheroids versus single-cell suspension in cell survival and arrest of osteoarthritis progression. J Biomed Mater Res A. 2021 Jun;109(6):869-878.

[2] Maeng SW, Ko JY, Park TY, Yun J, Park SH, Han SH, Joo KI, Ha S, Jee N, Im GI, Cha HJ. Adipose stem cell transplantation using adhesive protein-based viscous immiscible liquid for cartilage reconstruction. Chemical Engineering Journa. 2023 May;463(1):142379.

Acknowledgements: This research was supported by a grant of the Korea Korean Fund for Regenerative Medicine (RS-2022-00070271 and RS-2023-00215015).

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.