Biomedicines, 18/09/2025
Introduction
Mesenchymal stem cells (MSCs) offer promising prospects for novel treatment modalities in cellular therapies and artificial organ production, due to their remarkable abilities to self-renew and differentiate into various specialized cell types. MSCs are widely distributed in numerous tissues, including the umbilical cord, bone marrow, cartilage, adipose tissue, placenta, and dental pulp, among others. Among these sources, the placenta stands out as an especially valuable reservoir of MSCs. In the context of cartilage regeneration, chondrocytes play a pivotal role in maintaining the structure and function of cartilage tissues within joints by synthesizing collagen and extracellular matrix (ECM).
Method
After isolating the placenta tissue, it was dissected and minced. To isolate the MSC, the tissue then undergoes enzymatic digestion using two different protocols, including trypsin with collagenase and the trypsin-only protocol. For subculture and expansion of MSC, 2D expansion method in T-75 flasks was applied and MSCs were cultured until reaching 80–90% confluence at a density of 5 × 104 cells/cm2. Every 5-7 days, cell characterization was performed by analyzing positive and negative surface markers. Then, 2D and 3D chondrogenic differentiation of MSCs were conducted. Finally, static and dynamic 3D chondrocyte cultures were performed using PLGA scaffolds.
Results
- MSC isolation and attachment
MSCs were successfully attached to culture flasks within 48 h of plating, exhibiting the typical fibroblast-like morphology characteristic of MSCs. No residual red blood cells or significant debris were observed, indicating effective isolation. Both the Trypsin + Collagenase and Trypsin-only protocols isolated MSCs; however, the Trypsin + Collagenase method yielded a significantly higher cell count, consistent with enhanced enzymatic digestion facilitating better tissue dissociation.
- MSC characterization
Flow cytometry resulted in high expression of CD73, CD90, and CD105 and negligible expression of hematopoietic markers CD14, CD34, CD45, and HLA-DR. These markers confirm the purity of the MSC population and their mesenchymal lineage, critical for ensuring the correct cellular phenotype for chondrogenic differentiation.
- Chondrocyte differentiation in 2D and 3D cultures
In the 2D monolayer culture system, MSCs underwent successful differentiation into chondrocytes over a 30-day period. In 3D pellet cultures, Alcian Blue staining after 21 days revealed glycosaminoglycan (GAG) deposition, indicating the presence of cartilage-like extracellular matrix (ECM).
- PLGA biopolymer degradation timeline
PLGA scaffolds in both static and dynamic 3D cultures began degrading by day 6 and were largely degraded by day 9. Notably, PLGA pieces cultured in PBS alone showed no degradation over highlighting the role of cellular activity and media components in polymer breakdown.
The Alcian Blue and Toluidine Blue staining predominantly target sulfated GAGs and cartilage-specific ECM molecules. While ECM components were detected as early as day 6, the majority of ECM deposition and scaffold remodeling occurred over the subsequent days, with optimal ECM maturity expected closer to day 21.
- RCCS-Mediated PLGA Biopolymer Degradation
Similar degradation kinetics were observed in the RCCS system, with PLGA degradation commencing by day 6 and being completed by day . The dynamic culture environment may accelerate degradation through enhanced nutrient diffusion and mechanical forces. Despite scaffold degradation, cells largely remained within 3D aggregates or attached to residual scaffold fragments, maintaining a 3D microenvironment.
- Assessing ECM Formation in Chondrogenic Tissue
H&E and Toluidine Blue staining of samples after 9 days in RCCS culture revealed isogenous groups of chondrocytes surrounded by ECM, including visible collagen fibers, which are consistent with cartilage tissue morphology. Chondrogenic differentiation was primarily confirmed through morphological changes and ECM-specific staining. Also, collagen type II as a cartilage-specific marker was used to validate differentiation.
- Biopolymer Degradation in 3D Cell Cultures
Segmented PLGA pieces cultured in 6-well plates exhibited degradation by day 6 and complete degradation by day 9 in media containing cells, while no degradation occurred in PBS-only controls. These results confirm that cellular metabolism and media components contribute to PLGA degradation.
The PLGA pieces cultured in the RCCS-STLV bioreactor with PBS alone did not degrade over 21 days, contrasting with static culture observations and suggesting that mechanical conditions and medium composition both influence degradation rates.
Discussion
This study successfully isolated MSCs using two protocols, with the Trypsin and Collagenase method providing more efficient in terms of yield. Characterization confirmed the expression of MSC markers (CD73, CD90, CD105). Although telomerase activity was not assessed, previous studies have demonstrated that placenta-derived MSCs generally exhibit robust proliferative capabilities and may possess longer telomere lengths compared to MSCs derived from other sources. The study also confirmed the successful chondrogenic differentiation of MSCs into chondrocytes in both 2D and 3D culture systems, with morphology changes from the fibroblast-like shape to a rounder chondrocyte morphology. However, an unexpected rapid degradation of the PLGA biopolymer was observed in both static and dynamic 3D cultures, potentially affect the structural integrity of the final tissue construct. Moreover, TGF-β1 was shown to influenced MSC differentiation and contributed to cartilage formation, as evidenced by the formation of isogenous groups and collagen fiber within the extracellular matrix. Notably, the dynamic culture system in the RCCS-4HD bioreactor played a key role in supporting chondrocyte phenotypic expression and ECM formation, despite the challenges posed by PLGA degradation. the 3D aggregation of cellular elements was confirmed through multilayered cell structures embedded in a dense extracellular matrix and spatial organization typical of 3D tissue constructs.
Conclusion
In conclusion, our study highlights the potential of placenta-derived MSCs as a valuable cell source for regenerative medicine applications, especially in cartilage tissue engineering. The combination of these MSCs with 3D culture systems and bioreactor technology holds great promise for improving the efficacy of tissue engineering strategies. To fulfil the potential of therapeutic interventions for cartilage repair, future studies should focus on optimizing biopolymer compositions, exploring various growth factor protocols, and refining bioreactor parameters to enhance tissue development.
References
Mujde, C., & Bisgin, A. (2025). Three-Dimensional Cartilage Tissue Engineering Using Placenta-Derived Extra-Embryonic Mesenchymal Stem Cells: From Isolation to Differentiation. Biomedicines, 13(9), 2291. https://doi.org/10.3390/biomedicines13092291
Source: Biomedicines
Link: https://www.mdpi.com/2227-9059/13/9/2291
