Introduction

Articular cartilage is a highly specialised connective tissue characterised by its unique zonal architecture and the absence of vasculature, nerves, and lymphatic drainage. These structural constraints severely limit its intrinsic self-repair capacity. Consequently, whether precipitated by mechanical trauma or degenerative disease, cartilage injuries often initiate a deleterious cascade of pathological events. The initial injury triggers a robust inflammatory response, marked by elevated pro-inflammatory mediators, including tumour necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and various chemokines.

This inflammatory milieu further drives the expression of matrix-degrading enzymes, specifically matrix metalloproteinases (MMPs) and the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family. These enzymes facilitate progressive disruption of the extracellular matrix (ECM) by depleting essential collagen fibres and proteoglycans. The resulting degradation products act as "damage-associated molecular patterns", creating a feed-forward loop that exacerbates inflammation and accelerates ECM loss. This progressive impairment eventually compromises subchondral bone integrity, leading to pathological remodelling and the formation of osteophytes, hallmarks of osteoarthritis.

This study explores the development of a three-dimensional (3D) microcarrier system derived from decellularised ECM (dECM). By utilising dECM as a reliable seeding carrier within the 3D FloTrix™ platform, the study demonstrates how a biomimetic microenvironment can support high cell viability and promote functional cartilage regeneration in a minimally invasive manner.

Challenges in Cartilage Regeneration

Current therapeutic strategies in regenerative medicine face significant hurdles. Traditional scaffold-based transplantations frequently necessitate large surgical incisions, which are associated with donor-site morbidity, postoperative pain, and heightened risks of infection. To address these limitations, minimally invasive implantation of microtissues has emerged as a promising alternative. Microtissues—small, functional tissue clusters—allow for the flexible repair of irregularly shaped cartilage defects through injectable delivery systems.

While scaffold-free cell aggregation is one method of microtissue construction, these dense clusters often suffer from limited nutrient diffusion, leading to the formation of a necrotic core. To circumvent this, the integration of biological microcarriers is essential. The natural cartilage ECM remains as the ideal source for such carriers, as it provides not only mechanical scaffolding but also the specific biochemical cues required for chondrogenic maintenance.

(Image: Article, published by Materials Today Bio, titled “High paracrine activity of hADSCs cartilage microtissues inhibits extracellular matrix degradation and promotes cartilage regeneration”.)

Recently, a study titled “High paracrine activity of hADSCs cartilage microtissues inhibits extracellular matrix degradation and promotes cartilage regeneration” was published online in Materials Today Bio [IF: 10.2]. This study demonstrated that ADSC microtissues (ADSCs-MT) constructed using the 3D FloTrix™ microSPIN 6-channel stirred rotary bioreactor achieved excellent cartilage regeneration outcomes, potentially advancing the autologous application of cartilage microtissues.
Decellularisation of ECM removes cellular components and other antigenic substances, thereby reducing the risks of inflammatory reactions and immune rejection while preserving the biological, mechanical, and biochemical properties of the ECM. Consequently, a decellularised ECM can serve as a reliable three-dimensional (3D) carrier for cell seeding.

Research Background

Functional tissue engineering relies heavily on the successful recellularization of scaffolds with cell populations capable of robust proliferation and targeted differentiation. Ethical complexities and prolonged differentiation timelines hinder the clinical translation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), despite their superior potency. Consequently, mesenchymal stem cells (MSCs) have emerged as the preferred candidate for cartilage engineering, owing to their potent immunomodulatory properties and well-characterised chondrogenic potential. Furthermore, MSCs offer a significant advantage over primary chondrocytes by allowing for autologous harvesting via minimally invasive procedures. While allogeneic MSCs are commercially attractive, recent evidence suggests that autologous MSCs exhibit superior survival rates and avoid the risk of inducing T-cell memory phenotypes associated with allogeneic rejection.
This study utilised two primary autologous MSC sources—adipose-derived stem cells (ADSCs) and bone marrow-derived stem cells (BMSCs)—to construct microtissues and systematically compare them. 
Both ADSCs and BMSCs can be ethically obtained and have well-documented application prospects. Among autologous sources, Bone Marrow-Derived Stem Cells (BMSCs) and Adipose-Derived Stem Cells (ADSCs) represent the most viable clinical options. ADSCs can be harvested in greater quantities with significantly lower donor-site morbidity, giving them a key advantage over the longer-established BMSCs. Crucially, the regenerative efficacy of these two cell types is highly dependent on the supporting scaffold and the biomechanical environment. Despite extensive research into their individual chondrogenic capabilities, a systematic, head-to-head comparison within a standardised 3D microtissue framework remains sparse.

(Image: 3D FloTrix™ microSPIN Multiplex System) 

In this study, 3D FloTrix™ technology was employed to establish a standardised microcarrier environment for the comparative evaluation of homologous adipose-derived stem cells (ADSCs) and bone marrow-derived stem cells (BMSCs). By leveraging the 3D FloTrix™ microSPIN 6-channel stirred rotary bioreactor, the researchers constructed high-uniformity microtissues (ADSCs-MT and BMSCs-MT), thereby ensuring that the observed biological variances were attributable to intrinsic cellular phenotypes rather than technical inconsistencies in the culture process.
The findings revealed that the microcarrier microenvironment specifically enhanced the proliferative and migratory capacities of ADSCs. Subsequent in vitro and in vivo analyses demonstrated that ADSCs-MT delivered cartilage regeneration outcomes significantly superior to those of BMSCs-MT. Transcriptomic profiling further elucidated this therapeutic efficacy, revealing that ADSCs-MT actively suppressed the expression of markers associated with:

• ECM degradation 
• Osteogenic differentiation (preventing unwanted calcification)
• Fibrocartilage formation (ensuring high-quality hyaline-like repair)

Furthermore, the study validated the protective paracrine effects of these microtissues on inflammatory chondrocytes. By utilising the precision of the 3D FloTrix™ system, this research establishes a definitive case for the application of ADSC-derived microtissues as a superior modality for autologous cartilage regeneration.

Study Design

Figure 1: Overall Study Design Diagram

The schematic illustrates the process of preparing ADSCs-MT and BMSCs-MT using cartilage-derived decellularised extracellular matrix (cECM) microcarriers, as well as the in vitro and in vivo validation experiments for cartilage regeneration achieved through chondrogenic induction of ADSCs-MT.

Research Content

Following the initial preparation of cECM and its corresponding microcarriers, ADSCs-MT and BMSCs-MT were constructed utilising a 6-channel stirred rotary bioreactor. This system facilitated uniform cell-carrier assembly and optimal nutrient diffusion, preventing the formation of necrotic cores standard in static aggregate cultures.

Figure 2: In Vitro Evaluation of ADSCs-MT and BMSCs-MT

(A) Schematic diagram of microtissue preparation; 

(B) Key timepoint images of microtissue preparation; 

(C) SEM observation of microtissue microstructure; 

(D) mRNA expression levels of SOX9, COL2, and ACAN in the two microtissue groups;

(E) Immunofluorescence detection of SOX9 protein in the two microtissue groups; 

(F) Quantitative analysis of SOX9 protein expression;

(G) Western blot analysis of COL2 and ACAN proteins in the two microtissue groups; 

(H) H&E staining and toluidine blue staining results of microtissues; 

(I) Transwell migration assay under three chemotactic gradients.

To further elucidate the disparities in gene expression profiles and regulatory signalling pathways between ADSCs-MT and BMSCs-MT, mRNA-seq analysis was performed on the two microtissue groups after differentiation.

Figure 3: mRNA Sequencing and Differential Expression Gene (DEG) Analysis of the Two Microtissue Groups

(A) Venn diagram of expressed genes in the two microtissue groups; 

(B) Volcano plot of RNA sequencing data from the two microtissue groups; 

(C) Heatmap of differentially expressed genes; 

(D) GO enrichment analysis of differentially expressed genes; 

(E) KEGG enrichment analysis of differentially expressed genes.

To explore the development and differentiation processes of ADSCs-MT cells in depth, RNA sequencing was performed at key timepoints (Day 0, Day 7, and Day 14) during ADSCs-MT culture.

Figure 4: mRNA Sequencing and Differential Expression Gene (DEG) Analysis at Key Timepoints During hADSCs-MT Culture

(A) Venn diagram of expressed genes in ADSCs-MT at D0, D7, and D14; 

(B) Heatmap of differentially expressed genes; 

(C, F, I, L) Volcano plot, GO enrichment analysis, KEGG analysis, and PCR validation of differentially expressed genes for D7 vs. D0; 

(D, G, J, M) Volcano plot, GO enrichment analysis, KEGG analysis, and PCR validation of differentially expressed genes for D14 vs. D0; 

(E, H, K, N) Volcano plot, GO enrichment analysis, KEGG analysis, and PCR validation of differentially expressed genes for D14 vs. D7.

ADSCs-MT demonstrated more substantial paracrine effects and exhibited cartilage-protective properties.

Figure 5: Cartilage-Protective Effects of ADSCs-MT

(A) Schematic of the Transwell system for co-culture of microtissues and human chondrocytes; 

(B) Relative expression of matrix formation-related genes (COL2 and ACAN) and matrix degradation-related genes (MMP1, MMP13, ADAMTS4, and ADAMTS5) in chondrocytes across groups; 

(C) Protein expression of COL2 and ACAN in chondrocytes across groups; 

(D) Quantitative analysis of COL2 and ACAN protein expression; 

(E) Immunofluorescence analysis of MMP13; 

(F) Analysis of MMP13 protein expression based on immunofluorescence detection.

Based on macroscopic evaluation, histological staining, and immunofluorescence staining, tissue regeneration was assessed 8 weeks after subcutaneous implantation of different microtissues in nude mice.

Figure 6: Evaluation of Regenerated Tissues 8 Weeks After Subcutaneous Implantation in Nude Mice

(A) Macroscopic view of regenerated tissues 8 weeks after subcutaneous implantation in nude mice across groups; 

(B) Hematoxylin and eosin staining; (C) Toluidine blue staining; 

(D) Safranin O staining; 

(E) Immunohistochemical staining of COL2; 

(F) Immunohistochemical staining of COL1; 

(G) Immunofluorescence staining of SOX9.

Research Conclusion

This study bridges the gap between biomimetic materials science and industrialised cell manufacturing by integrating mesenchymal stem cells with cartilage-derived decellularised extracellular matrix (cECM) microcarriers within a 3D dynamic culture environment. Our systematic comparison reveals that ADSCs-MT exhibit a clear therapeutic advantage over BMSCs-MT, characterised by a distinct transcriptomic profile that suppresses fibrocartilage markers and prevents osteogenic transition, thereby ensuring a hyaline-like phenotype. This study also demonstrates that combining autologous ADSCs with cECM microcarriers in a 3D dynamic bioreactor environment significantly optimises cartilage regeneration. ADSCs-MT exhibited superior chondrogenic differentiation, potent paracrine-mediated chondroprotection, and high-quality in vivo tissue synthesis.
Furthermore, the utilisation of the 3D FloTrix™ automated bioreactor system demonstrates that achieving these complex biological outcomes is compatible with industrial scalability. By eliminating the technical variability inherent in manual processes, this research provides a definitive blueprint for the standardised, autologous production of cartilage microtissues. Ultimately, this work moves the field closer to a new generation of minimally invasive, patient-specific therapies that offer the precision of "off-the-shelf" products with the biological efficacy of native tissue.

Author Profiles

First Author: Liu Wei, female, joint master’s student at Shandong First Medical University (Shandong Academy of Medical Sciences) and the Chinese PLA General Hospital, primarily engaged in research on cartilage injury and tissue engineering repair.
Corresponding Author: Professor Qi Jianhong, Shandong First Medical University (Shandong Academy of Medical Sciences), School of Sports Medicine and Rehabilitation, Chief Physician, Doctoral Supervisor.
Corresponding Author: Professor Peng Jiang, Director of the Institute of Orthopedics, Chinese PLA General Hospital, Director of the Key Laboratory of Military Orthopedic Trauma, Chief Physician, Doctoral Supervisor.
Corresponding Author: Professor Wang Aiyuan, Researcher at the Institute of Orthopaedics, Chinese PLA General Hospital. Long-term engagement in basic and clinical translational research on bone and cartilage tissue engineering technologies.

References

Wei Liu, Hongyu Jiang, Jiajie Chen, Yue Tian, Ying He, Ying Jiao, Yanjun Guan, Zhibo Jia, Yanbin Wu, Cheng Huang, Yiben Ouyang, Wenjing Xu, Jianhong Qi, Jiang Peng, & Aiyuan Wang. (n.d.). High paracrine activity of hADSCs cartilage microtissues inhibits extracellular matrix degradation and promotes cartilage regeneration. In Science Direct. Materials Today Bio. https://www.sciencedirect.com/science/article/pii/S2590006424004332