For the last three decades, various tissue engineering approaches have been investigated to heal focal cartilage defects. Scientists and engineers have utilized numerous biomaterials, ranging from hydrogels to synthetic scaffolds (Chu et al., 1995, Crawford et al., 2009, Jutila et al., 2015), as well as biofabrication methods, ranging from injection molding to 3D printing (Chang et al., 2003, Guo et al., 2017, Yan et al., 2023) to manufacture tissue-engineered cartilage. Multiple in vitro and in vivo studies have shown that functionally competent tissue-engineered constructs can be manufactured regardless of specific biofabrication methods (Kock et al., 2012, Little et al., 2011, Patel et al., 2019). Despite the tremendous success in cartilage biofabrication, identifying critical parameters that are predictive of product performance in vivo remains a major hurdle for the field (see Fig. 1).

Notably, the Food and Drug Administration guidance in the past three decades suggests that the mechanical properties of the engineered cartilage tissue should be measured prior to implantation as they could affect the success of the implantation (Center for Biologics Evaluation, 2019). Interestingly, multiple studies have shown that the macro level mechanical properties are strongly correlated to the overall composition of the engineered tissue, suggesting composition as a potential non-destructive parameter to predict mechanical properties (Kim and Bonassar, 2023, Rotter et al., 2002, Vunjak-Novakovic et al., 1999). However, to date, no tissue-engineered cartilage constructs have been able to replicate all mechanical properties of native tissue at multiple scales (Griffin et al., 2015, Little et al., 2011, Patel et al., 2019).

One of the challenges of replicating the mechanical properties of native cartilage tissue arises from the fact that the architectural features and compositional distribution of tissue-engineered cartilage constructs do not match native tissue. Regardless of the biomaterials or fabrication methods, tissue-engineered cartilage constructs have extremely different architectural and compositional features compared to native tissue. Notably, clinically available tissue-engineered cartilage products or products that are in clinical trials all utilize collagen scaffolds (Crawford et al., 2012, Kon et al., 2012, Nurmukhametov et al., 2021). Previous studies have shown that collagen scaffold architecture creates compressive instabilities that are not observed in native tissue (Kim et al., 2023, 2022). More importantly, such local compositional thresholds are shown to have a strong correlation to the probability of instabilities for the tissue-engineered cartilage constructs (Middendorf et al., 2017). In addition, studies have shown that this local mechanical strain is predictive of cell death in native and tissue-engineered cartilage constructs (Bartell et al., 2015, Kim et al., 2023). These studies collectively indicate that identifying and monitoring the local compositional parameters are crucial for determining the quality of tissue-engineered cartilage.

Recent advances in a variety of scaffold-free technologies, such as pellet and micromass culture systems, have demonstrated to be a potential biofabrication approach for producing tissue-engineered cartilage constructs (Johnstone et al., 2013, Kim et al., 2011, Mohanraj et al., 2014). Notably, osteochondral tissue engineering technology, manufactured by fusing condensed mesenchymal stem cell bodies (CMBs) to trabecular bone scaffolds, have demonstrated promising in vivo results in the treatment of focal cartilage defects (Bhumiratana et al., 2014, Stefani et al., 2020). The grafts were then cultured for an extended period for stem cells differentiation and chondrogenic maturation. During the culture, CMBs integrate with each other and generate unique architectural features as well as cartilage matrix containing collagen and aggrecan. Remarkably, engineered cartilage tissue from CMBs have comparable bulk mechanical properties to that of native cartilage (Bhumiratana and Vunjak-Novakovic, 2015). Notably, the architecture of tissue grown from combining CMBs is distinct from native and tissue-engineered cartilage with collagen scaffold. Investigating the relationships among the architectural features, local composition, and the micromechanical environment within tissue-engineered cartilage from CMBs, is critical for identifying critical parameters that are predictive of in vivo performance.

As such, the objective of this study was to investigate the relationship among the architecture, local composition, and local micromechanical environment in tissue-engineered osteochondral grafts under unconfined compression testing. We obtained osteochondral grafts manufactured by fusing CMBs with trabecular bone scaffolds and investigated the relationship among architectural features, local composition, and local mechanical behavior. We (i) compared the architectural and compositional differences between the two different culture techniques, (ii) identified the global and local strain distribution, and (iii) found the correlation between the local composition and micromechanical behavior.