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for tendon organoids. Appropriate mechanical stimuli organoids and drive the field toward greater refinement
can induce cells to differentiate into tenocyte phenotypes, (Figure 2).
which is crucial for the maturation and functionalization of
tendon organoids. 4.4.1. Optimizing materials for omics and gene editing
technologies
Future organoid platforms should aim to model
both fibrotic and healthy tendon microenvironments, The optimization of cell sources, scaffolds, and biochemical
enabling interventions to disrupt degenerative cycles factors is critical for enhancing the functionality and
while promoting physiological collagen remodeling. This biomimicry of organoids. Single-cell RNA sequencing
approach allows stage-specific customization of therapies (RNA-seq) technology can resolve the heterogeneity of
tailored to the progression of tendon degeneration. To stem cell populations, 32,33 identifying the most suitable
fibrosis-specific organoids, although resembling native stem cell subpopulations for tendon differentiation as seed
fibril morphology, their disordered collagen cross-linking cells for tendon organoid construction. Single-cell analysis
results in compromised mechanical properties. Such can characterize the gene expression profiles of individual
models can evaluate fibrosis-associated molecular events cells isolated from tendon tissue, revealing subpopulations
33
+
or test therapeutic strategies. By comparing responses of nestin TSPCs with strong tenogenic potential, which
between healthy and fibrotic organoids—such as collagen can be utilized for tendon organoid development. Studies
reorganization efficiency under mechanical loading— using RNA-seq have demonstrated that changes in the
critical thresholds in pathological progression can be microenvironment during 3D culture of human TSPCs
identified, offering precise targets for clinically reversing affect the proportions of different cell subpopulations,
66
fibrosis. By optimizing the mechanical environment, the thereby regulating stem cell function. Analyzing epigenetic
physical properties of the ECM, the selection of scaffold modifications in stem cells, such as DNA methylation
materials, and the application of biomechanical stimuli, it and histone modifications, can reveal key epigenetic
is possible to replicate the physiological and pathological mechanisms governing tendon differentiation, optimizing
processes of tendons, promoting the formation and the directed differentiation of stem cells. By examining
functionalization of tendon organoids. However, the gene expression profiles under different biochemical
widespread implementation of these approaches faces treatments, the most effective combinations of small
significant challenges. A major limitation lies in the lack molecules for promoting tendon differentiation can be
of standardized engineering platforms tailored for tendon identified. Similarly, analyzing protein expression profiles
organoid culture, which require seamless integration on scaffold surfaces can help select materials that enhance
of scaffold customization and dynamic loading of cell adhesion, proliferation, and differentiation. 145,146 For
biomechanics. Current systems often depend on bulky example, proteomic studies have demonstrated that lower
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bioreactors or 2D stretching devices that fail to recapitulate material stiffness promotes tenogenic differentiation in
the 3D multiaxial mechanical cues (e.g., tension, shear, stem cells, providing evidence for optimizing the design
146
and compression) experienced by tendons in vivo. 141-143 of bioactive scaffolds in tendon tissue engineering. The
Moreover, scalable and user-friendly technologies for application of novel genome editing technologies and
applying spatially resolved mechanical stimuli within high-throughput screening methods further supports the
high-throughput organoid arrays are still underdeveloped, optimization of stem cell culture conditions.
hindering systematic exploration of mechanobiological Organ-on-a-chip technology is a biomimetic
responses. Future research should further explore the platform based on microfluidics that can simulate the
144
synergistic mechanisms of these physical factors while microenvironment of human tissues, 61,62 offering a novel tool
addressing these engineering challenges, providing more for the construction and investigation of tendon organoids.
comprehensive theoretical and technical support for the By integrating cells, scaffold materials, and mechanical stimuli
construction of tendon organoids. into a microchip, this technology precisely regulates the
microenvironment of tendon organoids, enabling dynamic
4.4. Engineering strategies research on their development, function, and pathological
As outlined above, the construction of tendon organoids processes. Leveraging microfluidic technology, this platform
involves the multidimensional synergy of cells, materials, facilitates accurate drug delivery and mimics in vivo fluid
mechanical environments, and biochemical signals. transport, while also applying dynamic stretching or
Advanced engineering technologies enable precise shear forces to replicate the mechanical environment
design and optimization from the molecular level to the of tendons within the body. This approach accurately
macroscopic scale. Through interdisciplinary collaboration simulates the biochemical and physical conditions of actual
and technological innovation, engineering strategies organs, supporting high-throughput screening and drug
provide the technical foundation for constructing tendon testing. 147-149 In addition, organ-on-a-chip technology can

Volume 1 Issue 3 (2025) 11 doi: 10.36922/OR025170016