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Three dimensional (3D) microenvironments more accurately replicate native microenvironments for stem cell maintenance and function compared with two dimensional (2D) microenvironments. However, the molecular mechanisms by which 3D microenvironments regulate stem cell function remain largely unexplored at the single-cell level. Here, using a single-cell analysis and functional analysis, we found not all cell-subpopulations respond to 3D microenvironments based on a systematically 3D gelatin microcarrier culture system we developed for the expansion and function maintenance of hTSPCs. 3D microenvironments alter the cell-subpopulation distribution of human tendon stem/progenitor cells (hTSPCs) by improving the proportion of ICAM1+ITGB8+ and FGF7+CYGB+ subpopulations. We also revealed the activated FGF7 signaling in the two subpopulations is responsible for the enhanced tenogenesis of hTSPCs through cell-cell interactions. The hTSPCs cultured in 3D niche with a specific cell-subpopulation structure exhibited superior stem-cell characteristics and functions both in vitro and in vivo. Together, our study demonstrates that 3D microenvironments can regulate stem-cell function by modulating the critical cell subpopulation and identifies FGF7 as a novel regulator for tenogenic differentiation and tendon regeneration.

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Although being of utmost importance for human health and mobility, stem cell identity and hierarchical organization of musculoskeletal progenitors remain largely unexplored. Here, cells from E10.5, E12.5, and E15.5 murine limbs are analyzed by high throughput single-cell RNA sequencing to illustrate the cellular architecture during limb development. Single-cell transcriptional profiling demonstrates the identity and differentiation architecture of musculoskeletal stem cells (MSSC), soft and hard tissue progenitors through expression pattern of musculoskeletal markers (scleraxis [Scx], Hoxd13, Sox9, and Col1a1). This is confirmed by genetic in vivo lineage tracing. Moreover, single-cell analyses of Scx knockout mice tissues illustrates that Scx regulates MSSC self-renewal and proliferation potential. A high-throughput and low-cost multi-tissues RNA sequencing strategy further provides evidence that musculoskeletal system tissues, including muscle, bone, meniscus, and cartilage, are all abnormally developed in Scx knockout mice. These results establish the presence of an indispensable limb Scx+Hoxd13+ MSSC population and their differentiation into soft tissue progenitors (Scx+Col1a1+) and hard tissue progenitors (Scx+Sox9+). Collectively, this study paves the way for systematically decoding the complex molecular mechanisms and cellular programs of musculoskeletal tissues morphogenesis in limb development and regeneration.

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The problem of tendon and ligament (T/L) regeneration in musculoskeletal diseases has long constituted a major challenge. In situ injection of formable biodegradable hydrogels, however, has been demonstrated to treat T/L injury and reduce patient suffering in a minimally invasive manner. An injectable hydrogel is more suitable than other biological materials due to the special physiological structure of T/L. Most other materials utilized to repair T/L are cell-based, growth factor-based materials, with few material properties. In addition, the mechanical property of the gel cannot reach the normal T/L level. This review summarizes advances in natural and synthetic polymeric injectable hydrogels for tissue engineering in T/L and presents prospects for injectable and biodegradable hydrogels for its treatment. In future T/L applications, it is necessary develop an injectable hydrogel with mechanics, tissue damage-specific binding, and disease response. Simultaneously, the advantages of various biological materials must be combined in order to achieve personalized precision therapy.

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Tendon injuries are common musculoskeletal system disorders, but the tendons have poor regeneration ability. To address this issue, tendon tissue engineering provides potential strategies for future therapeutic treatment. Elements of the physical microenvironment, such as the mechanical force and surface topography, play a vital role in regulating stem cell fate, enhancing the differentiation efficiency of seed cells in tendon tissue engineering. Various inducible scaffolds have been widely explored for tendon regeneration, and scaffold-enhancing modifications have been extensively studied. In this review, we systematically summarize the effects of the physical microenvironment on stem cell differentiation and tendon regeneration; we also provide an overview of the inducible scaffolds for stem cell tenogenic differentiation. Finally, we suggest some potential scaffold-based therapies for tendon injuries, presenting an interesting perspective on tendon regenerative medicine.

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