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The importance of skeletal muscle tissue is undoubted being the controller of several vital functions including respiration and all voluntary locomotion activities. However, its regenerative capability is limited and significant tissue loss often leads to a chronic pathologic condition known as volumetric muscle loss. Here, we propose a biofabrication approach to rapidly restore skeletal muscle mass, 3D histoarchitecture, and functionality. By recapitulating muscle anisotropic organization at the microscale level, we demonstrate to efficiently guide cell differentiation and myobundle formation both in vitro and in vivo . Of note, upon implantation, the biofabricated myo‐substitutes support the formation of new blood vessels and neuromuscular junctions—pivotal aspects for cell survival and muscle contractile functionalities—together with an advanced muscle mass and force recovery. Altogether, these data represent a solid base for further testing the myo‐substitutes in large animal size and a promising platform to be eventually translated into clinical scenarios. Synopsis The regenerative capability of skeletal muscle tissue is limited and significant tissue loss often leads to a chronic pathologic condition known as volumetric muscle loss. By exploiting the potentials of our biofabrication approach, one can manufacture advanced cell‐laden myo‐substitutes that ultimately may restore the functionalities of severely damaged skeletal muscles in vivo . Biofabricated myo‐substitutes may represent a valid candidate for volumetric muscle loss treatment. Upon implantation, biofabricated myo‐substitutes support the formation of new blood vessels and neuromuscular junctions together with an advanced muscle mass and force recovery. The employed biofabrication approach is compatible with human primary stem cells ‐ namely pericytes. Graphical Abstract The regenerative capability of skeletal muscle tissue is limited and significant tissue loss often leads to a chronic pathologic condition known as volumetric muscle loss. By exploiting the potentials of our biofabrication approach, one can manufacture advanced cell‐laden myo‐substitutes that ultimately may restore the functionalities of severely damaged skeletal muscles in vivo .

Skeletal stem and progenitor cell populations are crucial for bone physiology. Characterization of these cell types remains restricted to heterogenous bulk populations with limited information on whether they are unique or overlap with previously characterized cell types. Here we show, through comprehensive functional and single-cell transcriptomic analyses, that postnatal long bones of mice contain at least two types of bone progenitors with bona fide skeletal stem cell (SSC) characteristics. An early osteochondral SSC (ocSSC) facilitates long bone growth and repair, while a second type, a perivascular SSC (pvSSC), co-emerges with long bone marrow and contributes to shape the hematopoietic stem cell niche and regenerative demand. We establish that pvSSCs, but not ocSSCs, are the origin of bone marrow adipose tissue. Lastly, we also provide insight into residual SSC heterogeneity as well as potential crosstalk between the two spatially distinct cell populations. These findings comprehensively address previously unappreciated shortcomings of SSC research.

Bone marrow mesenchymal stromal cells (BMSCs), identified as pericytes comprising the hematopoietic niche, are a group of heterogeneous cells composed of multipotent stem cells, including osteochondral and adipocyte progenitors. Nevertheless, the identification and classification are still controversial, which limits their application. In recent years, by lineage tracing and single-cell sequencing, several new subgroups of BMSCs and their roles in normal physiological and pathological conditions have been clarified. Key regulators and mechanisms controlling the fate of BMSCs are being revealed. Cross-talk among subgroups of bone marrow mesenchymal cells has been demonstrated. In this review, we focus on recent advances in the identification and classification of BMSCs, which provides important implications for clinical applications.

Bone formation starts near the end of the embryonic stage of development and continues throughout life during bone modeling and growth, remodeling, and when needed, regeneration. Bone-forming cells, traditionally termed osteoblasts, produce, assemble, and control the mineralization of the type I collagen-enriched bone matrix while participating in the regulation of other cell processes, such as osteoclastogenesis, and metabolic activities, such as phosphate homeostasis. Osteoblasts are generated by different cohorts of skeletal stem cells that arise from different embryonic specifications, which operate in the pre-natal and/or adult skeleton under the control of multiple regulators. In this review, we briefly define the cellular identity and function of osteoblasts and discuss the main populations of osteoprogenitor cells identified to date. We also provide examples of long-known and recently recognized regulatory pathways and mechanisms involved in the specification of the osteogenic lineage, as assessed by studies on mice models and human genetic skeletal diseases.

Aging is associated with impaired tissue regeneration. Stem cell number and function have been identified as potential culprits. We first demonstrate a direct correlation between stem cell number and time to bone fracture union in a human patient cohort. We then devised an animal model recapitulating this age-associated decline in bone healing and identified increased cellular senescence caused by a systemic and local proinflammatory environment as the major contributor to the decline in skeletal stem/progenitor cell (SSPC) number and function. Decoupling age-associated systemic inflammation from chronological aging by using transgenic Nfkb1KO mice, we determined that the elevated inflammatory environment, and not chronological age, was responsible for the decrease in SSPC number and function. By using a pharmacological approach inhibiting NF-κB activation, we demonstrate a functional rejuvenation of aged SSPCs with decreased senescence, increased SSPC number, and increased osteogenic function. Unbiased, whole-genome RNA sequencing confirmed the reversal of the aging phenotype. Finally, in an ectopic model of bone healing, we demonstrate a functional restoration of regenerative potential in aged SSPCs. These data identify aging-associated inflammation as the cause of SSPC dysfunction and provide mechanistic insights into its reversal.

Chondrocytes in the resting zone of the postnatal growth plate are characterized by slow cell cycle progression, and encompass a population of parathyroid hormone-related protein (PTHrP)-expressing skeletal stem cells that contribute to the formation of columnar chondrocytes. However, how these chondrocytes are maintained in the resting zone remains undefined. We undertook a genetic pulse-chase approach to isolate slow cycling, label-retaining chondrocytes (LRCs) using a chondrocyte-specific doxycycline-controllable Tet-Off system regulating expression of histone 2B-linked GFP. Comparative RNA-seq analysis identified significant enrichment of inhibitors and activators for Wnt signaling in LRCs and non-LRCs, respectively. Activation of Wnt/β-catenin signaling in PTHrP + resting chondrocytes using Pthlh-creER and Apc -floxed allele impaired their ability to form columnar chondrocytes. Therefore, slow-cycling chondrocytes are maintained in a Wnt-inhibitory environment within the resting zone, unraveling a novel mechanism regulating maintenance and differentiation of PTHrP + skeletal stem cells of the postnatal growth plate.

Tissue-resident stem cells are essential for development and repair, and in the skeleton, this function is fulfilled by recently identified skeletal stem cells (SSCs). However, recent work has identified that SSCs are not monolithic, with long bones, craniofacial sites, and the spine being formed by distinct stem cells. Recent studies have utilized techniques such as fluorescence-activated cell sorting, lineage tracing, and single-cell sequencing to investigate the involvement of SSCs in bone development, homeostasis, and disease. These investigations have allowed researchers to map the lineage commitment trajectory of SSCs in different parts of the body and at different time points. Furthermore, recent studies have shed light on the characteristics of SSCs in both physiological and pathological conditions. This review focuses on discussing the spatiotemporal distribution of SSCs and enhancing our understanding of the diversity and plasticity of SSCs by summarizing recent discoveries.

Calvarial suture skeletal stem cells (Su-SSCs) are a distinct stem cell population for craniofacial bone formation by intramembranous ossification, compared to long bone periosteal SSCs (LB-PSSCs) with endochondral (osteochondrogenic) ossification. However, whether SSC intrinsic or extrinsic factors affect their differentiation process has not been well elucidated. Here, using an inducible Prx1-CreER-EGFP+/−;Rosa26-tdTomato mouse model, we observed that endogenous Prx1+ Su-SSCs and their orthotopic transplantation into calvarial injury do not form cartilage intermediates at the injury sites, while the transplantation of Prx1+ LB-PSSCs into LB injury induces osteochondrogenic differentiation, respectively. However, the heterotopic transplantation of Prx1+ Su-SSCs (Su-SSCs into LB injury) showed some surprising findings that the transplanted Su-SSCs acquire new chondrocyte differentiation properties at the LB injury sites, although the heterotopic-transplanted Prx1+ LB-PSSCs maintained their endochondral ossification properties at the calvarial injury sites. Further, a comparative single-cell transcriptomic analysis of LB-PSSCs and Su-SSCs revealed that Su-SSCs express a higher set of anti-chondrogenic genes, such as Wnt5b, Twist1 while LB-PSSCs highly express chondrogenic Hoxa-9, Hoxc-9, Hoxa-10, Hoxc-10, and Comp genes. We also found that the heterotopic transplantation of LB-PSSCs into calvarial injury enhances bone healing in vivo. Taken together, these findings suggest that LB-PSSCs have high regenerative capability with invariable endochondral ossification even after the heterotopic transplantation but Su-SSCs are more flexible and regulated by the local bone environment. The transplantation of periosteal SSCs will be a promising method for large craniofacial bone defects.

The periosteum is the major source of cells involved in fracture healing. We sought to characterize progenitor cells and their contribution to bone fracture healing. The periosteum is highly enriched with progenitor cells, including Sca1 + cells, fibroblast colony-forming units, and label-retaining cells compared to the endosteum and bone marrow. Using lineage tracing, we demonstrate that alpha smooth muscle actin (αSMA) identifies long-term, slow-cycling, self-renewing osteochondroprogenitors in the adult periosteum that are functionally important for bone formation during fracture healing. In addition, Col2.3CreER-labeled osteoblast cells contribute around 10% of osteoblasts but no chondrocytes in fracture calluses. Most periosteal osteochondroprogenitors following fracture can be targeted by αSMACreER. Previously identified skeletal stem cell populations were common in periosteum but contained high proportions of mature osteoblasts. We have demonstrated that the periosteum is highly enriched with skeletal progenitor cells, and there is heterogeneity in the populations of cells that contribute to mature lineages during periosteal fracture healing.

The growth plate is a specialized cartilage structure near the ends of long bones that orchestrates longitudinal bone growth during fetal and postnatal stages. Within this region reside a dynamic population of growth plate skeletal stem cells (gpSSCs), primarily located in the resting zone, which possess self-renewal and multilineage differentiation capacity. Recent advances in cell-lineage tracing, single-cell transcriptomics, and in vivo functional studies have revealed distinct subpopulations of gpSSCs, which are defined by markers such as parathyroid hormone-related protein (PTHrP), CD73, axis inhibition protein 2 (Axin2), forkhead box protein A2 (FoxA2), and apolipoprotein E (ApoE). These stem cells interact intricately with their niche, particularly after the formation of the secondary ossification center, through stage-specific regulatory mechanisms involving several key signaling pathways. This review summarizes the current understanding of gpSSC identity, behavior, and regulation, focusing on how these cells sustain growth plate function through adapting to biomechanical and molecular cues.