Inhibition of PI3K/AKT signaling pathway prevents blood-induced heterotopic ossification of the injured tendon.

Objective: It is a common clinical phenomenon that blood infiltrates into the injured tendon caused by sports injuries, accidental injuries, and surgery. However, the role of blood infiltration into the injured tendon has not been investigated. Methods: A blood-induced rat model was established and the impact of blood infiltration on inflammation and HO of the injured tendon was assessed. Cell adhesion, viability, apoptosis, and gene expression were measured to evaluate the effect of blood treatment on tendon stem/progenitor cells (TSPCs). Then RNA-seq was used to assess transcriptomic changes in tendons in a blood infiltration environment. At last, the small molecule drug PI3K inhibitor LY294002 was used for in vivo and in vitro HO treatment. Results: Blood caused acute inflammation in the short term and more severe HO in the long term. Then we found that blood treatment increased cell apoptosis and decreased cell adhesion and tenonic gene expression of TSPCs. Furthermore, blood treatment promoted osteochondrogenic differentiation of TSPCs. Next, we used RNA-seq to find that the PI3K/AKT signaling pathway was activated in blood-treated tendon tissues. By inhibiting PI3K with a small molecule drug LY294002, the expression of osteochondrogenic genes was markedly downregulated while the expression of tenonic genes was significantly upregulated. At last, we also found that LY294002 treatment significantly reduced the tendon HO in the rat blood-induced model. Conclusion: Our findings indicate that the upregulated PI3K/AKT signaling pathway is implicated in the aggravation of tendon HO. Therefore, inhibitors targeting the PI3K/AKT pathway would be a promising approach to treat blood-induced tendon HO.

A Cd9+Cd271+ stem/progenitor population and the SHP2 pathway contribute to neonatal-to-adult switching that regulates tendon maturation.

Tendon maturation lays the foundation for postnatal tendon development, its proper mechanical function, and regeneration, but the critical cell populations and the entangled mechanisms remain poorly understood. Here, by integrating the structural, mechanical, and molecular properties, we show that post-natal days 7-14 are the crucial transitional stage for mouse tendon maturation. We decode the cellular and molecular regulatory networks at the single-cell level. We find that a nerve growth factor (NGF)-secreting Cd9+Cd271+ tendon stem/progenitor cell population mainly prompts conversion from neonate to adult tendon. Through single-cell gene regulatory network analysis, in vitro inhibitor identification, and in vivo tendon-specific Shp2 deletion, we find that SHP2 signaling is a regulator for tendon maturation. Our research comprehensively reveals the dynamic cell population transition during tendon maturation, implementing insights into the critical roles of the maturation-related stem cell population and SHP2 signaling pathway during tendon differentiation and regeneration.

Niloticin inhibits osteoclastogenesis by blocking RANKL-RANK interaction and suppressing the AKT, MAPK, and NF-κB signaling pathways.

Dysregulation of osteoclasts or excessive osteoclastogenesis significantly -contributes to the occurrence and development of osteolytic diseases, including osteoporosis, inflammatory bone erosion, and tumor-induced osteolysis. The protein-protein interaction between the receptor activator of nuclear factor (NF)-κB (RANK) and its ligand (RANKL) mediates the differentiation and activation of osteoclasts, making it a key therapeutic target for osteoclastogenesis inhibition. However, very few natural compounds exerting anti-osteoclastogenesis activity by inhibiting the RANKL-RANK interaction have been found. Niloticin is a natural tetracyclic triterpenoid compound with anti-viral, antioxidative, and mosquitocidal activities. However, its role in osteoclastogenesis remains unknown. The present study found that niloticin directly binds to RANK with an equilibrium dissociation constant of 5.8 µM, blocking RANKL-RANK interaction, thereby inhibiting RANKL-induced AKT, MAPK (p38, JNK, and ERK1/2), and NF-κB (IKKα/ß, IκBα, and p65) pathways activation, and reducing the expression of key osteoclast differentiation-related regulatory factors (NFATc1, c-Fos, TRAP, c-Src, ß3-Integrin, and cathepsin K) in osteoclast precursors, ultimately negatively regulating osteoclastogenesis. These findings suggest that niloticin could serve as a novel osteoclastogenesis inhibitor and might have beneficial effects on bone health.

Single cell analysis reveals inhibition of angiogenesis attenuates the progression of heterotopic ossification in Mkx-/- mice.

Tendon heterotopic ossification (HO) is characterized by bone formation inside tendon tissue, which severely debilitates people in their daily life. Current therapies fail to promote functional tissue repair largely due to our limited understanding of HO pathogenesis. Here, we investigate the pathological mechanism and propose a potential treatment method for HO. Immunofluorescence assays showed that the Mohawk (MKX) expression level was decreased in human tendon HO tissue, coinciding with spontaneous HO and the upregulated expression of osteochondrogenic and angiogenic genes in the tendons of Mkx-/- mice. Single-cell RNA sequencing analyses of wild-type and Mkx-/- tendons identified three cell types and revealed the excessive activation of osteochondrogenic genes during the tenogenesis of Mkx-/- tendon cells. Single-cell analysis revealed that the gene expression program of angiogenesis, which is strongly associated with bone formation, was activated in all cell types during HO. Moreover, inhibition of angiogenesis by the small-molecule inhibitor BIBF1120 attenuated bone formation and angiogenesis in the Achilles tendons of both Mkx mutant mice and a rat traumatic model of HO. These findings provide new insights into the cellular mechanisms of tendon HO and highlight the inhibition of angiogenesis with BIBF1120 as a potential treatment strategy for HO.

Extracellular Matrix Remodeling in Stem Cell Culture: A Potential Target for Regulating Stem Cell Function.

Stem cells (SCs) hold great potential for regenerative medicine, tissue engineering, and cell therapy. The clinical applications of SCs require both high quality and quantity of transplantable cells. However, during conventional in vitro expansion, SCs tend to lose properties that make them amenable for cell therapies. Extracellular matrix (ECM) serves an essential regulatory part in the growth, differentiation, and homeostasis of all cells in vivo, and when signals are transmitted to cells, they do not respond passively. Many cell types can remodel pericellular matrix to meet their specific needs. This reciprocal cell-ECM interaction is crucial for the conservation of cell and tissue functions and homeostasis. In vitro ECM remodeling also plays a key role in regulating the lineage fate of SCs. A deeper understanding of in vitro ECM remodeling may provide new perspectives for the maintenance of SC function. In this review, we critically examined three ways that cells can be used to influence the pericellular matrix: (1) exerting tensile force on the ECM, (2) secreting a variety of ECM proteins, and (3) degrading the surrounding matrix, and its impact on SC lineage fate. Finally, we describe the deficiencies of current studies and what needs to be done next to further understand the role of ECM remodeling in ex vivo SC cultures. Impact statement The effect of extracellular matrix (ECM) remodeling on physiological activities and disease progression has been extensively studied, but its effect on in vitro stem cell (SC) culture has received insufficient attention. More and more research has shown that in vitro ECM remodeling is critical for maintaining SCs function. This review will highlight how cells remodel ECM and the significance of ECM remodeling in ex vivo SC culture, as well as summarize the shortcomings of current research and what needs to be done next to further our understanding of the role of ECM remodeling in ex vivo SC culture.

Single-cell RNA-seq reveals functionally distinct biomaterial degradation-related macrophage populations.

Macrophages play crucial roles in host tissue reaction to biomaterials upon implantation in vivo. However, the complexity of biomaterial degradation-related macrophage subpopulations that accumulate around the implanted biomaterials in situ is not fully understood. Here, using single cell RNA-seq, we analyze the transcriptome profiles of the various cell types around the scaffold to map the scaffold-induced reaction, in an unbiased approach. This enables mapping of all biomaterial degradation-associated cells at high resolution, revealing distinct subpopulations of tissue-resident macrophages as the major cellular sources of biomaterial degradation in situ. We also find that scaffold architecture can affect the mechanotransduction and catabolic activity of specific material degradation-related macrophage subpopulations in an Itgav-Mapk1-Stat3 dependent manner, eventually leading to differences in scaffold degradation rate in vivo. Our work dissects unanticipated aspects of the cellular and molecular basis of biomaterial degradation at the single-cell level, and provides a conceptual framework for developing functional tissue engineering scaffolds in future.

3D printing of chemical-empowered tendon stem/progenitor cells for functional tissue repair.

Tendon injuries are the leading cause of chronic debilitation to patients. Tendon stem/progenitor cells (TSPCs) are potential seed cells for tendon tissue engineering and regeneration, but TSPCs are prone to lose their distinct phenotype in vitro and specific differentiation into the tenocyte lineage is challenging. Utilizing small molecules in an ex vivo culture system may be a promising solution and can significantly improve the therapeutic applications of these cells. Here, by using an image-based, high-throughput screening platform on small molecule libraries, this study established an effective stepwise culture strategy for TSPCs application. The study formulated a cocktail of small molecules which effected proliferation, tenogenesis initiation and maturation phases, and significantly upregulated expression of various tendon-related genes and proteins in TSPCs, which were demonstrated by high-throughput PCR, ScxGFP reporter assay and immunocytochemistry. Furthermore, by combining small molecule-based culture system with 3D printing technology, we embedded living, chemical-empowered TSPCs within a biocompatible hydrogel to engineer tendon grafts, and verified their enhanced ability in promoting functional tendon repair and regeneration both in vivo and in situ. The stepwise culture system for TSPCs and construction of engineered tendon grafts can not only serve as a platform for further studies of underlying molecular mechanisms of tenogenic differentiation, but also provide a new strategy for tissue engineering and development of novel therapeutics for clinical applications.