MSCs can be isolated from virtually every tissue in the body, with the most common source being adipose, followed by bone marrow, placenta, etc (Si, 2019). The extracted adipose tissue was finely minced and subsequently digested with Type I Collagenase at room temperature with permanent shaking for a few hours before isolating the primary MSC (Melzer et al., 2021). Primary bone marrow-derived MSCs were isolated using the whole bone marrow adherent method and non-adherent cells were subsequently removed after 24 h (Wei et al., 2023). MSCs highly express CD29, CD73, CD90, and CD105 but lack expression of the surface molecules CD45, CD34, CD14, CD11b, CD79a, CD19, and HLA-DR (Si, 2019; Wu, 2023; Goldberg et al., 2024). In vivo, the safety of autologous MSCs injections for treating AT has been well-documented; however, the underlying mechanisms of action remain not fully understood (Goldberg et al., 2024).

In vitro, MSCs are often used as comparison samples for characterisation of tendon stem cells and can express a diverse array of tenogenic differentiation markers, including scleraxis (SCX), tenomodulin (TNMD), collagen I, II, and III (Col I,II, and III), decolin (DCN) and biglycan (Bgn), but at lower levels than TDSCs (Costa-Almeida et al., 2019). The application of MSC therapy to treat AT is now widely used and validated. Preclinical study shows significant results of in situ implantation of autologous bone marrow mesenchymal stem cells for treatment of AT (Smith et al., 2013). In comparing various sources of MSCs, Myrto Bami et al. found that synovial-derived tenomodulin had the highest level of expression, had a high proliferative capacity, and showed superiority in tendon generation (Bami et al., 2020). In addition, MSCs are highly sensitive to their environment, including adaptation to the extracellular environment. In the Achilles tendinopathy microenvironment, this adaptation can increase cellular potency (Wang et al., 2020; Melzer et al., 2021).

In 2007, Bi et al. first identified and isolated TDSCs from human and mouse tendon tissues (Bi et al., 2007). TDSCs were derived from biopsies of the Achilles tendon tissue (Yin et al., 2020). The Achilles tendon was removed from the peritendinous tissue, cut up and digested with Type I Collagenase for several hours. Then, TDSCs were induced by resuspending the cell suspension obtained after filtration in a specialised medium for tendon stem cells (Mao et al., 2022). TDSCs stem cells have typical features of MSCs, such as typical surface antigens, self-renewal, clonality and the potential for differentiation into adipogenic, osteogenic, and chondrogenic lineages (Schneider et al., 2018; Guo et al., 2023). TDSCs expressed CD44, CD90, CD90.1, CD105, CD146, Stro-1, Oct-4, nucleostemin, and SSEA-1, but not CD31, CD45, CD34, CD18, CD106, CD117, CD144, and Flk-1 (Schneider et al., 2018). Our previous studies have cultured TDSC with uniform long spindle cell morphology (Mao et al., 2022; Mao et al., 2024; Zhang et al., 2024). We have verified that these cells expressed CD29 (100%), CD44 (98.8%) and CD90 (89.2%) and CD31 and CD34 were negative. In addition, tenogenic differentiation markers such as SCX, TNMD, COL Ⅰ and COL Ⅲ were upregulated during tenogenic differentiation (Mao et al., 2022). TDSCs are the ideal cell type for application in AT. It is homologous to tenocytes and expresses high levels of SCX, TNMD and tenascin-C (TNC) than MSCs during differentiation (Lu et al., 2023a). It has the potential to spontaneously differentiate into tendon cells including tendonocytes and ECM formation improve the cellular matrix, influence angiogenesis and collagen synthesis, as well as regulate the metabolism of the Achilles tendon microenvironment.

iPSCs can be derived through the chemical reprogramming of human somatic cells, such as fibroblasts. The earliest and most classical iPSCs were generated by using four transcription factors (OCT4, SOX2, KLF4 and c-Myc) (Takahashi et al., 2007). Owing to the intricate processes involved, such as transcription and translation, controlling transcription factors once they are delivered into cells presents a significant challenge. In contrast, strategies in which non-genetic and exogenous stimuli such as small molecules induce iPSCs regulation have been welcomed. Small molecules, typically less than 900 Da in molecular weight, are capable of permeating cell membranes. These molecules can directly interact with intracellular and cell-surface proteins, which are crucial for cell signaling pathways and epigenetic modifications, including changes to chromatin, histones, and DNA (Wang J. et al., 2023; Liuyang et al., 2023 optimised the original protocol using VTP50469, AKT kinase inhibitor, CX4945, and 5-iodotubercidin to rapidly and efficiently induce the generation of iPSCs (Liuyang et al., 2023). In repairing Achilles tendon, iPSCs are usually induced into iPSC-MSCs first, and then cytokines and Achilles tendon-related markers are added to match the microenvironment for tenogenic differentiation. Can Zhang et al. induced the generation of iPSCs from human foreskin fibroblasts. Then by changing the medium composition (including DMEM-low glucose, Glutamine, NEAA, human recombinant bFGF, ascorbic acid and 10% FBS), hiPSCs were differentiated towards MSCs. Ultimately, hiPSC-MSCs were cultured on scaffolds mimicking the structure of tendon ECM for further tenogenic differentiation (Zhang C. et al., 2015a). In addition, it was demonstrated that human iPSCs can directed differentiation of tenocytes by modeling human somite development in vitro (Nakajima et al., 2018; Nakajima et al., 2021). This process is characterized by the general expression of SCX, Mohawk (MKX), COL Ⅰ and POSTN (Nakajima et al., 2021).

DNA methylation at the fifth position of cytosine (5 mC), as a stable epigenetic mark, acts mainly as a transcriptional repressor (Kim and Costello, 2017; Thankam et al., 2019). DNA methylation is catalyzed by dNA methyl transferases (dNMTs), which removes methyl groups (-CH3) from cytosine residues on S-adenosyl methionine (SAM), particularly on CpG dinucleotide sequences, resulting in 5-methylcytosine residues (Figure 3) (Thankam et al., 2019). Approximately 70% of gene promoter sites are associated with CpG islands (CGIs) responsible for transcriptional initiation (Walewska et al., 2023).

Inflammatory responses can affect Achilles tendon healing by inducing epigenetic changes. Hypermethylation is the main methylation modification. cAMP Response Element-Binding Protein 1 (CREB1) upregulation induces TNF-α and IL-6-related pro-inflammatory responses. This involved 67 genes expressing hypomethylation/upregulation that were significantly enriched in multiple pathways, including gap junction formation, melanogenesis and PI3K-Akt signalling (Lee et al., 2022). Inflammatory cytokines inhibit the expression of genes and proteins, including SCX and TNMD, thereby impairing tenogenic differentiation of stem cells and affecting the quality of tendon healing (Dai et al., 2019). Five other genes (Leprel2, Foxf1, Mmp25, Igfbp6, Peg12) have also been identified as hypomethylated with strong correlation to multipotent mesenchymal cells, fibroblasts, tendon, and/or extracellular matrix (Trella et al., 2017). Among them, Leprel2, Foxf1, and Mmp25 exhibited functions closely related to collagen fibers. Igfbp6 is thought to play an important role in immunity and inflammation, including the regulation of proliferation, apoptosis, angiogenesis, cell migration and fibrosis progression (Liso et al., 2022). In addition, the PI3K/Akt signalling pathway is associated with maintaining the pluripotent state of stem cells and sustaining self-renewal capacity (Bechard et al., 2012). DNA methylation has also been demonstrated to inhibit the spontaneous differentiation of MSCs in culture, whereas removal of the methyl group resulted in a shift towards osteoblastic and adipogenic Differentiation (Webb et al., 2016).

Histones are the primary protein constituents of chromatin, playing a crucial role in condensing DNA into compact structures known as nucleosomes. Each nucleosome is formed by an octamer, comprising two molecules each of the four histone proteins (H2A, H2B, H3, and H4), which are wrapped with roughly 147 base pairs of DNA (Riasat et al., 2020). Amino acid residues at the N-terminus of histones are susceptible to post-transcriptional modification. To date, four mechanisms in histone modification have been best described and functionally characterised, namely, acetylation, methylation, ubiquitination and phosphorylation (Figure 3). Histone modifications are important for transcriptional regulation, and combinations of histone modifications in promoter regions can determine the epigenetic state of a cell, thereby promoting gene activation or repression (Thankam et al., 2019).

Over-activation of the NF-κB pathway leads to a low-grade inflammatory infiltrate, which can cause TDSCS senescence or aggravated cellular hypoplasia, whereas lowering the levels of H3K9me3 and H3K27me3 could reverse TDSCS aging by inhibiting the over-activation of NF-κB (Jin et al., 2023). Under normoxic conditions and/or in the absence of mechanical stimulation, amplification of TDSCs was accompanied by an overall decrease in histone methylation, a decrease in SCX nuclear binding, and spontaneous differentiation, with a gradual decrease in the stemness of TDSCs. Expression of “stemness” genes such as Oct4 and Sox2 is significantly reduced in successive generations of MSCs. Corresponding to these changes are epigenetic dysregulations, particularly histone H3 acetylation, which is closely associated with MSC senescence and spontaneous osteogenic differentiation (Li et al., 2011). Can Zhang et al. found that elevated levels of histone acetylation in the TGF-β signalling pathway (Tgfb1 and Tgfb2 genes) contribute to the maintenance of phenotypic properties of TDSCs (Zhang et al., 2018b). The expression of MKX, Eya1, HoxA11 and DCN was upregulated in TDSCs with elevated histone acetylation, whereas genes related to osteogenic (Runx2) and chondrogenic (Sox9) profiles were virtually unchanged (Zhang et al., 2018a).

In addition, H3K9MTase G9a may play an important role in regulating the differentiation and growth of TDSCs, and the proliferation of G9a-deficient TDSCs was significantly reduced. Furthermore, the expression levels of tendon transcription factor genes SCX, Mohawk (MKX), Egr1, Six1, and Six2), were significantly reduced. Also The tendon-related genes Col Ⅰ and TNMD periostin and periostin were also downregulated (Wada et al., 2015).

To date, approximately 2,600 mature miRNAs have been identified in humans, and they play a role in regulating over 60% of coding genes (Liu Q. et al., 2021b). MiRNAs primarily function as silencers of coding genes by binding to the 3′-untranslated region (3′-UTR) of the target mRNAs,it either inhibits the translation process or facilitates the degradation of the mRNA. This mechanism regulates gene expression in a negative manner and is believed to be inversely related to the regenerative capacity of damaged tissues (Liu et al., 2019). MiRNAs play an important role in mediating tenogenic differentiation, apoptosis or senescence in tenocytes. MiRNAs regulate cytokine networks and control the proliferation and differentiation of stromal cells that affect the composition of the extracellular matrix.

Collagen formation is an important indicator of tenogenic differentiation occurring in TDSCs. MiR-378a inhibits tenogenic differentiatio by suppressing the production of Col I and ECM (Liu et al., 2019). MiR-29a has been identified as a potent post-transcriptional regulator with the capability to target Col I and III, making it a promising candidate for facilitating the repair of Achilles tendon injuries (Watts et al., 2017). MiR-29a also enhances the tendonogenic potential of TDSCs by regulating PTEN/AKT signalling and inhibiting TDSCs lipogenic differentiation and tendon fat infiltration (Yao et al., 2021).

The oxidative stress environment in AT leads to indiscriminate upregulation of anabolic and pro-apoptotic factors, and when the balance between the two is dysregulated, it causes increased miR28 expression and elevated levels of acetylated p53, leading to impaired antioxidant defences as well as activation of the pro-apoptotic pathway (Poulsen et al., 2014). In addition, miR-217 can also inhibit tenogenic differentiation of TDSCs by decreasing EGR1 expression (Han et al., 2017).

Elevated expression of Achilles tendon-specific markers in stem cells is closely related to their differentiation into tenocytes. MiR-135a promotes differentiation of TDSCs to tenocytes by upregulating gene expression with SCX and TNMD (Chen et al., 2015). In addition, miRNAs can regulate stem cell senescence. Notably, miR-335 has been linked to senescence in human MSCs, where it inhibits chondrogenic differentiation in vivo and markedly diminishes the in vivo immunomodulatory capabilities of hMSCs (Tomé et al., 2014).

In recent years, widely transcribed lncRNAs in mammalian genomes have been found to have multiple biological functions. lncRNAs regulate a wide range of biological events by modulating gene expression, including cell differentiation (Liu et al., 2023). Inflammation in Achilles tendon injury is closely related to collagen repair. The lncRNA FOXD2-AS1/miR-21-3p/PTEN axis is involved in the regulation of AT, and inhibition of FOXD2-AS1 reduces collagen fibre breakage, increases Col I and TNMD expression, and reduces IL-1β and TNF-α by modulating the miR-21-3p/PTEN axis and promoting the activation of the PI3K/AKT signalling pathway (Ke and Zhang, 2023). After co-culture of ADSCs and injured tenocytes, the expression of lncRNA Morf4l1 was upregulated and targeted to 3′UofmiR-145-5p to promote the expression of TGF-β2, inhibit oxidative stress and increase the cell proliferation capacity as well as to promote collagen repair (Zhao H. et al., 2022a). This process may be related to the demethylation of lncRNA.

Furthermore, it has been demonstrated that several lncRNAs, including H19 and KCNQ1OT1, can control TDSC differentiation. lncRNA H19 regulates tenogenic differentiation in TDSCs by directly targeting miR-29b-3p. Overexpression of H19 significantly upregulated the mRNA levels of tenogenic formation markers and ECM markers, including SCX, MKX, Col I, Fmod, TNMD, and DCN, and genetic silencing H19 led to a significant reduction in collagen formation and a decrease in the levels of these tenogenic formation and ECM markers (Liu Y.-J. et al., 2021c). Erroneous TDSCs differentiation is implicated in the pathogenesis of calcific tendinopathy, and knockdown of the lncRNA KCNQ1OT1 protects TDSCs from lipogenic and osteogenic differentiation by down-regulating the miR-138 target genes PPARγ and RUNX2 (Yu et al., 2018).

lncRNA MALAT1 regulates tenogenic differentiation of TDSCs through the miR-378a-3p/MAPK1 axis, and the expression of Scx, MKX, and Col I was downregulated after knockdown of lncRNA MALAT1 (Zhao Z. et al., 2022b). Some lncRNAs promote osteogenic differentiation of stem cells and thus act as promoters of AT. LncRNA AC108925, lncRNA MALAT1 and lncRNA TUG1, LncRNA HIF1A-AS2 may respectively promote osteogenic differentiation of TDSCs by regulating miR-146a-3p levels, RUNX2 expression, miR-665/IL6/PI3K/Akt pathway (Yu et al., 2020; Liu et al., 2023).

Circular RNAs (circRNAs) has a covalently closed loop and high stability compared to Col I, II, and III linear analogues, and have been identified as having a regulatory role in a variety of physiological functions (Han et al., 2022; Yang et al., 2023). CircRNAs regulate gene expression predominantly at the translational and post-transcriptional levels and can fine-tune cell proliferation, senescence and differentiation by interacting with RNA-binding proteins and/or miRNAs (Han et al., 2022). circ_0005736 enhances TGF-β1-induced TDSCs tenogenic differentiation by increasing SCX, MKX, and FMO as well as ECM (Col I) production through the miR-636/MAPK1 axis to promote TDSCs proliferation, invasion, and migration (Yang et al., 2023). circPVT1 plays an important anti-aging role in TDSCs. circPVT1 knockdown significantly inhibited the migration of TDSCs and reduced the expression of TNMD, Col I, and SCX, suggesting that circPVT1 is necessary for the regulation of migration and tenogenic differentiation of TDSCs (Han et al., 2022). Upregulation of circRNA-Ep400 expression by M2 macrophages during the inflammatory phase of Achilles tendon injury and upregulation of Col I, Col III and α-SMA as well as TGF-β1 expression via miR-15b-5p/FGF1/7/9 axes in fibroblasts and tenocytes promote accelerated peritendinous fibrosis after tendon injury (Jin et al., 2021).