{'fraction_non_alphanumeric': 0.038360881542699725, 'fraction_numerical': 0.019421487603305785, 'mean_word_length': 5.368859649122807, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 2, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
TGFβ1-transfected tendon stem cells promote tendon fibrosis

Background In aged people, tendon injuries frequently occur during sporting and daily activities. In clinical practice, typical physiotherapeutic, pharmacotherapeutic, and surgical techniques do not result in the full recovery of injured tendons, which may lead to chronic degenerative disease. Methods We first isolated tendon stem cells (TSCs) from rats and transfected them with the TGFβ1 gene, resulting in TGFβ1-TSCs. The proliferation of TSCs was detected using the Cell Counting Kit 8, and TSCs were identified by immunofluorescence analysis and differentiation capacity analysis. Aggrecan, COL2A1, alpha smooth muscle actin (α-SMA), and p-Smad2 expression levels were detected using western blotting and quantitative reverse transcription polymerase chain reaction. Additionally, a tendon injury model was generated to explore the effect of TGFβ1 on the repair of the tendon by TSCs. Results Compared with fibrinogen treatment, TSC + fibrinogen or TGFβ1-TSC + fibrinogen treatment significantly promoted the fibrosis of injured tendons, as evidenced by histological analyses, with TGFβ1-TSC + fibrinogen having a greater effect than TSC + fibrinogen. In TGFβ1-TSCs, increased expression levels of aggrecan and COL2A1 indicated that TGFβ1 signaling induced chondrogenic differentiation. Meanwhile, the increased collagen and α-SMA protein levels indicated that TGFβ1 promoted fibrogenesis. Additionally, TGFβ1 stimulated the production of phosphorylated Smad2 in TSCs, which suggested that the chondrogenic and fibrogenic differentiation of TSCs, as well as tissue regeneration, may be associated with the TGFβ1/Smad2 pathway. Conclusion TGFβ1-TSC therapy may be a candidate for effective tendon fibrosis.


Introduction
Aged people frequently experience tendon injuries while performing sports or even daily activities. Tendon injuries account for > 40% of musculoskeletal diseases [1,2]. In clinical practice, physiotherapy, pharmacotherapy, and surgery are the typical choices for tendon repair [3][4][5]. However, these treatments do not usually result in a fully recovered tendon, which may result in a chronic degenerative disease [6]. During recovery in adults, injured tendons do not rebuild normal tissue, but mainly produce scar tissue, which lacks tensile strength [7]. The failure to completely recover is due to the low regeneration capability of tenocytes, which is associated with the hypovascularity and low metabolism of tendon tissue [8,9]. Therefore, the development of new therapies capable of stimulating the regeneration of injured tendons has been encouraged in recent decades.
Uncommitted stem cells are endowed with a high proliferation potential; they can differentiate into all types of cells under the appropriate conditions [10]. Stem cell-based therapies have attracted increasing interest for tendon healing [11]. In contrast to adult tendons, fetal tendon tissue is capable of regeneration after Open Access impairment, and injured tendons fully regain their former performance [6]. However, embryonic stem cellbased therapies have a great risk of tumor generation, in addition to the ethical issues regarding the harvesting of cells from embryos [12]. Induced pluripotent stem cells are an alternative for tendon healing without ethical issues, but tumorigenesis cannot be avoided [13]. In recent years, mesenchymal stem cells have been shown to be safer because their self-renewal and/or differentiation potential are relatively restricted compared with those of embryonic and induced pluripotent stem cells [11]. Mesenchymal stem cells were first identified in tendon tissue in 2007 and were named tendon stem cells (TSCs) [14]. Several studies have shown that TSC-based therapies substantially promote tendon healing and tissue regeneration [15][16][17]. Stem cell-based treatment is the most promising therapeutic strategy for tendon healing [18,19].
Previous studies have shown that tendon development and healing are highly associated with growth factors, such as fibroblast growth factor, epidermal growth factor, bone morphogenetic protein, and transforming growth factor (TGF) [11,20]. Of these, TGFβ1 is one of the most attractive bioactive factors because it plays multiple roles in tendon healing [20]. Specifically, TGFβ1 is essential for tendon formation as it induces the expression of tendonspecific proteins and stimulates chondrogenic differentiation [21,22]. TGFβ1 is also involved in the production of mesenchymal stem cells, it stimulates the production of collagen types I and III, and it participates in cell migration and mitogenesis [23,24]. The application of stem cell-based therapies, in combination with growth factor supplementation, is being increasingly applied to tendon healing [11]. Nonetheless, information on the effects of TSC-based therapy in combination with TGFβ1 supplementation on tendon healing remains limited. Therefore, we isolated TSCs and overexpressed TGFβ1 in these cells to explore the effect of TGFβ1 on TSC-induced fibrogenesis and the expression of related molecules.

TSC isolation and culture
All animal experiments were approved by the animal research ethics committee of the First People's Hospital of Jiujiang City. TSCs were collected from male Sprague-Dawley rats weighing approximately 50 g. Prior to TSC collection, the rats were anesthetized by intraperitoneal injection of 0.03% pentobarbital sodium (30 mg/kg), and the tendons were separated from the paratendon, fat, and muscle tissues. The tendon samples were sectioned into 1 mm slices and digested in a 3 mg/mL collagenase solution (Sigma-Aldrich, St. Louis, MO, USA). The digested solution was filtered through a 70 μm cell strainer and centrifuged at 300 × g for 5 min to collect the tendon cells. The cells were cultured in Dulbecco's modified Eagle's medium (Gibco, Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Beyotime Biotechnology, Shanghai, China) and 1% penicillinstreptomycin antibiotic (Beyotime Biotechnology), and were sub-cultured after reaching 80% confluence.

Cell proliferation assay
Cells at passage three were seeded into 96-well plates (10 4 cells per well) and incubated with Cell Counting Kit-8 solution (Beyotime Biotechnology) according to the manufacturer's instructions. After 4 h, cell proliferation was assessed by measuring absorbance at 450 nm (Spec-traMax; Molecular Devices, San Francisco, CA, USA).

Differentiation capacity analysis
To evaluate multipotency, cells at passage three were subcultured into adipogenic, chondrogenic, or osteogenic media (Thermo Scientific, Pittsburgh, PA, USA). After 21 days of induction, the cells were fixed with 4% paraformaldehyde, washed with deionized water, and stained with alizarin red (osteogenic potential), alcian blue (chondrogenic potential), or oil red O (adipogenic potential). The stained cells were observed under a light microscope (Olympus, Tokyo, Japan).

Generation of a TSC line stably overexpressing TGFβ1
TGFβ1 cDNA was cloned and inserted into the pCDH-MCS-T2A-puro lentiviral vector (XIAMEN Anti-hela Biological Technology Trade Co. Ltd., Xiamen, China). The following primers were used: TGFβ1 forward primer, 5′-TAG AGC TAG CGA ATT CGC CAC CAT GAT GCC GCC CTC GGG GCT GCG-3′ and TGFβ1 reverse primer, 5′-CAG CGG CCG CGG ATC CGC TGC ACT TGC AGG AGC GCA C-3′. The TGFβ1 expression plasmid (pCDH-TGFβ1) or the pCDH-MCS-T2A-puro lentiviral vector (negative control) was transfected into HEK293T cells together with psPAX2 (a packaging plasmid) and pMD2.G (an envelope plasmid) using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). Cell supernatants containing lentivirus were harvested at 48 h posttransfection. Subsequently, the lentivirus was purified and concentrated and the median tissue culture infectious dose (TCID 50 ) of the lentivirus was determined, as previously described [25]. For stable transfection, TSCs were incubated with 10 8 TCID 50 /mL lentiviral particles to which 8 mg/mL polybrene had been added. At 3 days post-infection, the cells were treated with 1 mg/ mL puromycin for 2 weeks. TGFβ1 overexpression was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the resulting cell line was named TGFβ1-TSCs.

Total RNA extraction and first-strand cDNA synthesis
Total RNA was isolated from TSCs using TRIzol reagent (Takara, Dalian, China), following the manufacturer's instructions. RNA quality and quantity were determined using 1% agarose gel electrophoresis and spectrometry (Nanodrop 2000, Thermo Scientific), respectively. RNA samples were treated with DNase I and reverse transcribed with random primers, a dNTP mix, and M-MLV reverse transcriptase (Takara) to synthesize first-strand cDNA.

Quantitative PCR
qPCR was used to determine the expression levels of aggrecan, COL2A1, TGFβ1, and RNA18S5N (internal control). Reactions were performed on a QuantStudio 7 Flex (Thermo Scientific) using a SYBR Green PCR premix (Takara). The qPCR program was set as follows: a preheating step at 95 °C for 60 s; followed by 40 cycles

Surgical procedure and treatment
Adult male Sprague-Dawley rats weighing approximately 200 g were anesthetized by intraperitoneal injection of 0.03% pentobarbital sodium (30 mg/kg), and one-third of the patellar tendon was removed to mimic tendon injury following a well-established protocol [16,26]. Fibrinogen, TSCs and fibrinogen (TSCs + fibrinogen), or TGFβ1-TSCs and fibrinogen (TGFβ1-TSCs + fibrinogen), in combination with thrombin, were injected into the defect area. Four weeks after surgery, the rats were killed for histological examination.

Histological examination
Tendon samples were fixed with 4% paraformaldehyde for 24-48 h. The fixed samples were dehydrated, embedded in paraffin, and sectioned. The sections were stained with hematoxylin and eosin (H&E) and observed under a light microscope (BX51, Olympus).

Statistical analysis
Statistical analyses were performed using Graph Pad Prism 5.0 (San Diego, CA, USA). An independent Student's t-test was used for comparisons between groups, and data normality was verified using the Shapiro-Wilk test. Statistical significance was set a p < 0.05.

TSC identification
Cells obtained from rat tendons were large, flat, and fibroblastic (Fig. 1A) and cell proliferation increased from 0 to 72 h of culture (Fig. 1B). Subsequently, we detected the surface markers on the TSCs. The isolated cells were negative for the hematopoietic stem cell-like marker, CD34 (0.40%). However, they were positive for the mesenchymal stem cell-like markers, CD73 (99.51%) and CD90 (99.77%, Fig. 2A). In addition, the isolated cells were positive for collagen type I expression, indicating that they were derived from tendons (Fig. 2B). To confirm the identity of the isolated cells as TSCs, we performed differentiation experiments. Osteogenic differentiation was confirmed by the presence of calcium deposits and alkaline phosphatase activity using alizarin red staining. Chondrogenic and adipogenic differentiation were determined by the presence of sulfated glycosaminoglycan using alcian blue staining and the presence of oil droplets using oil red O staining (Fig. 3). These results showed

TGFβ1 overexpression in TSCs increases the expression of chondrogenic and fibrogenic markers
After the transfection of pCDH-TGFβ1, the expression of TGFβ1 was successfully detected in TSCs (Fig. 4A).

TGFβ1 overexpression enhances TSC-mediated tendon fibrosis
H&E staining showed that rupture and macrophage infiltration were still observed in tendons treated with  fibrinogen (Fig. 5). Macrophage infiltration was observed in TSCs + fibrinogen-treated tendons. In contrast, the tendons treated with TGFβ1-TSCs + fibrinogen were continuous, without macrophage infiltration. These findings suggest that TGFβ1 enhanced the tendon-fibrosis ability of TSCs.

Discussion
Tendon recovery was promoted by treatment with TSCs + fibrinogen or TGFβ1-TSCs + fibrinogen, both of which had a greater effect than treatment with fibrinogen alone. The observed improvement in tendon recovery confirmed that TSC-based therapies substantially promote tissue regeneration after injury, which is in agreement with previous findings [15][16][17]. Moreover, fibrosis was significantly higher in the TGFβ1-TSCs + fibrinogen group than in the TSCs + fibrinogen group. Our findings are consistent with those of a previous study of TSC-based therapies supplemented with exosomes or extracellular matrix containing TGFβ1 [27,28]. These findings suggest that TGFβ1-TSC therapy may be a candidate for effective tendon healing. Furthermore, during the process of tissue injury healing, an appropriate amount of TGFβ1 promotes healing, whereas excessive TGFβ1 leads to scar hyperplasia and adverse effects on healing [29]. The amount of TGFβ1 produced by TGFβ1-TSCs was not determined, which is a limitation of this study. TGF-β1 signaling is regulated at multiple levels to avoid detrimental outcomes for cells [29]; thus, it is understandable that TGFβ1-TSCs promoted tendon repair, and this result indicates that the amount of TGFβ1 produced by TGFβ1-TSCs was appropriate.
Compared with TSCs, TGFβ1-TSCs showed higher expression levels of aggrecan and COL2A1. The increased expression levels of these two chondrogenic markers indicated that TGFβ1 signaling promoted the differentiation of TSCs into the chondrogenic lineage [30]. Similarly, the protein levels of collagen type II and α-SMA were also increased in TSCs upon TGFβ1 overexpression, which indicated that TGFβ1 promoted fibrogenesis in TSCs [31]. The increased protein levels of collagen type II and α-SMA suggested the increased production of extracellular matrix components that are dedicated to tendon structure formation [32]. Additionally, p-Smad2 levels were increased by TGFβ1 in TSCs, indicating that chondrogenesis and fibrogenesis in TSCs were induced via the TGFβ1/Smad2 pathway [33,34]. These findings suggest that the TGFβ1/Smad2 pathway induced chondrogenic and fibrogenic differentiation of TSCs and promoted tissue regeneration and tendon healing.

Conclusions
This study describes the molecular mechanism whereby TGFβ1 enhances the reparative effect of TSCs on tendons in vivo. Our results showed that TGFβ1 increased the levels of aggrecan, COL2A1, α-SMA, and p-Smad2 in TSCs. Furthermore, we demonstrated that TGFβ1-TSCs have a positive effect on the fibrosis of damaged tendons.