- Open Access
PEDF-derived peptide promotes tendon regeneration through its mitogenic effect on tendon stem/progenitor cells
Stem Cell Research & Therapyvolume 10, Article number: 2 (2019)
Tendon stem/progenitor cells (TSPC) exhibit a low proliferative response to heal tendon injury, leading to limited regeneration outcomes. Exogenous growth factors that activate TSPC proliferation have emerged as a promising approach for treatment. Here, we evaluated the pigment epithelial-derived factor (PEDF)-derived short peptide (PSP; 29-mer) for treating acute tendon injury and to determine the timing and anatomical features of CD146- and necleostemin-positive TSPC in the tendon healing process.
Tendon cells were isolated from rabbit Achilles tendons, stimulated by the 29-mer and analyzed for colony-forming capacity. The expression of the TSPC markers CD146, Oct4, and nestin, induced by the 29-mer, was examined by immunostaining and western blotting. Tendo-Achilles injury was induced in rats by full-thickness insertion of an 18-G needle and immediately treated topically with an alginate gel, loaded with 29-mer. The distribution of TSPC in the injured tendon and their proliferation were monitored using immunohistochemistry with antibodies to CD146 and nucleostemin and by BrdU labeling.
TSPC markers were enriched among the primary tendon cells when stimulated by the 29-mer. The 29-mer also induced the clonogenicity of CD146+ TSPC, implying TSPC stemness was retained during TSPC expansion in culture. Correspondingly, the expanded TSPC differentiated readily into tenocyte-like cells after removal of the 29-mer from culture. 29-mer/alginate gel treatment caused extensive expansion of CD146+ TSPC in their niche on postoperative day 2, followed by infiltration of CD146+/BrdU− TSPC into the injured tendon on day 7. The nucleostemin+ TSPC were located predominantly in the healing region of the injured tendon in the later phase (day 7) and exhibited proliferative capacity. By 3 weeks, 29-mer-treated tendons showed more organized collagen fiber regeneration and higher tensile strength than control tendons. In culture, the mitogenic effect of the 29-mer was found to be mediated by the phosphorylation of ERK2 and STAT3 in nucleostemin+ TSPC.
The anatomical analysis of TSPC populations in the wound healing process supports the hypothesis that substantial expansion of resident TSPC by exogenous growth factor is beneficial for tendon healing. The study suggests that synthetic 29-mer peptide may be an innovative therapy for acute tendon rupture.
Tendons contain dense connective tissues and mediate the transmission of muscle force to the bone, which is crucial for the control of body movement. Tendon injuries are common and often caused by overstretching the tendon. However, tendons have limited ability for self-healing following severe injury, because of their avascularity and acellularity [1, 2]. Unlike another type of connective tissue, bone marrow mesenchymal stromal cells (BM-MSCs) are difficult to mobilize into the injured sites of tendons . The repair of tendons is therefore a slow and relatively difficult process. Recently, intensive efforts have been made to use cell therapy-based approaches to accelerate tendon regeneration and repair . Adult mesenchymal stromal cells (MSCs) can be obtained to provide an adequate cellular source for tendon regeneration . However, cell transplantation is a time-consuming process. In addition, cost and technical and safety issues are obstacles to providing benefits to patients [3, 4]. Growth factors injected into and around the injured site to stimulate tendon stem/progenitor cells (TSPC) proliferation may offer an alternative option for promoting tendon repair. Connective tissue growth factor (CTGF) can promote tendon wound healing by stimulating proliferation of a TSPC population marked by CD146. The CD146+ TSPC cluster the tendon periphery, mainly near the blood vessels . Platelet-rich plasma (PRP) that harbors platelet-derived growth factor (PDGF) is another means of treating tendon injury, although the effect is limited, possibly because of a low concentration of PDGF in the preparation . It has been reported that hydrogel combinations of fibroblast growth factor (FGF)-2, insulin-like growth factor (IGF)-1, and PDGF-BB can improve the survival of adipose-derived mesenchymal stem cells (ASCs) and aid tendon healing [6, 7]. The advantage of growth factor treatment is that it is readily available for acute tendon injury, avoiding the waiting period of cell therapy. In addition, several growth factors have the ability to induce TSPC proliferation in culture. CTGF can enhance the clonogenic capacity of CD146+ TSPC . FGF-2 promotes the growth of TSPC marked by expression of scleraxis (Scx) and SRY-box containing gene 9 (Sox9) . However, the mitogenic signaling stimulating TSPC proliferation remains largely unknown.
Pigment epithelial-derived factor (PEDF) has been reported to mediate the proliferation of several stem/progenitor cell populations. PEDF is effective in stimulating the proliferation of neuronal progenitor cells and human embryonic stem cells [9, 10]. Recent studies demonstrated further that PEDF and pigment epithelial-derived factor-derived short peptide (PSP) (29-mer; residues Ser93-Thr121) can stimulate the proliferation of limbal stem cells, muscle satellite cells, and hepatic stem cells [11,12,13]. Moreover, PSP has been suggested to have a potential for treating several types of tissue injury [11,12,13]. In this study, we investigated the potential application of the 29-mer to the healing of Achilles tendon ruptures in animals. The mitogenic activity of the 29-mer on TSPC in vitro was also explored.
The PSP 29-mer (Ser93-Thr121; SLGAEQRTESIIHRALYYDLISSPDIHGT) and 34-mer (Asp44-Asn77; DPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTN) were synthesized, modified by acetylation at the NH2 termini and amidation at the COOH termini for stability, and characterized by mass spectrometry (> 90% purity) at GenScript (Piscataway, NJ). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotic–antimicotic solutions, and trypsin were purchased from Invitrogen (Carlsbad, CA). 5-Bromo-2′-deoxyuridine (BrdU), insulin–transferrin–sodium selenite (ITSE) media supplement, Hoechst 33258 dye, Trichrome Stain (Masson) Kit, and all chemicals were from Sigma-Aldrich (St. Louis, MO). Dispase II and collagenase I were obtained from Roche (Indianapolis, IN). Anti-BrdU antibody (GTX42641) was from GeneTex (Taipei, Taiwan). Anti-CD146 (ab75769), anti-Oct4 (ab18976), anti-nestin (ab6142), anti-CD31 (ab24590), anti-nucleostemin (ab70346), anti-collagen I (ab6308), and anti-collagen III antibody (ab6310) antibodies were from Abcam (Cambridge, MA). Phospho-Stat3 (Tyr705), STAT3, phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), and ERK antibody were purchased from Cell Signaling Technology (Danvers, MA). SB203580, PD98059, and STAT3 inhibitor (No. 573096) were purchased from Calbiochem (La Jolla, CA, USA).
All animals were housed in an animal room under temperature control (24–25 °C) and a 12:12 light-dark cycle. Standard laboratory chow and tap water were available ad libitum. Experimental procedures were approved by the Mackay Memorial Hospital Review Board (New Taipei City, Taiwan) and were performed in compliance with national animal welfare regulations.
Isolation and culture of TSPC
New Zealand white rabbits (6–8 months old, 3.0–4.0 kg) were used for the isolation of tendon cells. Achilles tendons were removed from the rabbits, by cutting through their bony attachments, and washed two times in sterile phosphate-buffered saline (PBS) containing 50 μg/ml gentamicin. The tendon and tendon sheath were cut into small pieces (1–2 mm3). Each 100 mg of tissue was then digested in a solution containing 3 mg/ml of type I collagenase and 4 mg/ml of dispase II in 1 ml balanced salt solution (BSS; Alcone) at 37 °C for 4 h. The digested tissues were washed three times in PBS, collected by centrifugation (800g for 10 min), placed into tissue culture dishes (Falcon Labware, NJ, USA) and resuspended in high-glucose DMEM, supplemented with 10% FBS and 50 μg/ml gentamycin, and maintained at 37 °C with 5% CO2. After 5 days, the medium was changed to remove the loosened tissue residues. For passaging, the tendon cells were harvested with 0.25% trypsin/EDTA and counted using a hemocytometer.
Approximately 2 × 103 primary tendon cells were seeded into 25 T cell culture flasks (Corning), incubated with 10% FBS medium for 2 days, and their clonogenic capacity was determined in DMEM basal medium (2% FBS, 1% ITSE, 300 μg/ml l-glutamine, 1% antibiotic-antimicotic solutions), supplemented with 10 μM 29-mer, 34-mer, or peptide solvent (DMSO; dimethyl sulfoxide), for a further 10 days. The culture medium was changed every 3 days. Expression of TSPC markers by these expanded tendon cells was determined by immunostaining and western blot analysis.
Cells cultured on slides were fixed with 4% paraformaldehyde, treated at 4 °C with methanol for 1 min, and then blocked with 1% goat serum and 5% BSA for 1 h. The cells were stained with antibodies to CD146 (1:50 dilution), nucleostemin (1:100 dilution), or BrdU (1:100 dilution) at room temperature (RT) for 3 h. The slides were subsequently incubated with FITC-donkey anti-rabbit IgG or Alexa Fluor® 647 Goat anti-mouse IgG (1:500 dilution; BioLegend, San Diego, CA) for 20 min and then counterstained with Hoechst 33258 for 6 min. The slides were rinsed with PBS with Triton X100 (0.5%) three times, mounted with FluorSave™ reagent (Calbiochem) and viewed with a Zeiss epifluorescence microscope.
After 3 weeks, the repaired Achilles tendon (n = 16) and the contralateral control tendons (n = 16) were harvested for mechanical evaluation. The specimens were dissected to remove the gastrocnemius/soleus muscle complex and soft tissues, leaving the intramuscular tendinous fibers, Achilles tendon, and calcaneal bone intact. The specimens were kept moist with normal saline during the entire testing procedure. The sagittal and transverse diameters of the mid-part of the callus were measured using a caliper to estimate the cross-sectional area . Tendon fibers were fixed in a metal clamp by fine sandpaper. The calcaneal bone was fixed in a custom-made clamp at 30° dorsiflexion, relative to the direction of traction, as described previously [14, 15]. The mechanical testing machine (MTS Systems Corp., 14000 Technology Drive, Eden Prairie, MN) pulled the mounted tendon at a constant speed (0.1 mm/s) until failure, after a preload of 0.8 N had been applied. The data acquisition rate was 1/0.03 s. The ultimate tensile stress at tendon failure, expressed in newtons (N), was recorded during the process of failure testing. Young’s modulus (MPa) was calculated from the linear slope of the axial force–axial displacement curve.
BrdU labeling in vitro
BrdU (final concentration, 10 μM) was added to the culture for 4 h. After fixing with 4% paraformaldehyde, the cells were exposed to cold methanol for 1 min and then treated with 1 N HCl at RT for 1 h before performing immunocytochemistry. The phenotype of TSPC was determined by immunostaining of nucleostemin.
Western blot analysis
Cell lysis, SDS–PAGE, and antibodies used for immunoblotting were as described in a previous study . The band intensity in immunoblots was evaluated with a Model GS-700 imaging densitometer (Bio-Rad Laboratories, Hercules, CA) and analyzed using Labworks 4.0 software.
Quantitative real-time RT-PCR
Surgical procedure for rat Achilles tendon injury
To investigate the effects of the 29-mer peptide on tendon healing, a rat model of Achilles tendon injury was established, as reported previously . Ten-week-old adult male Sprague-Dawley rats (initial body weight = 312 ± 11 g) were anesthetized by an intraperitoneal injection of xylazine (10 mg/kg). The left tendo-Achilles injury was created by full-thickness insertion of an 18-G needle through the tendo-Achilles, 1 cm proximal to the calcaneum attachment site. This created a horizontal wound which was flanked by intact tendon tissue to prevent the retraction of the severed ends. The skin incision was closed after the wound was irrigated with sterile saline. Treatments were applied to the area around the tendon lesion by subcutaneous injection with 150 μl of alginate gel mixed with 100 μM 29-mer or DMSO vehicle.
Formalin-fixed, paraffin-embedded tendon specimens were cut into 5-μm longitudinal sections, deparaffinized in xylene, and rehydrated in a graded series of ethanol concentrations. Slides were blocked with 10% goat serum for 60 min and then incubated with primary antibody against CD146 (1:50 dilution), nucleostemin (1:50 dilution), and CD31 (1:100 dilution) at room temperature (RT) for 2 h. The slides were subsequently incubated with the FITC-donkey anti-rabbit IgG and Alexa Fluor® 647 Goat anti-mouse IgG (1:500 dilution) for 20 min and then counterstained with Hoechst 33258 for 6 min and viewed with Zeiss epifluorescence microscope.
Deparaffinized tendon specimens were also blocked with 10% goat serum for 60 min and then incubated with antibody against CD146, collagen I, and collagen III (1:100 dilution, 37 °C for 3 h). The slides were subsequently incubated with the appropriate peroxidase-labeled goat immunoglobulin (1:500 dilution; Chemicon, Temecula, CA) for 20 min and then incubated with chromogen substrate (3,3′-diaminobenzidine) for 2 min before counterstaining with hematoxylin.
In vivo detection of DNA synthesis
For the detection of cell expansion, BrdU was reconstituted in DMSO as stock (80 mM). One hundred fifty microliters of BrdU mixed with 350 μl of PBS was injected intraperitoneally into the rats at days 0, 3, and 5 after surgery. Tissue sections were treated with 1 N HCl at RT for 1 h, and DNA synthesis was assessed by anti-BrdU antibodies.
The tendon specimens were stained with hematoxylin and eosin (H&E). Photomicrographs of the tissue were captured through an Olympus IX71 light microscope and an Olympus XC10 camera (Japan). Four sections of each sample were selected and evaluated by two blinded observers to assess the tendon morphology according to a modified semi-quantitative grading score from 0 to 3. The score analyzed the fiber arrangement, fiber structure, nuclear roundness, cell density, infiltration of inflammatory cells and fibroblasts, and neovascularization [18,19,20]. According to this grading system, a perfectly normal tendon scored 0 and mild and moderate prevalence scored 1 and 2, whereas a score of 3 was assigned to a severely abnormal tendon.
The data were generated from three independent experiments. All numerical values are expressed as the mean ± SD. Comparisons of two groups were made using two-tailed Student’s t test. P < 0.05 was considered significant.
The 29-mer stimulates the proliferation of CD146+ TSPC in culture
To verify the function of PSP, primary rabbit tendon cells were treated with the 29-mer, control peptide (34-mer), or peptide solvent (DMSO) for 10 days, as described in the “Materials” and “Methods” sections. Tendon cells stimulated with the 29-mer formed colonies (consisting of about 30–350 cells; Fig. 1a). Quantitatively, 29-mer-treated tendon cells had a greater clonogenic capacity than those of the solvent and control peptide-treated cells (Fig. 1b, 9.0 ± 1.1% versus 1.5 ± 0.2% and 1.5 ± 0.2%). CD146 is a TSPC marker and has been shown to be correlated with clonogenic capacity in vitro [3, 21]. Immunostaining of colonies by CD146 showed that most of the 29-mer-treated cells had a CD146-positive phenotype, approximately 7-fold more than the tendon cells treated by solvent or the control peptide (Fig. 1b, c). Western blot analysis showed that the levels of CD146 protein were 3.8-fold higher than the solvent control when the primary tendon cells were stimulated with the 29-mer for 10 days (Fig. 1d). Oct4 and nestin have been identified as TSPC markers [3, 21,22,23]. The 29-mer treatment also can result in greater amounts of Oct4 and nestin proteins than the solvent control (2-fold and 2.4-fold, respectively). Collectively, the 29-mer stimulates TSPC expansion in culture. The expression of TSPC markers is enriched by 29-mer treatment, implying that the expanded TSPC may still sustain their stemness.
Sustained release of the 29-mer peptide promotes Achilles tendon healing
To investigate the therapeutic effect of the 29-mer on injured tendons, a rat model of Achilles tendon injury was established by full-thickness insertion of an 18-G needle horizontally through the tendo-Achilles . On day 2 post-surgery, the needle insertion had led to inflammatory responses and necrotic tissue formation (Fig. 2a). Alginate gel loaded with the 29-mer was coated onto the tendon lesion to treat the tendon rupture. The hydrogel releases 90% of the loaded 29-mer in 5 days, as described in a previous report . According to histological analysis of the H&E stained sections at 3 weeks postoperation, vehicle/alginate gel alone did not affect the self-healing, characterized by scar-like tissue, inflammatory matrix (marked by a red arrowhead), and numerous neovessels (marked by black arrows) in the healing region (HR; Fig. 2b). The 29-mer/alginate gel treatment improved wound-healing, with evidence of formation of densely aligned collagen fibers, less inflammatory cell invasion, and fewer neovessels in the HR (Fig. 2c). Masson’s trichrome staining confirmed that the 29-mer/alginate gel treatment led to dense and aligned collagen fibers parallel to the native tendon, in contrast to disorganized collagen formed in the vehicle/alginate group (Fig. 2d). Histological scoring analysis was performed by two blinded examiners. The average histology score for fiber arrangement, fiber structure, nuclear roundness, infiltration of inflammatory cells/fibroblasts, and neovascularization was significantly lower for the 29-mer/alginate gel group than the vehicle group 3 weeks after treatment (Fig. 2e; *P < 0.001). The higher cell density in the 29-mer/alginate gel group reflects accelerated TSPC proliferation. Collectively, the 29-mer displays efficacy in promoting tendon regeneration.
Type I collagen (Col I) fiber bundles are major tendon matrix components and endow tendons with the ability to support tensile loading. Type III collagen (Col III) is typically over-expressed during the early inflammatory and proliferative stages of tendon healing. The increased content of Col III at later stages of tendon healing can cause thinner collagen fibers with a lower tensile strength . IHC staining was used to compare the amounts of Col I and Col III at the tendon rupture site after healing for 3 weeks. As shown in Fig. 3, intact tendons featured predominantly Col I, with little Col III in the tendon matrix. The vehicle group displayed a weaker Col I deposition in the tendon matrix, but retained substantial Col III in the rupture site, reflecting the natural healing process. In contrast, the 29-mer/alginate gel treatment exhibited a strong expression of Col I in the tendon cells than the vehicle group. Col III expression in the 29-mer group was decreased to a level similar to intact tendons. The Col I/Col III staining and Masson’s trichrome staining (Fig. 2d) data imply that 29-mer treatment contributes to the tendon healing responses.
We examined the biomechanical properties of repaired tendons to determine the efficacy of the 29-mer treatment. Sixteen rats underwent Achilles tendon surgery were randomized to either the 29-mer/alginate gel or vehicle treatment groups. Mechanical testing 3 weeks after the surgery showed that the ultimate tensile stress (UTS) and Young’s modulus of the repaired tendon in the 29-mer/alginate gel group reached 89.1% and 89.3%, respectively, compared to the contralateral intact tendons (Fig. 4). In contrast, the values of the UTS and Young’s modulus of the vehicle group were 68.7% and 61.1%, respectively, significantly lower than the 29-mer/alginate gel group (P < 0.05). In summary, the 29-mer/alginate gel treatment leads to increased tendon strength after healing for 3 weeks.
Expanded TSPC induced by 29-mer retain the capacity for tenogenic differentiation
Next, we investigated whether the expanded TSPC, induced by the 29-mer, still preserve the capacity for tenogenic differentiation. Isolated primary tendon cells were treated with the 29-mer for 10 days to expand TSPC, followed by culture in basal medium without the 29-mer to induce tenogenic differentiation for a further 2 weeks. The 29-mer pretreatment increased collagen deposition, compared to solvent pretreatment (Fig. 5a; Masson’s trichrome staining). A real-time qPCR assay revealed that, during the 10 days of culture with the 29-mer, the levels of Oct4 and nestin mRNA were 3.5- and 3.1-fold greater than in solvent-treated cells (Fig. 5b). Following the withdrawal of 29-mer for 14 days, the levels of Oct4 and nestin mRNA declined substantially. Notably, the primary tendon cells pretreated by the 29-mer showed higher levels of expression of tenocyte lineage-related genes, including collagen type I (Col1a1), collagen type III (Col3a1), and tenascin-C (Tnc), than solvent-pretreated cells (Fig. 5b; approximately 4.6- to 6.5-fold; column 4 versus column 3). Two tendon lineage-related transcription factors, the zinc finger protein EGR1 (early growth response-1) and the Mohawk homeobox (Mkx) protein, have been shown to direct tendon differentiation and repair [25, 26]. Molecular analyses of ectopic Egr1 or Mkx expression in murine C3H10T1/2 mesenchymal stem cells have shown that exposure to the 29-mer leads to increased expression of tendon lineage-related genes, including Col1a1, Col3a1, and Tnc [26, 27]. As shown in Fig. 5b, 29-mer treatment for 10 days led to 2.1-fold and 2.3-fold lower levels of Egr1 and Mkx than the solvent controls, implying that the 29-mer prevents TSPC spontaneous tenogenic differentiation in vitro. However, inhibition of differentiation by the 29-mer is reversible when the peptide is withdrawn; a qPCR assay revealed significantly more Egr1 and Mkx expression than in solvent-pretreated cells (3.4- and 2.7-fold greater; column 4 versus column 3). A similar trend was observed with significantly higher levels of expression of Col1a1, Col3a1, and Tnc in cells following 29-mer pretreatment. The data suggest that TSPC expanded by the 29-mer retain the capacity for tenogenic differentiation in vitro.
The 29-mer promotes CD146+ TSPC proliferation in response to acute Achilles tendon injury
To seek evidence of the effect of the 29-mer on TSPC expansion in vivo, we monitored CD146+ TSPC expansion following tendon surgery combined with 29-mer/alginate gel delivery for 2 days. Immunofluorescence analysis revealed that the level of CD146+ TSPC was higher, at the peripheral region of the injured Achilles tendon, in the 29-mer/alginate gel group than the vehicle group (Fig. 6a; CD146+ TSPC per microscope field, 61.4 ± 6.0 versus 33.6 ± 7.3). In contrast, the levels of CD31+ vascular endothelial cells were not affected by the 29-mer or vehicle treatment (Fig. 6a). To investigate whether the accumulation of CD146+ TSPC is partly due to the mitogenic effect of the 29-mer, rats were injected intraperitoneally with BrdU to detect TSPC DNA synthesis immediately after tendon surgery. At day 2 after tendon wounding, immunofluorescence analysis confirmed that the 29-mer/alginate gel treatment resulted in higher levels of CD146/BrdU double-positive TSPC than the vehicle group (Fig. 6b, c; 32.4 ± 2.9% versus 10.9 ± 1.3%). The results imply that the 29-mer can impart mitogenic activity on CD146+ TSPC in injured Achilles tendons.
The 29-mer causes accumulation of nucleostemin+ TSPC in the healing region of the Achilles tendon
One week after wounding, CD146 immunostaining of the Achilles tendons revealed large amounts of CD146+ TSPC in the HR of the 29-mer/alginate gel-treated tendons. In contrast, the amounts of CD146+ TSPC in the vehicle group were significantly lower (Fig. 7a; CD146+ TSPC per microscope field, 59.6 ± 9.2 versus 38.6 ± 6.9). The 29-mer effectively enhances CD146+ TSPC expansion into the HR, supporting rapid tendon wound healing. Interestingly, almost all of these CD146+ TSPC at the HR were stained negative for BrdU in the 29-mer and vehicle groups, even though there were large amounts of BrdU+ cells throughout the HR (Fig. 7b). It has been suggested that different TSPC populations exhibit different anatomical and temporal expression during rat Achilles tendon healing, such as nucleostemin+ TSPC throughout the HR at 1~2 weeks postoperatively . Immunofluorescence staining indicated that almost all of the BrdU+ cells at the HR were stained positive for nucleostemin (Fig. 7c). More importantly, significantly more nucleostemin/BrdU double-positive cells were observed in the 29-mer/alginate gel-treated tendon than the vehicle control (15.8 ± 3.0% versus 4.0 ± 0.5%). Taken together, the data indicate that 29-mer not only stimulates the rapid expansion of CD146+ TSPC in both the tendon periphery and HR but also causes marked accumulation of nucleostemin+ TSPC at the HR to accelerate the wound-healing.
The mitogenic effect of the 29-mer on nucleostemin+ TSPC is modulated by ERK2 and STAT3 signaling
Primary tendon cells were expanded for 14 days in culture and then exposed to the 29-mer (10 μM) for 24 h. TSPC purity was verified to be approximately 98% by nucleostemin staining (Fig. 8a). This also indicates that nucleostemin+ TSPC can be expanded as the major TSPC population from isolated primary rabbit tendon cells cultivated in basal culture medium. Cell proliferation was detected by BrdU pulse-labeling (4 h) and analyzed by BrdU immunostaining (red color). The assay revealed that stimulation by the 29-mer increased BrdU+ TSPC levels by 5.2-fold, compared with solvent treatment (Fig. 8b). Next, we used pharmacological inhibitors to explore the molecular basis of nucleostemin+ TSPC proliferation induced by the 29-mer. BrdU pulse-labeling assays revealed that pretreatment with STAT3 or ERK inhibitor suppressed the 29-mer-induced cell proliferation from 19.1 ± 1.7% to 3.4 ± 0.6% and 3.1 ± 0.6%, respectively (Fig. 8b). p38 MAPK inhibitor had no such effect. Meanwhile, the 29-mer can induce phosphorylation of ERK2 and STAT3 in nucleostemin+ TSPC. The immunoblots revealed that phosphorylation of STAT3 and ERK2 caused by the 29-mer occurred 10~40 min after the treatment (Fig. 8c). However, the 34-mer control peptide did not show these effects. Collectively, the findings imply that the 29-mer induces nucleostemin+ TSPC expansion in vitro via activation of the ERK and STAT3 signaling pathways.
Insufficient numbers of resident TSPC are activated in response to an acute tendon injury, and this may limit the healing process. This study is the first to show that the PSP 29-mer can promote the clonogenicity of CD146+ TSPC and proliferation of nucleostemin+ TSPC among cells isolated from the rabbit Achilles tendon. Moreover, we provide evidence that a single injection of the 29-mer hydrogel into a damaged tendon can induce expansion of CD146+ TSPC and nucleostemin+ TSPC at the early regenerative stage (2~7 days postoperative). Our study has demonstrated, in vitro and in vivo, that the 29-mer is a newly identified mitogen for TSPC.
In this study, an injectable alginate gel was used to provide sustained release of the 29-mer in the damaged tendon, leading to improved tendon repair. Alginate gel as a vehicle displays biocompatible, nontoxic, and biodegradable properties . Our previous study showed that a single injection of the 29-mer/alginate gel can stimulate muscle satellite cell proliferation significantly to promote rat soleus muscle regeneration . Despite these advantages, this previous experiment revealed that ~ 90% of the 29-mer was released from the alginate gel within 5 days in vitro . Nonetheless, herein, our day 2 and day 7 histological data indicate that the sustained release of 29-mer for a few days was sufficient to augment CD146+ TSPC and nucleostemin+ TSPC in the niche and HR. The results support the hypothesis that the growth factor stimulates a sufficient level of CD146+ TSPC at the early phase of tendon wound healing, playing a crucial role in relieving disorganized collagen formation in the HR .
Healing of the tendons begins with the inflammatory phase that shows accumulation of hemorrhages and leukocytes and local synthesis of chemotactic and angiogenic factors . The reparative phase begins later, characterized by the initiation of TSPC proliferation and migration of TSPC/tenocytes into the wound [30, 31]. Due to the limited self-healing capacity of the tendons, the healed tendon shows disorganized fiber architecture and scar tissue with disrupted collagens [3, 30]. These result in reduced elasticity, reduced mobility, and an increased propensity for the recurrence of injury . In this study, the 29-mer hydrogel treatment accelerated TSPC proliferation that initially overlapped the inflammatory phase (day 2 to day 7), thereby leading to a decrease in fibrosis and scar tissue formation. The incomplete description of the role of 29-mer in the inflammatory phase of tendon healing is a limitation of this study. In this regard, PEDF reportedly can reduce inflammation in vitro and in vivo. For example, PEDF induces expression of the anti-inflammatory cytokine interleukin 10 (IL-10) by human macrophages . PEDF treatment can reduce retinal inflammation in a rat model of diabetes . Interestingly, our previous animal study found that PSP 44-mer (a sequence containing the 29-mer) can reduce inflammatory responses in acute liver injury through its protective effect on hepatocytes . Whether the 29-mer is able to regulate the properties of inflammatory cells during tendon injury warrants additional research.
It has been reported that the nucleostemin is a TSPC marker because its expression is eliminated in tenocytes . In this study, the 29-mer increased the number of nucleostemin+ TSPC that were observed on day 7 post-injury. Nucleostemin+ TSPC have been shown to retain a high self-renewal capacity in vitro and express tenocyte-related markers, such as collagen I, collagen III, and Tnc . Because collagen I and collagen III are important structural protein in tendons, the nucleostemin+ TSPC have a direct impact on tendon repair.
The relationship between CD146+ TSPC and nucleostemin+ TSPC remains elusive. There is still no evidence that CD146+ TSPC can differentiate to nucleostemin+ TSPC. A study discovered that stem cells derived from the human anterior cruciate ligament (hACL) and medial collateral ligament (hMCL) express nucleostemin, but not CD146 . Interestingly, a recent report indicates that rat tendon injury leads to a large number of nucleostemin+ TSPC throughout the HR at 1 and 2 weeks post-injury . The different TSPC populations have been suggested to be involved in a time-controlled tendon repair process [3, 28].
The present cell signaling study exploits nucleostemin+ TSPC rather than CD146+ TSPC. CD146+ TSPC are rare (~ 0.8%) in the rat patellar tendon . Our histological sections also revealed that CD146+ TSPC numbers were extremely low in the uninjured rabbit Achilles tendon, and there was a rapid loss of stemness during culture in basal medium. PSP, as full-length PEDF, has been shown to initiate signaling by binding to the cell surface receptor, patatin-like phospholipase domain-containing protein 2 (PNPLA2). PNPLA2 receptor is essential for PEDF/PSP to induce mitogenic signaling on human embryonic stem cells and neural stem cells, as well as antiapoptotic signaling on hepatocytes [13, 38, 39]. Further studies are warranted to determine the expression of PNPLA2 in TSPC and the involvement of PNPLA2 in mediating PEDF/29-mer mitogenic signaling. Our study found that STAT3 signaling was critical for the induction of nucleostemin+ TSPC proliferation by the 29-mer. Our previous findings show that PSP enhances the proliferation of limbal stem cells and satellite cells by activating STAT3 signaling [11, 12]. In addition, phosphorylation of STAT3 has been found to be crucial for the proliferation of myoblasts and satellite cells induced by bFGF, leukemia inhibitory factor (LIF), and IL-6 [40, 41]. STAT3 is a transcription factor that regulates several targets closely associated with cell cycle progression, including cyclin D1 and SOCS3 [12, 42]. To our knowledge, there has been no report addressing the role of STAT3 signaling on TSPC proliferation. Our finding also implies that ERK2 activation is crucial for TSPC proliferation in vitro. This multiple signaling is reminiscent of the proliferative responses induced in satellite cells by the 29-mer . ERK signaling as STAT3 is also involved in cyclin D1 expression by satellite cells .
Growth factors accelerate TSPC expansion in the early phase of tendon wound-healing as a critical mechanism for improving tendon repair. This study shows that the PSP 29-mer displays mitogenic activity and regulates the resident TSPC, in response to acute tendon rupture, and facilitates tendon recovery of a higher quality in the animal model. The 29-mer may be a novel therapeutic remedy for acute tendon injury.
Adipose-derived mesenchymal stem cells
Bone marrow mesenchymal stromal cells
Balance salt solution
- Col1a1 :
Collagen type I
- Col3a1 :
Collagen type III
Connective tissue growth factor
Dulbecco’s modified Eagle’s medium
Early growth response-1
Extracellular signal-regulated kinase
Fetal bovine serum
Fibroblast growth factor
Human anterior cruciate ligament
Medial collateral ligament
Insulin-like growth factor
Leukemia inhibitory factor
Mitogen-activated protein kinase
- Mkx :
Octamer-binding transcription factor 4
Polymerase chain reaction
Platelet-derived growth factor
Pigment epithelial-derived factor
Patatin-like phospholipase domain-containing protein 2
Pigment epithelial-derived factor-derived short peptide
SRY-box containing gene 9
Signal transducer and activator of transcription 3
- Tnc :
Tendon stem/progenitor cells
Ultimate tensile stress
Lui PP. Stem cell technology for tendon regeneration: current status, challenges, and future research directions. Stem Cells Cloning. 2015;8:163–74.
Liu W, Yin L, Yan X, Cui J, Liu W, Rao Y, Sun M, Wei Q, Chen F. Directing the differentiation of parthenogenetic stem cells into tenocytes for tissue-engineered tendon regeneration. Stem Cells Transl Med. 2017;6:196–208.
Lee CH, Lee FY, Tarafder S, Kao K, Jun Y, Yang G, Mao JJ. Harnessing endogenous stem/progenitor cells for tendon regeneration. J Clin Invest. 2015;125:2690–701.
Berebichez-Fridman R, Gómez-García R, Granados-Montiel J, Berebichez-Fastlicht E, Olivos-Meza A, Granados J, Velasquillo C, Ibarra C. The Holy Grail of orthopedic surgery: mesenchymal stem cells-their current uses and potential applications. Stem Cells Int. 2017;2017:2638305.
Sánchez M, Anitua E, Azofra J, Andía I, Padilla S, Mujika I. Comparison of surgically repaired Achilles tendon tears using platelet-rich fibrin matrices. Am J Sports Med. 2007;35:245–51.
Farnebo S, Farnebo L, Kim M, Woon C, Pham H, Chang J. Optimized repopulation of tendon hydrogel: synergistic effects of growth factor combinations and adipose-derived stem cells. Hand (N Y). 2017;12:68–77.
Manning CN, Schwartz AG, Liu W, Xie J, Havlioglu N, Sakiyama-Elbert SE, Silva MJ, Xia Y, Gelberman RH, Thomopoulos S. Controlled delivery of mesenchymal stem cells and growth factors using a nanofiber scaffold for tendon repair. Acta Biomater. 2013;9:6905–14.
Tokunaga T, Shukunami C, Okamoto N, Taniwaki T, Oka K, Sakamoto H, Ide J, Mizuta H, Hiraki Y. FGF-2 stimulates the growth of tenogenic progenitor cells to facilitate the generation of tenomodulin-positive tenocytes in a rat rotator cuff healing model. Am J Sports Med. 2015;43:2411–22.
Ramírez-Castillejo C, Sánchez-Sánchez F, Andreu-Agulló C, Ferrón SR, Aroca-Aguilar JD, Sánchez P, Mira H, Escribano J, Fariñas I. Pigment epithelium-derived factor is a niche signal for neural stem cell renewal. Nat Neurosci. 2006;9:331–9.
Silva Dos Santos D, Coelho de Oliveira VC, Asensi KD, Vairo L, Carvalho AB, Campos de Carvalho AC, Goldenberg RC. Human menstrual blood-derived mesenchymal cells as new human feeder layer system for human embryonic stem cells. Cell Med. 2014;7:25–35.
Ho TC, Chen SL, Wu JY, Ho MY, Chen LJ, Hsieh JW, Cheng HC, Tsao YP. PEDF promotes self-renewal of limbal stem cell and accelerates corneal epithelial wound healing. Stem Cells. 2013;31:1775–84.
Ho TC, Chiang YP, Chuang CK, Chen SL, Hsieh JW, Lan YW, Tsao YP. PEDF-derived peptide promotes skeletal muscle regeneration through its mitogenic effect on muscle progenitor cells. Am J Physiol Cell Physiol. 2015;309:C159–68.
Shih SC, Ho TC, Chen SL, Tsao YP. Pigment epithelium-derived factor (PEDF) peptide promotes the expansion of hepatic stem/progenitor cells via ERK and STAT3-dependent signaling. Am J Transl Res. 2017;9:1114–26.
Hammerman M, Blomgran P, Ramstedt S, Aspenberg P. COX-2 inhibition impairs mechanical stimulation of early tendon healing in rats by reducing the response to microdamage. J Appl Physiol (1985). 2015;119:534–40.
Blomgran P, Hammerman M, Aspenberg P. Systemic corticosteroids improve tendon healing when given after the early inflammatory phase. Sci Rep. 2017;7:12468.
Ho TC, Chen SL, Yang YC, Chen CY, Feng FP, Hsieh JW, Cheng HC, Tsao YP. 15-deoxy-Delta(12,14)-prostaglandin J2 induces vascular endothelial cell apoptosis through the sequential activation of MAPKS and p53. J Biol Chem. 2008;283:30273–88.
Orhan Z, Ozturan K, Guven A, Cam K. The effect of extracorporeal shock waves on a rat model of injury to tendo Achillis. A histological and biomechanical study. J Bone Joint Surg Br. 2004;86:613–8.
Chen L, Liu JP, Tang KL, Wang Q, Wang GD, Cai XH, Liu XM. Tendon derived stem cells promote platelet-rich plasma healing in collagenase-induced rat Achilles tendinopathy. Cell Physiol Biochem. 2014;34:2153–68.
Manning CN, Havlioglu N, Knutsen E, Sakiyama-Elbert SE, Silva MJ, Thomopoulos S, Gelberman RH. The early inflammatory response after flexor tendon healing: a gene expression and histological analysis. J Orthop Res. 2014;32:645–52.
Wang T, Lin Z, Day RE, Gardiner B, Landao-Bassonga E, Rubenson J, Kirk TB, Smith DW, Lloyd DG, Hardisty G, et al. Programmable mechanical stimulation influences tendon homeostasis in a bioreactor system. Biotechnol Bioeng. 2013 May;110(5):1495–507.
Bi Y, Ehirchiou D, Kilts TM, Inkson CA, Embree MC, Sonoyama W, Li L, Leet AI, Seo BM, Zhang L, et al. Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nat Med. 2007;13:1219–27.
Webb S, Gabrelow C, Pierce J, Gibb E, Elliott J. Retinoic acid receptor signaling preserves tendon stem cell characteristics and prevents spontaneous differentiation in vitro. Stem Cell Res Ther. 2016;7:45.
Yin Z, Hu JJ, Yang L, Zheng ZF, An CR, Wu BB, Zhang C, Shen WL, Liu HH, Chen JL, et al. Single-cell analysis reveals a nestin+ tendon stem/progenitor cell population with strong tenogenic potentiality. Sci Adv. 2016;2:e1600874.
Eriksen HA, Pajala A, Leppilahti J, Risteli J. Increased content of type III collagen at the rupture site of human Achilles tendon. J Orthop Res. 2002;20:1352–7.
Liu W, Watson SS, Lan Y, Keene DR, Ovitt CE, Liu H, Schweitzer R, Jiang R. The atypical homeodomain transcription factor Mohawk controls tendon morphogenesis. Mol Cell Biol. 2010;30:4797–807.
Guerquin MJ, Charvet B, Nourissat G, Havis E, Ronsin O, Bonnin MA, Ruggiu M, Olivera-Martinez I, Robert N, Lu Y, et al. Transcription factor EGR1 directs tendon differentiation and promotes tendon repair. J Clin Invest. 2013;123:3564–76.
Liu H, Zhang C, Zhu S, Lu P, Zhu T, Gong X, Zhang Z, Hu J, Yin Z, Heng BC, et al. Mohawk promotes the tenogenesis of mesenchymal stem cells through activation of the TGFβ signaling pathway. Stem Cells. 2015;33:443–55.
Runesson E, Ackermann P, Karlsson J, Eriksson BI. Nucleostemin- and Oct 3/4-positive stem/progenitor cells exhibit disparate anatomical and temporal expression during rat Achilles tendon healing. BMC Musculoskelet Disord. 2015;16:212.
Ching SH, Bansal N, Bhandari B. Alginate gel particles-a review of production techniques and physical properties. Crit Rev Food Sci Nutr. 2017;57:1133–52.
James R, Kesturu G, Balian G, Chhabra AB. Tendon: biology, biomechanics, repair, growth factors, and evolving treatment options. J Hand Surg Am. 2008;33:102–12.
Sharma P, Maffulli N. Biology of tendon injury: healing, modeling and remodeling. J Musculoskelet Neuronal Interact. 2006;6:181–90.
Liu CF, Aschbacher-Smith L, Barthelery NJ, Dyment N, Butler D, Wylie C. What we should know before using tissue engineering techniques to repair injured tendons: a developmental biology perspective. Tissue Eng Part B Rev. 2011;17:165–76.
Yang SL, Chen SL, Wu JY, Ho TC, Tsao YP. Pigment epithelium-derived factor induces interleukin-10 expression in human macrophages by induction of PPAR gamma. Life Sci. 2010;87:26–35.
Zhang SX, Wang JJ, Gao G, Shao C, Mott R, Ma JX. Pigment epithelium-derived factor (PEDF) is an endogenous antiinflammatory factor. FASEB J. 2006;20:323–5.
Zhang J, Wang JH. Characterization of differential properties of rabbit tendon stem cells and tenocytes. BMC Musculoskelet Disord. 2010;11:10.
Yang J, Zhao Q, Wang K, Liu H, Ma C, Huang H, Liu Y. Isolation and biological characterization of tendon-derived stem cells from fetal bovine. In Vitro Cell Dev Biol Anim. 2016;52:846–56.
Zhang J, Pan T, Im HJ, Fu FH, Wang JH. Differential properties of human ACL and MCL stem cells may be responsible for their differential healing capacity. BMC Med. 2011;9:68.
Castro-Garcia P, Díaz-Moreno M, Gil-Gas C, Fernández-Gómez FJ, Honrubia-Gómez P, Álvarez-Simón CB, Sánchez-Sánchez F, Cano JC, Almeida F, Blanco V, et al. Defects in subventricular zone pigmented epithelium-derived factor niche signaling in the senescence-accelerated mouse prone-8. FASEB J. 2015;29:1480–92.
Gonzalez R, Jennings LL, Knuth M, Orth AP, Klock HE, Ou W, Feuerhelm J, Hull MV, Koesema E, Wang Y, et al. Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency. Proc Natl Acad Sci U S A. 2010;107:3552–7.
Serrano AL, Baeza-Raja B, Perdiguero E, Jardí M, Muñoz-Cánoves P. Interleukin-6 is an essential regulator of satellite cell-mediated skeletal muscle hypertrophy. Cell Metab. 2008;7:33–44.
Megeney LA, Perry RL, LeCouter JE, Rudnicki MA. bFGF and LIF signaling activates STAT3 in proliferating myoblasts. Dev Genet. 1996;19:139–45.
Brender C, Lovato P, Sommer VH, Woetmann A, Mathiesen AM, Geisler C, Wasik M, Ødum N. Constitutive SOCS-3 expression protects T-cell lymphoma against growth inhibition by IFNalpha. Leukemia. 2005;19:209–13.
We thank Dr. Tim J Harrison for kindly reading this manuscript. The authors would like to acknowledge Chu-Ping Ho and Yi-Cheng Huang for their assistance with sample collection and histological preparations.
The funding support from the Ministry of Science and Technology, Taiwan (MOST 104-2314-B-195-006-MY3) and Mackay Memorial Hospital (MMH-E-107-006).
Availability of data and materials
Experimental procedures were approved by the Mackay Memorial Hospital Review Board (MMH-A-S-103-38 and MMH-A-S-106-47) (New Taipei City, Taiwan), which was in accordance with MOST Guide for the Care and Use of Laboratory Animals.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.