PEDF-derived peptide promotes tendon regeneration through its mitogenic effect on tendon stem/progenitor cells
Tsung-Chuan Ho et al. Stem Cell Res Ther. 2019.
Abstract
Background: 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.
Methods: 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.
Results: 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.
Conclusions: 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.
Keywords: PEDF; Peptide; Signaling; Tendon stem/progenitor cell.
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Conflict of interest statement
Ethics approval
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.
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Not applicable.
Competing interests
The authors declare that they have no competing interests.
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Figures Fig. 1
The 29-mer induces TSPC expansion among primary tendon cells. a, b Colony-forming efficiency (CFE). 2 × 103 primary rabbit tendon cells were continuously cultivated in basal medium supplemented with 10 μM 29-mer, 34-mer (control peptide), or peptide solvent for 10 days. Representative images from three independent experiments. Three independent experiments were performed (n = 3, *P < .0005). c, d Immunofluorescence staining of colonies by CD146 antibody. Nuclei were stained by Hoechst 33258 (blue). The micrographs are representative of at least three independent experiments. The percentage of CD146+ cells per total cells in a colony was quantified. *P < 0.0001 versus solvent-treated cells. e, f Western blot analysis with antibodies as indicated. Representative immunoblots and densitometric analysis from three independent experiments are shown. The immunoblots were scanned and quantitated at individual sites and normalized to GAPDH. *P < 0.001 versus solvent-treated primary tendon cells Fig. 2
The 29-mer/alginate gel promotes Achilles tendon healing. a Representative H&E-stained longitudinal tendon sections 2 days after surgery. Rat Achilles tendon rupture was created by inserting an 18-G needle (the injured region is marked by a dashed line). The sections at higher magnification show normal tendon with a relative scarcity of cells among the collagen fibers. The injured region shows inflammation, marked by numerous blue-stained nuclei. b, c Representative H&E-stained sections 3 weeks after surgery. The 29-mer hydrogel treatment led to a dense, regular tendon tissue at the healing region (HR) compared to the disorganized tissue in tendons treated with vehicle hydrogel. Asterisk (*)—uninjured region. Arrowhead (red)—inflammatory matrix formed by high inflammatory cell invasion. Black arrow—neovessel. d Representative Masson’s trichrome-stained longitudinal tendon sections 3 weeks after surgery. The 29-mer-induced dense alignment of collagen fibers. e Histology scores of tendon healing 3 weeks after treatment. The histological scoring was performed double-blind. Fiber arrangement—scale of 0–3, 0 represents compacted and parallel, and 3 represents no identifiable pattern. Fiber structure—scale of 0–3, 0 represents continuous, long fibers, and 3 represents severely fragmented. Rounding of nuclei—scale of 0–3, 0 represents long spindle shape, and 3 represents severe rounding. Cell density—scale of 0–3, 0 represents normal pattern, and 3 represents severely increased. Fibroblasts/PMN/monocytes—1 (< 50 per field), 2 (51–100 per field), and 3 (101–150 per field). Vascularity—1 (< 5 per field), 2 (6–10 per field), and 3 (> 10 per field). High powered field (× 20). Results are expressed as mean ± SE of three independent experiments. *P < 0.001 versus vehicle-treated group. NS, not significant, P = 0.08 Fig. 3
Immunohistochemical staining of type I and type III collagen after tendon healing for 3 weeks following vehicle or 29-mer/alginate gel treatment. Representative micrographs from two independent experiments (n = 6) are shown. Magnification, × 200. Note the strong expression of Col III in tendon peripheral tissue (marked with an asterisk) Fig. 4
Biomechanical tests of normal tendon and regenerated tendons 3 weeks after surgery. Ultimate tensile stress (a) and Young’s modulus (b) of the 29-mer/alginate group (n = 8) and vehicle group (n = 8) were calculated from the axial force–axial displacement curve. The contralateral intact tendon was used as the control (n = 16). *P < 0.05 versus vehicle-treated group Fig. 5
Tenogenic differentiation of TSPC expanded by the 29-mer. Primary isolated tendon cells were treated with the 29-mer or solvent for 10 days, and the cells were cultured in medium without the 29-mer or solvent for a further 14 days. a Representative Masson’s trichrome-stained micrographs of primary tendon cells pretreated with the 29-mer or solvent and stained on day 24. Data represent three independent experiments. b Real-time qPCR analysis of the levels of TSPC marker genes (Oct4 and nestin) and teno-lineage marker genes (Col1a1, Col3a1, Tnc, Egr1, and Mkx) among primary tendon cells on days 10 and 24. Gapdh was used as a loading control. Data are representative of three independent experiments. *P < 0.001 versus solvent-treated cells. **P < 0.0001 versus solvent-treated cells Fig. 6
The 29-mer/alginate gel induces CD146+ TSPC proliferation in the peripheral region of the injured Achilles tendon. a Immunofluorescence analysis of the levels of CD146+ TSPC (green) and blood vessels (marked by CD31; red) at 2 days postoperation with the delivery of the 29-mer/alginate gel. Nuclei were visualized with Hoechst 33258 staining. Representative images are from six sections per rat tendon, with six rats per group. The digital image analysis of CD146+ TSPC was performed blinded on an average of six randomly selected × 400 magnification fields from each section, using a Zeiss epifluorescence microscope and Zeiss software. *P < 0.01 versus vehicle-treated group. b Double-immunofluorescence analysis of CD146 (green) and BrdU (red). Insets are the expanded TSPC detected by superimposing CD146, BrdU, and nuclear images using a digital program. Representative images are from three independent experiments with six rats per group. c Percentages of CD146/BrdU double-positive cells per total CD146+ cells. *P < 0.01 versus vehicle/alginate gel treatment Fig. 7
Immunohistochemical analysis of the distribution of various TSPC populations at 1 week postoperation. Tendons were treated with vehicle or 29-mer/alginate gel immediately after surgery. BrdU was injected intraperitoneally on days 0, 3, and 5. Tendons were harvested on day 7. a CD146 immunostaining (brown) and counterstaining with hematoxylin. Representative images are from three independent experiments. HR, healing region. The digital image analysis of CD146+ TSPC was performed blinded on an average of six randomly selected × 400 magnification fields from each section using a Nikon Eclipse 80i microscope equipped with a Leica DC 500 camera. *P < 0.05 versus vehicle-treated group. b CD146 and BrdU double immunostaining. Insets are the images before superimposition. Representative images are from three independent experiments. c Nucleostemin and BrdU double-immunostaining. Arrows indicate nucleostemin/BrdU double-positive TSPC. Representative images from three independent experiments with six rats per group are shown. Percentages of nucleostemin/BrdU double-positive cells per total nucleostemin+ cells were evaluated. *P < 0.002 versus vehicle group Fig. 8
The 29-mer induces nucleostemin+ TSPC proliferation in an ERK- and STAT3-dependent manner. a BrdU-labeling assay. Primary rabbit tendon cells were cultured to near confluence in culture flasks for 14 days and then verified by nucleostemin immunostaining (> 98%). Cells were pretreated with PD98059 (10 μM; ERK inhibitor), 50 μM STAT3 inhibitor, or SB203580 (10 μM; p38 MAPK inhibitor) for 2 h before treatment with 10 μM 29-mer and 10 μM BrdU for another 4 h. BrdU (red)-labeled nuclei were detected by immunofluorescence microscopy. Representative images are from three independent experiments. b The percentages of BrdU+ cells per total cells (counterstained by Hoechst 33258; blue) were calculated from ten randomly selected microscopic fields in each treatment. Results are expressed as mean ± SD of three independent experiments. *P < 0.05 versus solvent-treated; **P < 0.05 versus 29-mer-treated cells. c Representative immunoblots and densitometric analysis of the effect of the 29-mer on phosphorylation of ERK2 and STAT3 in nucleostemin+ TSPC; the immunoblots were scanned and quantitated at individual sites and normalized to STAT3 and ERK2, respectively
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