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. 2015 Dec 17;19(5):555–561. doi: 10.1007/s12603-014-0576-3

The effects of substance P on tendinopathy are dose-dependent: An in vitro and in vivo model study

Y Zhou 1, B Zhou 1, Kanglai Tang 1
PMCID: PMC12877608  PMID: 25923486

Abstract

Objectives

Substance P (SP) is known to be involved in neuropathic pain, chronic inflammation, and tendinopathy. The present study evaluated the effects of different doses of SP on tendon-derived stem cells (TDSCs) in vitro and tendons in vivo.

Methods

For the in vitro study, TDSCs cultured in growth medium with different concentrations of SP (negative control, 0.1 nM, and 1.0 nM). The effects of SP on TDSCs were examined with respect to their ability to proliferate and differentiate. For the in vivo study, we injected different doses of SP (saline control, 0.5 nmol, and 5.0 nmol) into rat patella tendons to investigate the effects of SP on tendons.

Results

Low and high doses SP significantly enhanced the proliferation ability of TDSCs. Low-dose of SP induced the expression of tenocyte-related genes; however, high-dose of SP induced the expression of non-tenocyte genes, which was evident by the high expression of PPARγ and collagen type II. In the in vivo study, only high-doses of SP (5.0 nmol) induced the tendinosis-like changes in the patella tendon injection model. Low doses of SP (0.5 nmol) enhanced the tenogenesis compared with saline injection and the high-dose SP group.

Conclusion

SP enhances the proliferation of TDSCs in vitro and the effects of SP on tendinopathy are dose-dependent in vivo..

Keywords: Tendons, stem cell, substance P, patella tendon, tendinopathy

Introduction

Traditionally, tendons are thought to contain only tenocytes. Tendon stem/progenitor cells were reported firstly in human and mouse tendons in 2007 and then subsequently confirmed to exist in rat and rabbit tendons (1, 2, 3). TDSCs differ from tenocytes in term of their colony-forming ability, self-renew ability, and multi-differentiation potential, which enables them to differentiate into tenocytes and non-tenocytes (2, 3). TDSCs are responsible for tendon maintenance and tendinopathy development (4, 5, 6).

SP is a neuropeptide that is widely distributed in the central and peripheral nervous systems. SP has been found to exert trophic effects in different cell types (7, 8). An increased amount of SP was previously reported in greater trochanteric pain syndrome (9), in the subacromial bursa of rotator cuff diseases (10), and in human rheumatoid arthritis synovial tissue (11). A previous study reported that tenocytes produced SP; and SP enhanced the proliferation of tenocytes (12). Our recent study also showed that TDSCs produced SP and high concentrations of SP induced non-tenocyte differentiation in TDSCs (13). However, it is still not known whether different concentrations of SP have different effects on TDSCs. We hypothesize that the effects of SP on TDSCs are concentration-dependent; low concentrations of SP enhance tenogenesis and high concentrations of SP induce TDSCs into non-tenocyte differentiation and tendinopathy.

Although the etiology of tendinopathy is considered to be multifactorial, the underlying mechanisms of tendinopathy are not clear. The role of neuropeptides in tendon pathology has been increasingly discussed in recent years (12). Previous studies have shown that tendinopathy was associated with SP-positive nerve fibers, the presence of granulation tissue, and haemosiderophages (12, 14). Recent studies further confirmed that SP accelerated the development of tendinopathy-like changes (15, 16). However, the temporal increasing expression of SP has been demonstrated in the healing of ruptured rat Achilles tendons. Injection of SP into the paratendinous region of ruptured and subsequently sutured rat Achilles’ tendons improved tendon healing (17). Therefore, we hypothesize that the effects of SP on tendons in vivo are dose-dependent, as low-does SP enhances tenogenesis and high-dose of SP induces tendinopathy.

This study was designed to determine the effects of different concentrations of SP on TDSCs in vitro and different doses of SP on tendons in vivo. In the in vitro study, we examined the proliferation and differentiation of TDSCs in response to different concentrations of SP. In the in vivo study, we administered different doses of SP injections in a rat patella tendon model and assessed the structural changes of tendons.

Materials and Methods

Study design

The ethics committee of the Southwest hospital approved the experimental protocols for the use of rat tendon samples and the tendon injection model in this study. Male Sprague–Dawley (SD) rats (n = 24; 6-8 weeks old) were followed using the Third Military Medical University Animal Experiment Centre’s protocol for the care and use of experimental animals. The rats had free access to food and water, and were housed in a 12-hour light/12-hour dark cycle. Six rats were used for isolating TDSCs in the in vitro study. TDSCs were seeded in growth medium (Dulbecco’s Modified Eagle’s Medium supplemented with 20% fetal bovine serum; Shanghai Juncheng Biotechnology Co, Ltd, People’s Republic of China) with different concentrations of SP respectively (negative control, 0.1 nM, and 1.0 nM). Another 18 rats were divided into three groups and underwent injection in the right patella tendon for the in vivo test. In group I, six rats received 50 ul of 100 nM SP injection (5.0 nmol); In group II, six rats underwent 50 ul of 10 nM SP injection (0.5 nmol); and in group III, six rats injected with 50 ul of saline. Our selected concentrations of SP were derived from other studies (18, 19). All rats were sacrificed by intramuscular injection with 10 mg / kg of ketamine for histology testing 14 days after injection.

Isolation of TDSCs and cell culture

TDSCs isolation procedures were carried out as previously described (3, 20). Briefly, the midsubstance patellar tendons were harvested after the rats were sacrificed. Peritendinous connective tissues were then carefully removed and surrounding adipose tissue and blood were cleaned off using phosphate buffered saline (PBS; pH 7.4). Next, the tendon samples were minced into fine pieces (1.0 x 1.0 x 1.0 mm3) and 10.0 mg of tendon tissue was digested with 3.0 mg of type I collagenase (Worthington Biochemical Corp, Lakewood, NJ, USA) and 4.0 mg of dispase (Stem Cell Technologies, Vancouver, BC, Canada) in 1 mL of PBS at room temperature for 2 hours. The cell suspensions were then centrifuged at 1500 g for 5 minutes. The supernatants were discarded and the cells were resuspended in growth medium (DMEM + 20% fetal bovine serum [FBS]). Cells were cultured separately in 6-well culture plates. The TDSCs formed adherent colonies after 9–12 days in culture. We detached cell colonies using trypsin and visually examined the cells under a microscope; we then used a micropipette to collect each individual detached cell colony. Finally, 100 cells from the detached colonies were subcultured in 25 cm2 dishes with different concentrations of SP (1.0 nM, 0.1 nM, negative control) for colony tests and further studies. A minimum of triplicates of cells from six rats was used for each experimental condition; we used passage numbers one to four TDSCs for all in vitro tests.

Immunostaining of Stem Cell Markers

To characterize and confirm TDSCs, we performed immunostaining to detect the expressions of nucleostemin (NS), octamer-binding transcription factor 4 (Oct-4), and Nanog. TDSCs at passage two were seeded in a 24-well dish at a density of 1.0 x 104 / well and were cultured for another 24 hours. Next, 4% paraformaldehyde was then applied for 10 minutes at room temperature to fix the cells. TDSCs were treated with 0.1% Triton X-100 in PBS for 10 minutes and washed three times with PBS. They were then incubated with rabbit anti-rat NS, rabbit anti-rat Oct-4, or rabbit anti-rat Nanog (1:350; Santa Cruz Biotechnology, Santa Cruz, CA, USA) respectively overnight at 4°C. Cultures were then washed three times with PBS, followed by the application of Cy3-conjugated goat anti-rabbit immunoglobulin G (IgG) antibodies (1:500, Santa Cruz Biotechnology) for 2 hours at room temperature. After washing the cells with distilled water, stained cells were observed under fluorescence microscopy and digital images were recorded.

Flow cytometric analysis

Flow cytometric analysis was used to confirm TDSCs and to examine the gene expressions of stem cell in TDSCs. TDSCs at passage one were harvested by trypsinization, then centrifuged at 500 g for 5 minutes at room temperature, and resuspended in microcentrifuge tubes at a density of 1 x 104 cells in 20 μL of staining buffer (Becton, Dickinson and Co, Franklin Lakes, NJ, USA). Next, 20 μL of the appropriate primary antibody was added to each vial and the cells were then incubated at 4°C for 2 hours. The cells were then washed and incubated with a phycoerythrin (PE) or fluorescein isothiocyanate (FITC) conjugated secondary antibody at 4°C for an additional hour. The cells were again washed with PBS and analyzed in the Guava Personal Cytometry system using the protein expression software package, analyzing at least 50,000 cells per sample. Antibodies were used according to the manufacturers recommended dilutions.

TSC Proliferation

To determine the effects of SP on TDSC’s proliferation, we performed both the populations doubling time (PDT) test and cell counting Kit-8 (CCK-8) tests. To measure PDT, TDSCs were seeded onto a 6-well dish at a density of 5 x 104/well. Growth medium was changed with different concentrations of SP (negative control, 0.1 nM, and 1.0 nM) after 12 hours of culture. Then, the medium was changed with different concentrations of SP each day. The number of the cells was counted at 36, 48, and 72 hours respectively using the cellometer Auto T4 automated cell counter (Nexcelom Bioscience LLC, Lawrence, MA, USA) and the PDT was calculated. For the CCK test, TDSCs were seeded onto 96-well culture dishes at a density of 4,000 cells / well in 100 μL of growth medium in regular 20% O2 culture conditions for 4 hours. Then, the growth medium was changed and replaced with different concentrations of SP (negative control, 0.1 nM, and 1.0 nM). Next, 10 μL of CCK-8 solution was added to each well of the dish after seeding for 24 hours and incubating for another 2 hours. Finally, the absorbance was measured at 450 nm using a microplate reader (SpectraMax Plus384 Absorbance Microplate Reader; Molecular Devices, LLC, Sunnyvale, CA, USA).

Gene Expression Analysis

To characterize the differentiation of TDSCs to different concentrations of SP, we determined the expression of tenocyte-related and non-tenocyte related genes in TDSCs using quantitative real-time polymerase chain reaction (qRT-PCR). Briefly, 2 x 105 TDSCs were seeded in a T25 flask and cultured in growth medium with three different concentrations of SP (negative control, 0.1 nM, and 1.0 nM) respectively for 72 hours. Total RNA was extracted from TDSCs using the RNeasy Mini Kit (Qiagen, Limburg, the Netherlands). Collagen (Col) types I and Col II, osteogenic transcription factor runt-related transcription factor 2 (Runx2) and peroxisome proliferator-activated receptorγ (PPARγ) were examined. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. Forward and reverse primers for all genes were designed based on previously published sequences (4, 21, 22), and were synthesized by Life Technologies (Carlsbad, CA, USA). At least three replicates were performed for each gene and each experimental condition.

Rat injection model and histology

A BD syringe with a 32-gauge needle was inserted from distal to proximal and from anterior to posterior at a 30° angle to the skin. The insertion site was approximately 5.0 mm above the patella tendon insertion of the tibia. After the skin was penetrated, the needle was moved forward slightly, and the solutions were slowly injected. Afterwards, the rats were allowed to move freely in their cage with unrestricted access to water and food. Patella tendons were harvested 2 weeks after SP injection and quickly snap-frozen in tissue-freezing medium (Triangle Biosciences, Durham, NC, USA) and stored at -80°C. Frozen tendon tissues were sectioned at a thickness of 8.0 mm in a cryostat and subjected to hematoxylin and eosin (H&E) staining or Masson staining.

Statistical Analysis

We used the statistical package of social sciences for Windows, version 21.0 (IBM Corporation, Armonk, NY, USA) for statistical analyses. All data are presented as the mean ± standard deviation (SD). The Shapiro-Wilk test was used for normality of the data before one-way analysis of variance (ANOVA) was used for multiple comparisons. Differences were considered significant when the p-value was less than 0.05.

Results

Characterization of TDSCs

To characterize TDSCs, TDSCs were first identified by their colony-forming ability, immunostaining of the stem cell markers, and flow cytometry analysis. The cells isolated from rat patella tendons formed the colony after 8-10 days of culture (Fig. 1). TDSCs treated with 1.0 nM of SP formed significantly more colonies compared with 0.1 nM of SP and the negative control (P < 0.05). The number of colonies treated with 1.0 nM of SP, 0.1nM of SP and negative control was 38 ± 4.27, 56 ± 5.13, and 84 ± 9.04, respectively. TDSCs were positively stained by stem cell markers OCT-4, Nanog, and NS (Fig. 2). The results of flow cytometry showed that TDSCs were negative for CD34, CD45 (cell surface marker for the hematopoietic cells), CD44 (cell surface marker for the mesenchymal stem cells) and CD31 (cell surface marker for the endothelial cells). However, TDSCs were positive for CD90 (cell surface marker for the fibroblasts) (Fig. 3).

Figure 1.

Figure 1

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Number of colonies

Figure 2.

Figure 2

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Figure 3.

Figure 3

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Flow cytometry analysis of TDSCs

SP significantly enhanced the proliferation of TDSCs

We tested the proliferative ability in TDSCs at passage three by PDT and CCK test. Results showed that SP significantly enhanced the proliferation of TDSCs; The PDT of TDSCs cultured with negative control or 0.1 nM or 1.0 nM of SP was 29.03 ± 3.73 hours, 19.49 ± 1.44 hours, and 16.73 ± 1.93 hours, respectively (Fig. 4). The CCK-8 test showed that the absorbance values of TDSCs cultured in the negative control or 0.1 nM or 1 nM of SP for 24 hours were 0.46 ± 0.05, 0.61 ± 0.05, and 0.82 ± 0.04, respectively. The absorbance value of TDSCs cultured in the negative control or 0.1 nM or 1 nM of SP for 48 hours were 0.67 ± 0.06, 0.84 ± 0.05, and 1.14 ± 0.06, respectively (Fig. 5).

Figure 4.

Figure 4

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The PDT of TDSCs

Figure 5.

Figure 5

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SP significantly enhanced the proliferation of TDSCs

SP-induced the differentiations of TDSCs were concentration-dependent

The multi-differentiation potentials of TDSCs toward osteogenesis, adipogenesis and chondrogenesis were determined by qRT-PCR. Treatment with high concentrations of SP (1.0 nM) significantly enhanced the expression of adipocyte and chondrocyte genes. The expression of PPARγ and Col II in the TDSCs treated with 1.0 nM SP had 19.1-fold and 0.4-fold increases compared with the negative controls, respectively. However, the expression of Col II and PPARγ in the TDSCs treated with lower concentrations of SP (0.1 nM) had 84% and 75% decreases compared with the negative controls, respectively. Additionally, the expression of Runx2 in TDSCs treated with 1.0 nM of SP and 0.1 nM of SP had a 48% and 88% decrease, respectively, as compared with the negative control. Conversely, the treatment of lower concentrations of SP (0.1 nM) significantly increased the expression of Col I. The expression of Col I in TDSCs treated with 0.1 nM of SP and 1.0 nM had 16-fold and 4.4-fold increases, respectively, compared with the negative control (Fig. 6).

Figure 6.

Figure 6

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SP-induced tendinopathy was dose-dependent

We further performed in vivo experiments to examine the effects of different doses of SP injections into patella tendons. Patella tendons exhibited tendinosis-like changes with high-dose SP injection (5.0 nmol) via H&E and masson staining two weeks after injection. H&E staining showed that the alignment of collagen fibers was disorganized and more cells appeared in the injection location in the high-dose SP group. No obvious tendinopathy change was found in low-dose SP group (0.5 nmol) or the saline control group. However, there were more cells in the low-dose SP group compared with the saline control group (Fig. 7A-I). Masson staining results showed that with the treatment of high SP does treatment (5 nmol), the presence of tenogenesis formation was less and more disorganized compared with low SP does treatment (0.5 nmol) and the saline control injection (Fig. 7J-R).

Figure 7.

Figure 7

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H&E and Masson staining of patella tendons tissue

Discussion

TDSCs play a major role in maintaining metabolic balance and in the development of tendinopathy (3, 23). Recent studies have shown that SP accelerated the development of tendinosis-like changes (14, 16, 24). Considering that SP appears under both normal physiological condition and tendinopathy tendon (12, 25), It is essential to define the effects of different dose of SP on TDSCs and tendons in order to gain a better understanding of tendinopathy. Our study showed that both low concentrations of SP and high concentrations of SP significantly enhanced the proliferation of TDSCs in vitro. However, the effects of SP on tendinopathy were dose-dependent. Low doses of SP enhanced the tenogenesis and high doses of SP induced tendinopathy changes in vivo.

SP is known to be involved in chronic inflammation, neuropathic pain, and tendinopathy (26, 27). Recent studies have reported that tenocytes produce SP and are involved in the tendinopathy (28, 29). Tissue concentrations of SP in the Achilles tendon and paratenon and bony insertion, as assessed by RIA was increased by 22% (30). Endogenous SP production in tendons was enhanced under various conditions, including loading (31), pilocarpine (32), and induced tendon injury (33). There are two possible mechanisms for the increased SP’ production. One is local stimulation of neuropeptidergic nerves. The number of nociceptive SP-positive nerve fibers was found to be significantly higher in Achilles tendinosis samples than in tendon samples (14). The booster effect of endogenous SP for fibroblast proliferation via autocrine/paracrine stimulation is another probable mechanism (18). Three weeks of exercise lead to both obvious proliferation of tenocytes and increased endogenous SP’ production in a rabbit model (31). Moreover, inhibitors of endopeptidase and angiotensin-converting enzyme led to increased SP concentrations, and a pronounced myositis change (34). Finally, the SP antagonist, capsaicin blocks both neurogenic and non-neurogenic mediators of inflammation (35) and reduced concentrations of SP by ~60% (36). Taken together, the observed SP up-regulation in the tendon suggests a refined balance, which moderated by the autocrine and central nervous systems.

The underlying tissue changes during tendinopathy mainly constitute hypercellularity (37). In our study, we found that SP significantly enhanced the proliferation of TDSCs in vitro and in tenocytes in vivo. These results were consistent with those of previous studies in which SP accelerated hypercellularity in rabbit Achilles tendon and human Achilles tendon cells (16, 25, 38, 39). The probable mechanism for these changes include that SP reduces tumor necrosis factor-α (TNF-α)-induced cell death (40) and SP fostered interleukin -6 (IL-6) and IL-8 production in rheumatoid arthritis (41). The mitogenic cell signaling pathway, namely phosphorylation of ERK1/2 might be another probable mechanism (19). Moreover, SP stimulated DNA synthesis in fibroblasts (42). Collectively, SP enhances the proliferative ability of TDSCs and may play a critical role in tendinopathy.

In addition, we investigated the effects of SP on the differentiation of TDSC. Our in vitro experiments showed that high concentrations of SP enhanced the expression of PPARγ and collagen type II, but decreased the tenogenic differentiation. In contrast, low concentrations of SP did not induce the differentiation of TDSCs into non-tenocytes in vivo, but enhanced tenogenesis and Col I expression. Wang et al reported that prostaglandin E2 (PGE2) exerted biphasic effects on human tendon stem cells; high levels of PGE2 induced hTSCs to differentiate aberrantly into non-tenocytes (43). In our in vivo testing, high doses of SP induced tendinopathy-like changes, while low doses enhanced the tenogenesis. These results have never previously been shown for SP. SP was reported to enhances collagen remodeling and MMP-3 expression by human tenocytes (28, 29), which mediated macrophage activation (44). On one hand, SP accelerated the development of tendinosis-like changes (16, 25). On the other, SP supply in tendon repair promoted early tissue proliferation for the healing of ruptured rat Achilles tendons (17). Therefore, the administration and monitoring of the SP concentration might be of interest as a potential new approach to for the treatment of Achilles tendon rupture and tendinopathy. Collectively, it is reasonable to assume that the effects of SP on tendinopathy are in a dose-dependent manner.

The limitations of this study were that we did not show the probable molecular mechanisms by which SP affected the proliferation and differentiation of TDSCs. However, this study focused on characterizing the effects of different doses of SP on tendons and tendinopathy. Thus, the limitation does not undermine our conclusion that the effects of SP on tendinopathy are dose-dependent. Further studies are needed to clarify the mechanisms of different doses of SP on tendons.

In summary, this study was designed to define the effects of different doses of SP on TDSCs in vitro and tendons in vivo. The findings reported here show that SP enhanced the proliferation of TDSC and that the effects of SP on tendinopathy are dose-dependent; low concentrations of SP is beneficial to tendons due to enhanced tenogenesis; However, high concentrations of SP may be detrimental, as it induced tendinopathy changes in vivo.

Acknowledgments: This research is supported by grants from the National Natural Science Foundation of China (81230040) and China Scholarship Council (CSC) sponsors Binghua Zhou.

Conflict of Interest Statement: We declare no conflicts of interest.

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