Perivascular cells of the supraspinatus tendon express ... - Springer Link

Histochem Cell Biol (2009) 131:733–741 DOI 10.1007/s00418-009-0581-5

ORIGINAL PAPER

Perivascular cells of the supraspinatus tendon express both tendon- and stem cell-related markers Herbert Tempfer · A. Wagner · R. Gehwolf · C. Lehner · M. Tauber · H. Resch · H. C Bauer

Accepted: 19 February 2009 / Published online: 11 March 2009 © Springer-Verlag 2009

Abstract Tendons and ligaments are often aVected by mechanical injuries or chronic impairment but other than muscle or bone they possess a low healing capacity. So far, little is known about regeneration of tendons and the role of tendon precursor cells in that process. We hypothesize that perivascular cells of tendon capillaries are progenitors for functional tendon cells and are characterized by expression of marker genes and proteins typical for mesenchymal stem cells and functional tendon cells. Immunohistochemical characterization of biopsies derived from intact human supraspinatus tendons was performed. From these biopsies perivascular cells were isolated, cultured, and characterized using RT-PCR and Western blotting. We have shown for the Wrst time that perivascular cells within tendon tissue express both tendon- and stem/precursor cell-like characteristics. These Wndings were conWrmed by results from in vitro studies focusing on cultured perivascular cells isolated from human supraspinatus tendon biopsies. The results suggest that the perivascular niche may be considered a source for tendon precursor cells. This study provides further H. Tempfer and A. Wagner have contributed equally to this paper. H. Tempfer (&) · A. Wagner · R. Gehwolf · C. Lehner · H. C Bauer Division of Zoology and Functional Anatomy, Department of Organismic Biology, University of Salzburg, Hellbrunnerstr. 34, 5020 Salzburg, Austria e-mail: [email protected] H. Tempfer · R. Gehwolf · C. Lehner · H. Resch · H. C Bauer Paracelsus Private Medical University, Strubergasse 21, 5020 Salzburg, Austria M. Tauber · H. Resch Department of Traumatology and Sports Injuries, University Hospital of Salzburg, Müllner Hauptstr. 48, 5020 Salzburg, Austria

information about the molecular nature and localization of tendon precursor cells, which is the basis for developing novel strategies towards tendon healing and facilitated regeneration. Keywords Supraspinatus tendon · Tendon stem/ progenitor cells · Perivascular cells · RT-PCR · Regeneration

Introduction Tendons are bands of Wbrous connective tissue attaching muscle to bone and transmitting tensile forces generated by the muscle. They are characterized by low vascularisation and a low cell density. Tendon cells produce the extracellular matrix (ECM) which is responsible for the high tensile strength, consisting mainly of collagens type I and III and several proteoglycans (Kannus 2000). Since tendons are often aVected by mechanical injuries or degenerative impairment, the renewal of tendon cells and the reconstruction of functionally und structurally intact tendon tissue are of great medical interest. Tendon cells are poorly characterized so far and it is not clear whether they are Wbroblasts or a separate cell class. Based on various phenotypes tendon cells are sometimes described as tenoblasts and/or tenocytes, the latter being the diVerentiated form (Chuen et al. 2004; Kannus 2000). A variety of potential marker proteins, such as tenascin-C, biglycan, tenomodulin or decorin (Docheva et al. 2005; Oshima et al. 2006; Riley 2008; Shukunami et al. 2006; Trebaul et al. 2007; Tufvesson and Westergren-Thorsson 2003) have been related to tendon cells, however, none of these proteins is exclusively expressed in tendon tissue (Docheva et al. 2005; Oshima et al. 2006; Riley 2008; Shukunami

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et al. 2006; Trebaul et al. 2007; Tufvesson and WestergrenThorsson 2003). Recently, a population of stem/progenitor cells residing within tendons has been described, showing the potential of self renewal and the capability to give rise to various cell types like adipocytes and osteoblasts. The particular composition of the progenitors’ ECM, which contains constituents speciWc for their place of origin, gives a hint about the progenitors’ niche (Bi et al. 2007). Thus, it appears that the molecular environment provided by the niche is essential for the diVerentiation of mesenchymal progenitor cells. This has been shown, for example, for the adipose tissue-derived stroma cells which are associated with perivascular cells (Zannettino et al. 2008). These cells in the perivascular niche are considered to play a role in regeneration of various tissues. Such mechanism may also apply for other tissues like bone, muscle or tendon, the matrix of which is also produced by cells of mesenchymal origin. Stem/progenitor cells of mesenchymal origin are supposed to be important in tendon formation and healing and are of great interest as a possible tool for accelerating the healing process (Bi et al. 2007). In this study, we have shown for the Wrst time that perivascular cells within tendon tissue express tendon- and stem/precursor cell-like characteristics. In order to further substantiate our Wndings from in vivo studies, cultured tendon-derived perivascular cells were subjected to molecular and functional analyses. In summary, results from our in vivo and in vitro studies suggest that perivascular cells of human supraspinatus tendons exhibit speciWc properties usually attributed to both, mature tendon tissue and mesenchymal stem/precursor cells.

Histochem Cell Biol (2009) 131:733–741

Biopsies were taken according to a standardized protocol. The location of tissue harvesting was close to the bony footprint at the greater tuberosity, where usually degenerative alterations and tendon tears occur. For biopsy, an arthroscopic punch was used, which creates a tissue cube with an edge length of about 3–4 mm. Immunohistochemistry For immunohistochemistry, the specimens were Wxed in cold 4% paraformaldehyde (PFA), paraYn embedded and sectioned (thickness 5–7 m). Sections were deparaYnized in xylene, rehydrated through a graded ethanol series, and incubated with 5% normal goat serum in phosphate buVered saline (PBS) for 30 min. Sections were incubated with antibodies recognizing Musashi-1 (Msi-1) (Chemicon, Billerica, MA, USA), Nestin (Santa Cruz Biotechnology, Billerica, MA, USA), alpha smooth muscle actin (SMA) (Sigma, St Louis, MO, USA) and Prominin/CD133 (Abcam, Cambridge, UK). Incubation was performed at 4°C overnight in a humidiWed chamber. Endogenous peroxidase activity was quenched with ddH2O containing 3% peroxide for 20 min at room temperature. After treatment with a peroxidase-conjugated secondary antibody (Chemicon, Billerica, MA, USA) for 1 h at room temperature, the sections were treated with DAB (3,3⬘diaminobenzidine tetra hydrochloride, ready to use tablets, Sigma, Vienna, Austria), counterstained with May–Gruenwald dye (Merck, Darmstadt, Germany) for 2 min, dehydrated, and mounted in Eukitt (Sigma, Vienna, Austria). As control, primary antibodies were omitted and staining was done as described above. In situ hybridization

Materials and methods Eleven biopsies of intact human supraspinatus tendons were obtained during posttraumatic surgical interventions not involving the rotator cuV (open Bankart repairs and open glenoid fractures) with patients’ informed consents. The patients’ ages ranged from 19 to 65 years (mean 42 years). Before undergoing shoulder surgery, all patients were subjected to magnetic resonance imaging (MRI) as additional diagnostic tool to exclude rotator cuV pathology. Any radiological sign on MRI of tendinopathy, either tendinitis or partial or complete tear formation was a deWnite exclusion criterion for biopsy harvesting. In addition, intraoperatively the supraspinatus tendon was evaluated macroscopically for integrity. In the case of macroscopic degenerative changes, as tendon fraying for example, biopsy was not performed.

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For in situ hybridization, paraYn sections were treated according to standard procedures. Sense and antisense Scleraxis cRNA probes were transcribed in vitro from cDNA templates and labelled with Digoxigenin-, or BiotinUTP (RNA labelling mix, Roche Applied Sciences, Vienna, Austria), using Sp6, T3 or T7 polymerase, according to the manufacturers’ instructions. After over night probe hybridization, sections were incubated with an alkaline phosphatase-conjugated anti-Digoxygenin antibody (anti-Dig-Fab fragments, Roche Applied Sciences, Vienna, Austria) at 4°C overnight for detection of hybridized Scleraxis-probe. Alkaline phosphatase activity was assessed by NBT/BCIP staining (Nitro blue tetrazolium chloride/ 5-Bromo-4-chloro-3-indolyl phosphate, toluidine salt; Roche Applied Sciences, Vienna, Austria). Peroxidase activity was visualized using DAB as described above. Sections were counterstained with Nuclear Fast Red™ (Sigma, Vienna, Austria).


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RNA preparation and reverse transcription (RT)-PCR Total RNA from tendon biopsies and cultured tendon cells was isolated using the PureZol™ RNA isolation kit (Biorad, Vienna, Austria). RNA yield and integrity were evaluated by spectrophotometry and by agarose gel electrophoresis. To avoid cross-contamination by genomic DNA, 1 g total RNA was digested with DNase I (Fermentas, St LeonRoth, Germany) prior to Wrst strand cDNA synthesis according to manufacturer’s instructions. After inactivation of DNase I RNA samples were subjected to Wrst strand cDNA synthesis in 20 l reaction volumes using 40 units of RevertAid™ RNase H¡ M-MuLV-Reverse Transcriptase (Fermentas, St Leon-Roth, Germany) and 25 pmol oligo(dT)18-primer, 0.25 mM dNTPs, 1£ Wrst strand synthesis buVer and 50 units of RiboLock™ RNase inhibitor (Fermentas, St Leon-Roth, Germany) at 42°C for 30 min, 50°C for 45 min and a Wnal termination step at 72°C for 5 min. If possible, intron spanning primers for RT-PCR were designed according to the annotated human sequences of Smooth-muscle -actin2 (SMA; NM_001613), Musashi-1 (Msi-1, NM_002442), Scleraxis (Scx, NM_001080514), Prominin-1 (CD133; NM_006017), Nestin (NM_006617), Collagen type I (Col-I, NM_000088), Collagen type III (Col-III, NM_000090), Smad8/Smad9 (Smad9 is referred to as Smad8 in the literature, NM_005905), CD29 (NM_033666), and CD44 (NM_001001392). As internal control for RT-PCR, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; NM_002046) and Hypoxanthine phosphoribosyl transferase 1 (HPRT; NM_000194) transcripts were ampliWed (all primer sequences are listed in Table 1). A 30 l PCR reaction mixture contained 1–3 l cDNA, 0.5 units of TrueStart™ Taq DNA polymerase (Fermentas,

St Leon-Roth, Germany), 1£ TrueStart™ Taq DNA polymerase reaction buVer, 3.5 mM MgCl2, 0.2 mM dNTPs and 300 nM each of the primers. The thermal cycling program included an initial denaturation step of 3 min at 94°C followed by 40 cycles of 30 s at 94°C, 30 s at 55°C, 30 s at 72°C and a Wnal extension step of 2 min at 72°C. AmpliWed PCR fragments were analyzed by agarose gel electrophoresis and sequence veriWcation of the ampliWcation products was performed according to standard procedures. Isolation and culture of tendon-derived cells Freshly obtained biopsies from human supraspinatus tendons (wet weight: 0.5 g each) were cut into small pieces under sterile conditions followed by a digestion step (4 h up to 12 h, depending on tissue quality and enzyme batch) in Dulbecco’s ModiWed Eagle’s Medium (DMEM), supplemented with 30 mg/ml Collagenase II (Gibco, Invitrogen, Lofer, Austria) at 37°C, 95% humidity and 5% CO2. After digestion, small pieces of microvessels were transferred to uncoated 25 cm2 cell culture Xasks and cultured for up to 2 weeks. The outgrowing cells were expanded until subconXuence in DMEM supplemented with 10% fetal bovine serum. This culture medium hampers growth and proliferation of the slowly dividing capillary endothelial cells which disappeared entirely after feeding procedures and passages of cultures. In the following, the outgrowing cells will be referred to as tendon-derived perivascular cells (TPVCs). Bandeiraea simplicifolia lectin (BSL) staining was performed with unWxed cells using a Rhodamine-conjugated BSL Isolectin B4 (Sigma-Aldrich, Vienna, Austria). In short, TPVCs were washed with phosphate-buVered saline (PBS) and incubated at 4°C for 90 min in 5 g/ml BSLRhodamine in PBS/10% FCS. Cells were then washed twice for 15 min with PBS at room temperature and treated with DAPI (4⬘-6-diamidino-2-phenylindole) (Invitrogen,

Table 1 Primer sequences for RT-PCR Sense primer

Antisense primer

Acc.No.

GAPDH 5⬘-AACATCATCCCTGCCTCTAC-3⬘

5⬘-CTGCTTCACCACCTTCTTG-3⬘

NM_002046

HPRT

5⬘-TGCTTTCCTTGGTCAGGCAGTATA-3⬘

5⬘-GCGATGTCAATAGGACTCCAGAT-3⬘

NM_000194

SMA

5⬘-TGTTCCAGCCATCCTTCATCGG-3⬘

5⬘-TGGAGCCACCGATCCAGACAGAG-3⬘

NM_001613

Msi-1

5⬘-AGCTTACAGCCATTCCTCTCACTGC-3⬘

5⬘-TGGTGAAGGCTGTGGCAATCAAAG-3⬘

NM_002442

Scx

5⬘-TGCGCCTGGCCTCCAGCTACATC-3⬘

5⬘-GTTGCTGAGGCAGAAGGTGCAGA-3⬘

NM_001080514

CD133

5⬘-TCACTGAGCACTCTATACCAAAGCG-3⬘

5⬘-TTGCACGATGCCACTTTCTCACTG-3⬘

NM_006017

Nestin

5⬘-TCTGAGGAAGTGGGGCAAGGAAT-3⬘

5⬘-TTAAGAGTGCTGCTCCTGAGCAGG-3⬘

NM_006617

Col-I

5⬘-CCAGTCACCTGCGTACAGAA-3⬘

5⬘-GAGACCACGAGGACCAGAAG-3⬘

NM_000088

Col-III

5⬘-AGTGCCAATCCTTTGAATG-3⬘

5⬘-TATGTGATGTTCTGGGAAGC-3⬘

NM_000090

Smad8

5⬘-AACGCCACCTA(CT)CCTGACTCTTTCCAG-3⬘ 5⬘-TTCCAGGC(GCT)TCCTCCCG(AG)AG(CT)GTG-3⬘ NM_005905

CD29

5⬘-AATGAAGGGCGTGTTGGTAG-3⬘

5⬘-CGTTGCTGGCTTCACAAGTA-3⬘

NM_033666

CD44

5⬘-GCAATGCTTCTCAGACCACA-3⬘

5⬘-CTGGCCAATGTAGTTCACAG-3⬘

NM_001001392

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Lofer, Austria) at a Wnal concentration of 1 g/ml at room temperature for 5 min. After washing with PBS, cells were analyzed with a Zeiss Axioplan™ Xuorescence microscope and images were taken using the Zeiss Axiovision™ software. As a positive control, BSL/DAPI staining was done with rat brain endothelial cells (RBECs), kindly provided by I. Krizbai (Inst. Biophysics, Biol. Res Ctr., Szeged, Hungary). Western blotting, detection of collagen type I secretion Collagen type I secretion was determined as described by Yang et al. (2004). BrieXy, cells were grown in two culture Xasks at equal densities; one Xask was then incubated with 40 g/ml ascorbic acid for 24 h. One millilitre of cell supernatant was digested with 100 g/ml pepsin under acid conditions, precipitated with 3 M NaCl, dialyzed against 0.05 M NH4CO3, and vacuum dried. The protein pellet was resolved in lysis buVer containing 20 mM Tris–Cl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM Pefabloc SC, 5 g/ml aprotinin, and 5 g/ml leupeptin. Protein contents were quantiWed spectrophotometrically using a BCA™ protein assay kit (Pierce, Rockford, USA). Proteins of the supernatant were separated by native PAGE and blotted onto PVDF membranes as previously described (Yang et al. 2004). Immunodetection was performed using a primary antibody directed against collagen type I (rabbit polyclonal, # ab292, Abcam, Cambridge, UK).

Results To identify the occurrence of precursor-like cells in human tendon tissue we used various methodological approaches, including immunohistochemistry, ISH, and RT-PCR. Immunohistochemistry and in situ hybridisation on histological sections derived from human supraspinatus tendons revealed an overlapping expression pattern of Musashi-1, Scleraxis, Nestin, SMA, and Prominin-1/CD133 in perivascular areas (Figs. 1, 2, 3, 4). Interestingly, part of the spindle-shaped Wbroblasts in the dense collagenous tissue also expressed Musashi-1, Nestin, Scleraxis or SMA, but deWnitely lacked Prominin-1/CD133. RT-PCR on RNA isolated from whole supraspinatus tendon tissue basically conWrmed the expression of the marker proteins mentioned (not shown). To verify our results from in vivo studies perivascular cells were isolated from tendon biopsies. After the third passage, this procedure yielded a homogenous cell population, referred to as tendon-derived perivascular cells, TPVCs, which was further biochemically and immunologically characterized. As evidenced by BSL staining, endothelial cells were successfully eliminated from the TPVC

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population (Fig. 5a). Light microscopic inspection of TPVCs has shown that most of the cells released from adherent microvessels exhibit a polygonal to Wbroblastic phenotype (Fig. 5b). RT-PCR on RNA isolated from subconXuent TPVCs revealed expression of SMA, Musashi-1, Scleraxis, Prominin-1/CD133, Nestin, Collagen type I and type III, Smad8, CD29, and CD44 (Fig. 5c). Western blotting conWrmed the expression of collagen type I in TPVCs and additionally showed an increase in collagen type I secretion after treatment with 40 g ascorbic acid, a substance which is known to increase collagen type I expression in all collagen I producing cells (Fig. 5d). Generally, there was no association observed between donor age or gender and expression levels of the markers examined in this work.

Discussion In this study, we have focused on the tendon- and stem cellrelated marker proWle of tendon tissue-derived perivascular cells in vivo and in vitro. The supraspinatus tendon is the most frequently aVected tendon in rotator cuV injury and thus regeneration after injury is an important issue in orthopaedic treatment. Regeneration is considered to be induced by tendon cells which are sparsely distributed in tendon tissue and which produce large amounts of matrix components, mostly collagen type I, following a complex process of matrix remodelling (Lo et al. 2004). The nature of tendon cells is a matter of controversy and so is the origin of these cells. Several lines of evidence have indicated that tendon precursor cells originate from mesenchymal stem cells which maturate and diVerentiate into matrix producing tenocytes (Chuen et al. 2004; Salingcarnboriboon et al. 2003). This diVerentiation process takes place in speciWc non-cellular niches which determine the Wnal phenotype of tendon cells. How these precursor cells populate tendon tissue and where they reside precisely is unknown. Here, we have investigated the distribution of tendon precursor cells in the human supraspinatus tendon, using the classical stem cell markers Musashi-1, Nestin, Prominin-1/CD133, CD29, and CD44 as well as the tendon-speciWc markers Scleraxis and Smad8. In addition, expression of collagen type I and III and the pericyte-associated marker SMA were monitored. Musashi-1, a member of an evolutionarily conserved family of RNA-binding proteins, is preferentially expressed in fetal and adult neural stem cells (Okano et al. 2005). Its mammalian homologue has attracted much interest because of its occurrence in human muscle-derived stem cells (Tamaki et al. 2007). The latter were shown to be capable of giving rise to neurons, endothelial cells and smooth


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Fig. 1 In situ hybridization on 7 m paraYn sections derived from a human supraspinatus tendon. Arrows indicate the localization of Scleraxis mRNA in perivascular areas (a) (bar 100 m, insert: bar 50 m) and in tendon Wbroblasts of the dense collagenous tissue (b) (bar 100 m). c, d: Hybridization with a Scleraxis sense probe (controls). Counterstaining: Nuclear Fast Red (bar 100 m)

muscle cells. Thus, Musashi-1 may be considered a relevant marker not only for neural stem cells, but also for cells derived from the mesenchymal stem cell lineage. Nestin, an intermediate Wlament protein is, like Musashi1, a common marker for neural progenitor cells, though it is also found in early embryonic muscle (Sejersen and Lendahl 1993) and in hair follicle sheath progenitor cells (Li et al. 2003). Prominin-1/CD133+ cells represent a population of noncommitted early progenitors, capable of self-renewing and diVerentiating into blood cells and other cell types. Antibodies against Prominin-1/CD133 are suitable for selecting and isolating cells from umbilical cord blood. Such cells give rise to mesenchymal stem cells in culture after four passages (Tondreau et al. 2005). Using immunohistochemical staining we have demonstrated that Musashi-1, Scleraxis, Nestin, and Prominin-1/ CD133 are expressed in perivascular cells (PVCs) in tendon tissue in vivo, suggesting a precursor-like nature of these cells which are obviously not yet committed to a deWned lineage. However, the occurrence of the tendon-related Scleraxis protein in these cells might be considered the

result of a Wrst lineage restriction directing these cells towards a tendogenic fate. Scleraxis encodes a b-HLH transcription factor which plays an important role in tendon development and formation (Brent et al. 2003; Murchison et al. 2007; Schweitzer et al. 2001). It is expressed mainly in tendon progenitor cells, but also in functional tendon cells. Here we show for the Wrst time that Scleraxis is expressed in perivascular cells of adult human tendon tissue. The strong expression of SMA, a cytoskeletal constituent, usually associated with pericytes, suggests that PVCs may well be of pericytic nature. In muscle, bone marrow, and dental pulp similar cells, also classiWed as “pericytes”, were observed previously (Dellavalle et al. 2007; Shi and Gronthos 2003). In order to further substantiate and to augment our histological Wndings derived from in vivo studies with tendon biopsies we established an in vitro system to characterize perivascular cells in culture. To this end, microvessels were carefully isolated from tendon biopsies and kept in a culture medium which suppresses endothelial growth and proliferation but, in parallel,

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Fig. 2 Immunohistochemical staining on 7 m paraYn sections derived from a human supraspinatus tendon. Arrows indicate expression of Musashi1 in perivascular cells (a, b) and in tendon Wbroblasts (c, d) in the dense collagenous tissue. Counterstaining: May–Gruenwald dye (bar a, c: 100 m; b, d: 50 m)

favours growth and diVerentiation of rapidly proliferating, non-endothelial cells of any kind (including Wbroblasts, astrocytes and tumour epithelial cells). As shown in Fig. 5b, the morphological appearance of the outgrowing cells, strongly resembled that of pericytes, which were described by us and others previously (Ehler et al. 1995; Shepro and Morel 1993). However, it has to be kept in mind that pericytes are morphologically and biochemically heterogeneous, which in general makes it diYcult to categorize perivascular cells in culture (Shepro and Morel 1993). Importantly, we have found that part of the tendon Wbroblasts (tenocytes) of the dense collagenous tissue express stem/precursor cell-related markers such as Nestin and Musashi-1 but lack Prominin-1/CD133. Although experimental evidence is still missing, we are tempted to speculate that these cells represent a less diVerentiated population of tendon cells residing in mature tendon tissue, probably contributing to an early response in tendon growth and/or regeneration. The observation that only perivascular cells but not tendon Wbroblasts express the early stem cell marker Prominin1/CD133 could be a Wrst clue towards a “maturation gradient” of tendon precursor cells, being lowest in the perivascular niche.

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Analysis of gene expression in cultured TPVCs largely conWrmed the results from in vivo studies. By use of RTPCR, SMA, Musashi-1, Scleraxis, Prominin-1/CD133, Nestin, CD29, and CD44 transcripts were detectable in TPVCs. Even a weak expression of Smad8 appeared to be retained in these cells. Smad8 is a member of the receptorregulated Smads (R-Smads), a family of intracellular proteins which transmit TGF- signals from cell surface receptors to the nucleus. Smad8 promotes tendon cell diVerentiation from mesenchymal progenitor cells by inhibiting the osteogenic pathway induced by the bone morphogenetic protein 2 (BMP2) (HoVmann et al. 2006; Towler and Gelberman 2006). CD29 and CD44 are commonly used as surface markers to characterize mesenchymal stem cells (Meirelles Lda and Nardi 2003). Thus, the expression of CD29 and CD44 together with Musashi-1, Prominin-1/ CD133 further emphasizes the stem/precursor cell-like nature of TPVCs. As for tendons, no reliable marker proWle for mature tendon cells has yet been established. Tenomodulin is considered to be a marker for diVerentiated tendon cells, however, it was also found in other, non-tendinous tissues (Oshima et al. 2006; Shukunami et al. 2006). Other potential markers for mature and immature tendon cells such as tenascin-C, biglycan, and decorin, are also expressed in non-tendinous


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Fig. 3 Immunohistochemical staining of 7 m paraYn sections from a human supraspinatus tendon. Arrows indicate expression of Nestin (a–d) and smooth muscle actin (e, f) in perivascular cells (a, b, e) and in some tendon Wbroblasts (c, d, f) in the dense collagenous tissue. Counterstaining: May–Gruenwald dye (a–c, e, f: bar 100 m; insert and d: bar 50 m)

Fig. 4 Immunohistochemical staining on 7 m paraYn sections derived from a human supraspinatus tendon. Arrows indicate expression of Prominin-1/CD133 in perivascular cells (a) Tendon Wbroblasts of the dense collagenous tissue are free from Prominin-1/CD133 (b) Counterstaining: May– Gruenwald dye (a, b bar 100 m, insert: bar 50 m)

tissues and cells, such as cartilage, smooth muscle cells, glial cells or lung Wbroblasts (Chiquet-Ehrismann and Tucker 2004; Tufvesson and Westergren-Thorsson 2003).

Therefore, in this study we additionally focused on tendon-associated functional properties of TPVCs. As a result, TPVCs were found to synthesize and secrete collagen type

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Fig. 5 a Cell culture of tendon perivascular cells at various time points: D1/P1 (day1/passage 1), D2/P1 (day2/passage 1), D3/P1 (day3, passage 1). Tendon perivascular cells growing out of the tendon-derived isolated vessel fragments (VF) exhibit a polygonal to Wbroblastic phenotype (scale bar 25 m; P0: passage 0). b Bandeiraea simplicifolia lectin (BSL) staining of tendon perivascular cells and rat brain endothelial cells (RBECs) (positive control). Nuclei are visual-

ized by DAPI staining. c Products of RT-PCR performed on RNA isolated from cultured tendon perivascular cells. Asterisks mark unspeciWc primer clouds. d. Western blotting of the supernatant from tendon perivascular cells shows the secreted fraction of Collagen type I (lane 1). Addition of 40 g/ml ascorbic acid causes an increase in secretion of Collagen type I (lane 2). Arrows indicate the 1 and 2 chain of collagen type I

I and were even susceptible to ascorbic acid treatment, responding with a strong increase of collagen type I secretion. Since the collagen has been extracted with pepsin, the ratio of the alpha 1 subunit to alpha 2 would be expected to be 2:1, other than seen in Fig. 5d. However, our result is in line with previously published data (Yang et al. 2004). Therefore, we assume that this minor deviation is speciWc to the antibody used. This Wnding further supports the notion that TPVCs have a distinct tenogenic diVerentiation potential. In summary, our results provide evidence to suggest that tendon-derived perivascular cells exhibit tendon- and mesenchymal stem cell-like characteristics. The observation that these cells show morphological features reminiscent of pericytes and even express the pericyte-related SMA leaves space for the speculation that we are dealing with a pericyte-like cell population, which is capable of giving rise to a new generation of mature tendon cells upon external or internal stimulation. This is in line with the literature (Caplan 2008; Crisan et al. 2008). It was of particular importance to demonstrate that these perivascular cells retain their stem cell characteristics in culture. In this respect, such cells may be of potenitial therapeutic use. The idea of precursor cells residing in perivascular niches has been described as a general pattern for virtually all postnatal organs and tissues, giving the chance for facilitated tissue regeneration.

Acknowledgments This work was funded by grant nr 08/07041 of the Paracelsus Private Medical University by grant nr. 03-08 of the Lorenz Boehler Foundation and by the Dr. Rainer Brettenthaler Stipendium 2008.

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