Immunohistochemical Characterization of Cells in Adult Human Patellar Tendons
Department of Orthopaedics and Traumatology, The Chinese University of Hong Kong, Shatin, NT, Hong Kong
Correspondence to: Chan Kai Ming, Dept. of Orthopaedics and Traumatology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong. E-mail: kaimingchan{at}cuhk.edu.hk
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Summary |
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Key Words: tenoblasts tenocytes MMP1 procollagen type 1 PCNA
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Introduction |
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Only a few studies have focused on the organogenesis of tendons in early development. A transcription factor, scleraxis, is a highly specific marker for all the connective tissues that mediate attachment of muscle to bone in chick and mouse (Schweitzer et al. 2001), and the development of tendon may involve signaling of BMP12 (Lou et al. 1999
) as well as FGF8 (Edom-Vovard et al. 2002
). However, the differentiation process of tendon in adults has not been studied. Tenoblasts and tenocytes may represent different statuses of differentiation with distinct roles in the tendon healing process in adults. They may contribute to the inherent healing potentials of tendons and the development of pathological conditions such as tendinosis (Rolf et al. 2001
).
Cells undergo proliferation and apoptosis during tissue differentiation. Therefore, the proliferation index and apoptosis index of tenoblasts may be higher than those of tenocytes, which are presumably terminally differentiated. In addition, tenoblasts and tenocytes may also exhibit different capacities in matrix remodeling. Matrix remodeling involves localized matrix formation and degradation, which is important to tendon healing, adaptation to exercise, and tendon growth. We propose that tenoblasts, identified as round tendon cells, are responsible for matrix remodeling in healthy tendons, in contrast to tenocytes.
Here we collected healthy patellar tendon samples from patients undergoing anterior cruciate ligament reconstruction for histological examination. An image analysis protocol was designed to distinguish tenocytes and tenoblasts according to cell shape, and the percentages of proliferating cells and apoptotic cells were measured. The expression of procollagen type I (Procol I), heat shock protein 47 (hsp47), matrix metalloproteinase 1 (MMP1), transforming growth factor beta 1 (TGFß1), and bone morphogenetic protein 12 (BMP12) and 13 (BMP13) were detected by immunohistochemistry (IHC).
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Materials and Methods |
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Sample Collection and Preparation
All subjects and controls were recruited from the authors' institution. Fourteen samples (10 male and 4 female, with an average age of 28, ranging from 18 to 41) from patients with anterior cruciate ligament deficiency undergoing bonepatellar tendonbone autograft were included in the current study. The subjects had no previous history or clinical signs of patellar tendon injury. Subjects were informed of the procedures and provided consent before surgery. A 0.5 x 0.2 x 0.2-cm piece of healthy patellar tendon was removed from the patellar tendon during anterior cruciate ligament reconstruction. All samples were taken within the deep layers of the tendon in its central portion. All specimens were cleansed in sterile saline, fixed in buffered formalin, and then used to prepare 5-µm-thick paraffin-embedded sections mounted on 3-aminopropyl-triethoxy-silane (Sigma-Aldrich; St Louis, MO) and dried overnight at 40C.
Immunohistochemical Staining
IHC staining was performed as described previously (Fu et al. 2003). In brief, after removal of paraffin and dehydration, two consecutive paraffin sections from each sample were quenched with 3% H2O2 in methanol for 20 min and transferred to warm 10 mmol/liter citrate buffer solution (pH 6) for 10 min. After brief digestion with 0.05% trypsin for 5 min, the sections were incubated with primary antibody in a humid chamber at 4C overnight. The primary antibodies and the titers used are listed in Table 1. Negative staining controls were prepared by omitting the primary antibody.
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TdT-mediated dUTP Nick-end Labeling (TUNEL)
TUNEL identifies cells undergoing apoptosis by labeling nuclear DNA fragments that have been cleaved during apoptosis (Gavrieli et al. 1992). Apoptotic cells were detected on paraffin-embedded sections using an Apoptosis Detection Kit (Intergen; Purchase, NY). After removal of paraffin and rehydration of the sections, sections were pretreated with 20 µg/ml protease K (Sigma Chemical; St Louis, MO) and quenched with 3% H2O2 in PBS for 5 min. The fragmented DNA in the apoptotic cells was labeled with digoxigenin-11-dUTP by terminal deoxinucleotidyl transferase (TdT) for 2 hr. Negative controls were prepared by replacing the enzyme with reaction buffer only. The digoxigenin-labeled cells were treated with the peroxidase-conjugated anti-digoxigenin antibody for 30 min and diaminobenzidine (DAB) was added for color development (Ito et al. 1999
). The sections were rinsed in distilled water, counterstained in Mayer's hematoxylin, dehydrated through graded alcohol to xylene, and mounted with p-xylene-bis-pyridinium bromide (DPX) permount (Sigma-Aldrich).
Image Analysis
After IHC staining, micrographs were taken from each slide using a CCD camera-assisted microscope (Leica Cambridge; Cambridge, UK) and analyzed with Metamorph image analysis software (Universal Imaging; Downingtown, PA). Under x400 magnification, 20 viewing fields (0.038 µm2) were systematically sampled to avoid allocation bias. Using classifying parameters including length, elliptical form factor, and standard area, tenoblasts and tenocytes can be distinguished on the basis of their morphology. Because cell morphology is the only discriminating feature, borderline cases between tenoblasts and tenocytes were excluded from further analysis, i.e., only "typical tenocytes" and "typical tenoblasts," defined by a non-overlapping set of classifying parameters, were included. Ovoid tendon cells and elongated tendon cells were distinguished using length and elliptical form factor. Length is defined as the span of the longest chord through the selected object, while the elliptical form factor is defined as the ratio of length to breadth. Breadth is defined as the caliper width that is perpendicular to the length. The reference values for elongated tendon cells are length >30 and elliptical form factor >1.7. The reference values for ovoid tendon cells are length <25 and elliptical form factor <1.5. Positive immunostaining signals were detected by color thresholding on the brown color of DAB peroxidation as described previously (Fu et al. 2002). For proliferation index and apoptosis index, the percentages of proliferating tenoblasts and apoptotic tenoblasts in all tenoblasts were calculated and the same was done for tenocytes. For the evaluation of protein expression in tenocytes and tenoblasts, an adapted scoring scheme was used (no detectable expression = 0; detectable expression with the percentage of positive cells less than 10% = 1; significant expression with the percentage of positive cells much higher than 10% = 2) (Gilardoni et al. 2003
), and the results were presented as median scores. Because a paired comparison was employed, detectable expression in score 1 and significant expression in score 2 also referred to a direct comparison in staining intensity, i.e., if an obvious difference in staining intensity between elongated tendon cells and ovoid tendon cells was consistently noted, the higher staining intensity was scored 2 and the lower staining intensity was scored 1, even when the percentages of positive cells were similar.
Statistical Analysis
Statistical analysis was done using the Statistical Package for Social Science (SPSS) 11.0 (SPSS; Chicago, IL). A paired t-test was employed to compare the proliferation index and the apoptosis index after checking for normal distribution by the Kolmogorov-Smirov test. A Wilcoxon sign-ranked test was used to compare the scores of protein expression in tenoblasts and tenocytes in a paired manner for all samples. Significant difference was determined at p<0.05.
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Results |
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Discussion |
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In contrast to tenocytes, which are scattered evenly on the collagen fibers in tendons, tenoblasts often appear in clusters with a localized pericellular region devoid of collagen fiber anchorage. This may explain why the expression of MMP1 in tenoblasts was higher, because MMP1 is one of the major extracellular proteases that can cleave native type I collagen fibers in tendons, and its activity is associated with enhanced cell migration and tendon response to injuries (Stamenkovic 2003). In addition to MMP1, we also found that the expression of hsp47 and procollagen type I was high in these clusters. Because hsp47 is a collagen-specific molecular chaperone that assists in collagen synthesis and production (Nagata 1998
), clustered tenoblasts may participate in active collagen matrix remodeling. The presence of localized matrix remodeling processes in normal tendons may be attributed to the cellular response to micro-injuries from daily activities. In fact, these sites may serve as nuclei for the intrinsic healing response as well as the origins of tendon non-healing. It is believed that tenoblasts normally undergo proliferation, matrix remodeling, apoptosis, and finally differentiation into tenocytes to resolve tendon healing. If this process is blocked or if tenoblasts deviate from their normal destination as tenocytes, active tenoblasts would accumulate at the expense of the extracellular matrix. This may result in progressive matrix disturbance and hence a long-standing status of non-healing known as tendinosis. Therefore, studies focused on active remodeling sites and tenoblasts will lead not only to a better understanding of tendon healing and tendon growth but also of pathological conditions such as overuse injuries and tendinosis.
Tenoblasts expressed higher levels of TGFß1, BMP12, and BMP13 compared with tenocytes. With respect to the ability to induce tendon formation (Wolfman et al. 1997) and promote tendon healing (Fu et al. 2003
), BMP12 and BMP13 may be involved in the activation of connective tissue progenitors and might trigger the differentiation processes of tenoblasts and tenocytes. It is likely that conversion between tenoblasts and tenocytes is regulated by the expression of these bone morphogenetic proteins. TGFß1 is one of the key regulatory cytokines for tendon healing (Molloy et al. 2003
). It can relay the signals from mechanical loading to modulate the adaptive response in tendons, including collagen synthesis (Heinemeier et al. 2003
) and proteoglycan synthesis (Robbins et al. 1997
). Therefore, the significant expression of TGFß1 in tenoblasts suggests that these cells are responsible for the maintenance of matrix integrity in response to injuries and mechanical stimuli. Increased TGFß1 levels associated with tendon adhesion (Khan et al. 2000
) and non-healing tendinosis (Fu et al. 2002
) may therefore indicate an accumulation of tenoblasts that leads to augmented collagen and proteoglycan deposition, respectively. Further characterization of tenoblasts, tenocytes, and active remodeling sites may help to explain how tendon respond to the needs of matrix degradation and deposition, such as exercise involving strain and stress, overuse injury, and pathological processes such as tendinosis.
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Footnotes |
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Literature Cited |
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Davidson CJ, Ganion LR, Gehlsen GM, Verhoestra B, Roepke JE, Sevier TL (1997) Rat tendon morphologic and functional changes resulting from soft tissue mobilization. Med Sci Sports Exerc. 29:313319[Medline]
Edom-Vovard F, Schuler B, Bonnin MA, Teillet MA, Duprez D (2002) Fgf4 positively regulates scleraxis and tenascin expression in chick limb tendons. Dev Biol. 247:351366[CrossRef][Medline]
Fu SC, Wang W, Pau HM, Wong YP, Chan KM, Rolf CG (2002) Increased expression of transforming growth factor-beta1 in patellar tendinosis. Clin Orthop 400:17413[Medline]
Fu SC, Wong YP, Chan BP, Pau HM, Cheuk YC, Lee KM, Chan KM (2003) The roles of bone morphogenetic protein (BMP) 12 in stimulating the proliferation and matrix production of human patellar tendon fibroblasts. Life Sci 76:965974
Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493501[Abstract]
Gilardoni MB, Ceschin DG, Sahores MM, Oviedo M, Gehrau RC, Chiabrando GA (2003) Decreased expression of the low-density lipoprotein receptor-related protein-1 (LRP-1) in rats with prostate cancer. J Histochem Cytochem. 51:15751580
Heinemeier K, Langberg H, Olesen JL, Kjaer M (2003) Role of TGF-{beta}1 in relation to exercise-induced type I collagen synthesis in human tendinous tissue. J Appl Physiol. 95:23902397
Ito H, Shimojo T, Fujisaki H, Tamamori M, Ishiyama S, Adachi S, Abe S, et al. (1999) Thermal preconditioning protects rat cardiac muscle cells from doxorubicin-induced apoptosis. Life Sci 64:755761[CrossRef][Medline]
Jozsa LG, Kannus P (1997) Structure and metabolism of normal tendons. In Jozsa LG, Kannus P, eds Human Tendons: Anatomy, Physiology, and Pathology. Champaign, IL, Human Kinetics, 4695
Kakar S, Khan U, McGrouther DA (1998) Differential cellular response within the rabbit tendon unit following tendon injury. J Hand Surg [Br] 23:627632[CrossRef][Medline]
Khan U, Kakar S, Akali A, Bentley G, McGrouther DA (2000) Modulation of the formation of adhesions during the healing of injured tendons. J Bone Joint Surg [Br] 82:10541058[CrossRef][Medline]
Lou J, Tu Y, Ludwig FJ, Zhang J, Manske PR (1999) Effect of bone morphogenetic protein-12 gene transfer on mesenchymal progenitor cells. Clin Orthop. 369:333339[Medline]
Molloy T, Wang Y, Murrell G (2003) The roles of growth factors in tendon and ligament healing. Sports Med 33:381394[Medline]
Muschler GF, Midura RJ (2002) Connective tissue progenitors: practical concepts for clinical applications. Clin Orthop Relat Res 1:6680
Nagata K (1998) Expression and function of heat shock protein 47: a collagen-specific molecular chaperone in the endoplasmic reticulum. Matrix Biol 16:379386[CrossRef][Medline]
Robbins JR, Evanko SP, Vogel KG (1997) Mechanical loading and TGF-beta regulate proteoglycan synthesis in tendon. Arch Biochem Biophys 342:203211[CrossRef][Medline]
Rolf CG, Fu BS, Pau A, Wang W, Chan B (2001) Increased cell proliferation and associated expression of PDGFRbeta causing hypercellularity in patellar tendinosis. Rheumatology (Oxford) 40:256261[CrossRef][Medline]
Salingcarnboriboon R, Yoshitake H, Tsuji K, Obinata M, Amagasa T, Nifuji A, Noda M (2003) Establishment of tendon-derived cell lines exhibiting pluripotent mesenchymal stem cell-like property. Exp Cell Res 287:289300[CrossRef][Medline]
Schweitzer R, Chyung JH, Murtaugh LC, Brent AE, Rosen V, Olson EN, Lassar A, et al. (2001) Analysis of the tendon cell fate using scleraxis, a specific marker for tendons and ligaments. Development 128:38553866
Stamenkovic I (2003) Extracellular matrix remodeling: the role of matrix metalloproteinases. J Pathol 200:448464[CrossRef][Medline]
Sulik KK, Dehart DB, Johnson CS, Ellis SL, Chen SY, Dunty WC Jr, Zucker RM (2001) Programmed cell death in extraocular muscle tendon/sclera precursors. Mol Vis 7:184191[Medline]
Wolfman NM, Hattersley G, Cox K, Celeste AJ, Nelson R, Yamaji N, Dube JL, et al. (1997) Ectopic induction of tendon and ligament in rats by growth and differentiation factors 5, 6, and 7, members of the TGF-beta gene family. J Clin Invest 100:321330