Calcified Tissue International

, Volume 76, Issue 5, pp 326–335 | Cite as

Localization of Matrix Metalloproteinases, (MMPs) Their Tissue Inhibitors, and Vascular Endothelial Growth Factor (VEGF) in Growth Plates of Children and Adolescents Indicates a Role for MMPs in Human Postnatal Growth and Skeletal Maturation

  • G. Haeusler
  • I. Walter
  • M. Helmreich
  • M. Egerbacher


Numerous studies have focused on the expression, regulation, and biological significance of matrix metalloproteinases (MMPs) in the growth plate. Findings in mouse knockout models and in vitro data from various species indicate that MMPs not only degrade extracellular matrix components but may regulate the activity of local growth factors. In this study we investigated the presence, distribution, and activity of various MMPs and inhibitors, tissue transglutaminase (tTG or TG2) and vascular endothelial growth factor (VEGF) in the human child and adolescent growth plates by means of immunohistochemistry and gelatin zymography. Tissue was derived during orthopedic surgery (epiphysiodesis) in two prepubertal and four pubertal patients.

MMP-2 and MMP-14 were present in reserve cell chondrocytes. MMP-14 was the most prominent MMP within all zones of the growth plate including proliferating chondrocytes. MMP-1 and MMP-13 (collagenases 1 and 3), MMP-9 (gelatinases B), MMP-10, and MMP-11 (stromelysins) and VEGF were positive in hypertrophic chondrocytes and osteoblasts. MMP-2 showed the same expression pattern but was negative in osteoblasts. Osteoclasts stained positive for MMP-9, MMP-2, and TG2. Tissue inhibitor of MMP (TIMP)-1 was present in all zones of the growth plate, osteoblasts, and osteoclasts; TIMP-2 was found in hypertrophic chondrocytes and osteoblasts. In summary, the presence of MMPs, TIMPs, TG2, and VEGF in our study indicated that the MMPs are relevant in growth plate physiology during the postnatal period in humans. The specific location of MMP expression within the growth plate may be the basis for further studies on the role of MMPs in the local regulation of chondrocyte differentiation, proliferation, and ossification at the chondroosseus junction.


Human growth plate Metalloproteinases VEGF Immunohistochemistry Gelatin zymography 



The authors are indepted to Prof. F. Grill and Dr. H. Manner from the Orthopedic Hospital Speising, Vienna, for their collaboration, and Waltraud Tschulenk for technical assistance.


  1. 1.
    Ballock, RT, O’Keefe, RJ 2003The biology of the growth plateJ Bone Joint Surg85715726PubMedGoogle Scholar
  2. 2.
    Birkedahl-Hansen, H, Moore, WG, Bodden, MK, Windsor, LJ, Birkedahl-Hansen, B, Carlo, A, Engler, JA 1993Matrix metalloproteinases: a reviewCrit Rev Oral Biol Med4197250PubMedGoogle Scholar
  3. 3.
    Birkedahl-Hansen, H 1995Proteolytic remodelling of extracellular matrixCurr Opin Cell Biol72835Google Scholar
  4. 4.
    Visse, R, Nagase, H 2003Matrix metalloproteinases and tissue inhibitors of metalloproteinasesCirc Res92827839CrossRefPubMedGoogle Scholar
  5. 5.
    Vu, TH, Werb, Z 2000Matrix metalloproteinase: effectors of development and normal physiologyGenes Dev1421232133CrossRefPubMedGoogle Scholar
  6. 6.
    Ortega, N, Behonick, D, Stickens, D, Werb, Z 2003How proteinases regulate bone morphogenesisAnn N Y Acad Sci995109116PubMedGoogle Scholar
  7. 7.
    Stamenkovic, I 2003Extracellular matrix remodelling: the role of matrix metalloproteinasesJ Pathol200448464CrossRefPubMedGoogle Scholar
  8. 8.
    Somerville, RP, Oblander, SA, Apte, SS 2003Matrix metalloproteinases: old dogs with new tricksGenome Biol42161–11CrossRefPubMedGoogle Scholar
  9. 9.
    Gomez, DE, Alonso, DF, Yoshiji, H, Thorgeirsson, UP 1997Tissue inhibitors of metalloproteinases: structure, regulation and biological functionsEur J Cell Biol74111122PubMedGoogle Scholar
  10. 10.
    Bord, S, Horner, A, Beeton, CA, Hembry, RM, Compston, JE 1999Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) distribution in normal and pathological human boneBone24229235CrossRefPubMedGoogle Scholar
  11. 11.
    Greenberg, CS, Birckbichler, PJ, Rice, RH 1991Transglutaminases: multifunctional cross-linking enzymes that stabilize tissuesFASEB J530713077PubMedGoogle Scholar
  12. 12.
    Aeschlimann, D, Wetterwald, A, Fleisch, H, Paulsson, M 1993Expression of tissue transglutaminase in skeletal tissues correlates with events of terminal differentiation of chondrocytesJ Cell Biol12014611470CrossRefPubMedGoogle Scholar
  13. 13.
    Nurminskaya, M, Magee, C, Faverman, L, Linsenmayer, TF 2003Chondrocyte-derived transglutaminase promotes maturation of preosteoblasts in periostal boneDev Biol263139152CrossRefPubMedGoogle Scholar
  14. 14.
    Johansson, N, Saarialho-Kere, U, Airola, K, Herva, R, Nissinen, L, Westermarck, J, Vuorio, E, Heino, J, Kähäri, VM 1997Collagenase-3 (MMP-13) is expressed by hypertrophic chondrocytes, periostal cells and osteoblasts during human fetal bone developmentDev Dyn208387397CrossRefPubMedGoogle Scholar
  15. 15.
    Stahle-Bäckdahl, MS, Sandstedt, B, Bruce, K, Lindahl, A, Jimenez, MG, Vega, JA, Lopez-Otin, C 1997Collagenase-3 (MMP-13) is expressed during human fetal ossification and re-expressed in postnatal bone remodelling and in rheumatoid arthritisLab Invest76717728PubMedGoogle Scholar
  16. 16.
    Bord, S, Horner, A, Hembry, RM, Compston, JE 1998Stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10) expression in developing human bone: potential roles in skeletal developmentBone23712CrossRefPubMedGoogle Scholar
  17. 17.
    Vu, TH, Shipley, JM, Bergers, G, Berger, JE, Helms, JA, Hanahan, D, Shapiro, SD, Senior, RM, Werb, Z 1998MMP-9/Gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytesCell93411422CrossRefPubMedGoogle Scholar
  18. 18.
    Holmbeck, K, Bianco, P, Caterina, J, Yamada, S, Kromer, M, Kuznetsov, SA, Mankani, M, Robey, PG, Poole, AR, Pidoux, I, Ward, JM, Birkedahl-Hansen, H 1998MT-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnoverCell998192CrossRefGoogle Scholar
  19. 19.
    Engsig, MT, Chen, QJ, Vu, TH, Pedersen, AC, Therkidsen, B, Lund, LR, Henriksen, K, Lenhard, T, Foged, NT, Werb, Z, Delaisse, JM 2000Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bonesJ Cell Biol151879889CrossRefPubMedGoogle Scholar
  20. 20.
    Balbin, M, Fueyo, A, Knauper, V, Lopez, JM, Alvarez, J, Sanchez, LM, Quesada, V, Bordallo, J, Murphy, G, Lopez-Otin, C 2001Identification and enzymatic characterization of two diverging murine counterparts of human interstitial collagenase (MMP-1) expressed at sites of embryo implantationJ Biol Chem2761025310262CrossRefPubMedGoogle Scholar
  21. 21.
    Billinghurst, RC, Dahlberg, L, Ionescu, M, Reiner, A, Bourne, R, Rorabeck, C, Mitchell, P, Hambor, J, Diekmann, O, Tschesche, H, Chen, J, Wart, H, Poole, AR 1997Enhanced cleavage of type II collagen by collagenases in osteoarthritic cartilageJ Clin Invest9915341543PubMedGoogle Scholar
  22. 22.
    Wu, CW, Tchetina, EV, Mwale, F, Hasty, K, Pidoux, I, Reiner, A, Chen, J, Wart, HE, Poole, AR 2002Proteolysis involving matrix metalloproteinase 13 (collagenase-3) is required for chondrocyte differentiation that is associated with matrix mineralizationJ Bone Mineral Res17639651Google Scholar
  23. 23.
    Mwale, F, Tchetina, E, Wu, CW, Poole, AR 2002The assembly and remodeling of the extracellular matrix in the growth plate in relationship to mineral deposition and cellular hypertrophy: An in situ study of collagens II and IX and proteoglycanJ Bone Miner Res17275283PubMedGoogle Scholar
  24. 24.
    Wu, W, Mwale, F, Tchetina, E, Kojima, T, Yasuda, T, Poole, R 2001Cartilage matrix resorption in skeletogenesisNovartis Found Symp232158166PubMedGoogle Scholar
  25. 25.
    Gack, S, Vallon, R, Schmidt, J, Grigoriadis, A, Tuckermann, J, Schenkel, J, Weiher, H, Wagner, EF, Angel, P 1995Expression of interstitial collagenase during skeletal development of the mouse is restricted to osteoblast-like cells and hypertrophic chondrocytesCell Growth Differ6759767PubMedGoogle Scholar
  26. 26.
    Freije, JM, Diez-Itza, I, Balbin, M, Sanchez, LM, Blasco, R, Tolivia, J, Lopez-Otin, C 1994Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomasJ Biol Chem2691676616773PubMedGoogle Scholar
  27. 27.
    Fowlkes, JL, Enghild, JJ, Suzuki, K, Nagase, H 1994Matrix metalloproteinases degrade insulin-like growth factor-binding protein-3 in dermal fibroblast culturesJ Biol Chem2692574225746PubMedGoogle Scholar
  28. 28.
    Sato, T, Foget, NT, Delaisse, JM 1998The migration of purified osteoclasts through collagen is inhibited by matrix metalloproteinase inhibitorsJ Bone Miner Res135966PubMedGoogle Scholar
  29. 29.
    Delaisse, JM, Eeckhout, Y, Neff, L, Francois-Gillet, C, Henriet, P, Su, Y, Vaes, G, Baron, R 1993(Pro)collagenase (matrix metalloproteinase-1) is present in rodent osteoclasts and in the underlying bone-resorbing compartmentJ Cell Sci10610711082PubMedGoogle Scholar
  30. 30.
    Hill, PA, Murphy, G, Docherty, AJP, Hembry, RM, Millican, A, Reynolds, JJ, Meikle, MC 1994The effects of selective inhibitors of matrix metalloproteinases (MMPs) on bone resorption and the identification of MMPs and TIMP-1 in isolated osteoclastsJ Cell Sci10730553064PubMedGoogle Scholar
  31. 31.
    Bord, S, Horner, A, Hembry, RM, Reynolds, JJ, Compston, JE 1997Distribution of matrix metalloproteinases and their inhibitor, TIMP-1, in developing human osteophytic boneJ Anat1913948CrossRefPubMedGoogle Scholar
  32. 32.
    Chambers, TJ, Darby, JA, Fuller, K 1985Mammalian collagenase predisposes bone surfaces to osteoclastic resorptionCell Tissue Res241671675CrossRefPubMedGoogle Scholar
  33. 33.
    Sato, H, Kinoshita, T, Takino, T, Nakayama, K, Seiki, M 1996Activation of a recombinant membrane type 1-matalloproteinase (MT1-MMP) by furin and its interaction with tissue inhibitor of metalloproteinases (TIMP)-2FEBS Lett393101104CrossRefPubMedGoogle Scholar
  34. 34.
    Kinoh, H, Sato, H, Tsunezuka, Y, Takino, T, Kawashima, A, Okada, Y, Seiki, M 1996MT-MMP, the cell surface activator of proMMP-2 (pro-gelatinase A) is expressed with its substrate in mouse tissue during embryogenesisJ Cell Sci109953959PubMedGoogle Scholar
  35. 35.
    Apte, SS, Fukai, N, Beier, DR, Olsen, BR 1997The matrix metalloproteinase-14 (MMP-14) gene is structurally distinct from other MMP genes and is co-expressed with the TIMP-2 gene during mouse embryogenesisJ Biol Chem2722551125517CrossRefPubMedGoogle Scholar
  36. 36.
    Gerber, HP, Vu, TH, Ryan, AM, Kowalski, J, Werb, Z, Ferrara, N 1996VEGF couples hypertrophic cartilage remodelling, ossification and angiogenesis during enchondral bone formationNature Med6623628Google Scholar
  37. 37.
    Carlevaro, MF, Cermelli, S, Cancedda, R, Cancedda, F 2000Vascular endothelial growth factor (VEGF) in cartilage neovascularization and chondrocyte differentiation: auto-paracrine role during enchondral bone formationJ Cell Sci1135969PubMedGoogle Scholar
  38. 38.
    Belotti, D, Paganoni, P, Manenti, L, Garofalo, A, Marchini, S, Taraboletti, G, Giavazzi, R 2003Matrix metalloproteinases (MMP9 and MMP2) induce the release of vascular endothelial growth factor (VEGF) by ovarian carcinoma cells: implications for ascites formationCancer Res6352245229PubMedGoogle Scholar
  39. 39.
    Horner, A, Bishop, NJ, Bord, S, Beeton, C, Kelsall, AW, Coleman, N, Compston, JE 1999Immunolocalisation of vascular endothelial growth factor (VEGF) in human neonatal growth plate cartilageJ Anat194519524CrossRefPubMedGoogle Scholar
  40. 40.
    Petersen, W, Tsokos, M, Pufe, T 2002Expression of VEGF 121 and VEGF 165 in hypertrophic chondrocytes of the human growth plate and epiphyseal cartilageJ Anat201153157CrossRefPubMedGoogle Scholar
  41. 41.
    Lin, R, Amizuka, N, Sasaki, T, Aarts, MM, Ozawa, H, Goltzman, D, Henderson, JE, White, JH 20021-Alpha, 25-dihydroxyvitamin D3 promotes vascularization of chondro-osseous junction by stimulating expression of vascular endothelial growth factor and matrix metalloproteinase 9J Bone Miner Res1716041612PubMedGoogle Scholar
  42. 42.
    Kawashima-Ohya, Y, Satakeda, H, Kuruta, Y, Kawamoto, T, Yan, W, Akagawa, Y, Hayakawa, T, Noshiro, M, Okada, Y, Nakamura, S, Kato, Y 1998Effects of parathyroid hormone (PTH) and PTH-related peptide on expressions of matrix metalloproteinase-2, -3, and -9 in growth plate chondrocyte culturesEndocrinology13921202127CrossRefPubMedGoogle Scholar
  43. 43.
    Sirum, KL, Brinckerhoff, CE 1989Cloning of the genes for human stromelysin and stromelysin-2: differential expression in rheumatoid synovial fibroblastsBiochemistry2886918698CrossRefPubMedGoogle Scholar
  44. 44.
    Fernandes, JC, Martel-Pelletier, J, Pelletier, JP 2002The role of cytokines in osteoarthritis pathophysiologyBiorheology39237246PubMedGoogle Scholar
  45. 45.
    Brown, CC, Hembry, RM, Reynolds, JJ 1989Immunolocalization of metalloproteinases and their inhibitors in the rabbit growth plateJ Bone Joint Surg Am71580593PubMedGoogle Scholar
  46. 46.
    Armstrong, AL, Barrach, HJ, Ehrlich, MG 2002Identification of the metalloproteinase stromelysin in the physisJ Orthop Res20289294CrossRefPubMedGoogle Scholar
  47. 47.
    Stamenkovic, I 2000Matrix metalloproteinases in tumor invasion and metastasisCancer Biol10415433CrossRefGoogle Scholar
  48. 48.
    Joronen, K, Salminen, H, Glumoff, V, Savontaus, M, Vuorio, E 2000Temporospatial expression of tissue inhibitors of matrix metalloproteinases-1, -2, and -3 during development, growth and aging of the mouse skeletonHistochem Cell Biol114157165PubMedGoogle Scholar
  49. 49.
    Geoffrey, V, Marty-Morieux, C, Le Goupil, N, Clement-Lacroix, P, Terraz, C, Frain, M, Roux, S, Rossert, J, Vernejoul, MC 2004In vivo inhibition of osteoblastic metalloproteinases leads to increased trabecular bone massJ Bone Miner Res19811822PubMedGoogle Scholar
  50. 50.
    Sobue, T, Hakeda, Y, Kobayashi, Y, Hayakawa, H, Yamashita, K, Aoki, T, Kumegawa, M, Noguchi, T, Hayakawa, T 2001Tissue inhibitor of metalloproteinases 1 and 2 directly stimulate the bone-resorbing activity of isolated mature osteoclastsJ Bone Miner Res1622052214PubMedGoogle Scholar
  51. 51.
    Johnson, KA, Etten, D, Nanda, N, Graham, RM, Terkeltaub, RA 2003Distinct transglutaminase 2-independent and transglutaminase-dependent pathways mediate articular chondrocyte hypertrophyJ Biol Chem211882418832CrossRefGoogle Scholar
  52. 52.
    Nanda, N, Iismaa, SE, Owens, WA, Huasain, A, Mackay Graham, F RM 2001Targeted inactivation of Gh/tissue transglutaminase IIJ Biol Chem2762067320678CrossRefPubMedGoogle Scholar
  53. 53.
    Uchide, K, Ueno, H, Takizawa, N, Okada, Y 2003Reduced levels of MMP-2 and TIMP-1 in dyssegmental dysplasiaJ Bone Miner Res18381382PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  • G. Haeusler
    • 1
  • I. Walter
    • 2
  • M. Helmreich
    • 2
  • M. Egerbacher
    • 2
  1. 1.Pediatric DepartmentMedical University of ViennaAustria
  2. 2.Pathobiology Department, Institute of Histology and EmbryologyUniversity of Veterinary MedicineAustria

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