Skip to main content
PMC home page
Journal of Anatomy logoLink to Journal of Anatomy
. 1999 Aug;195(Pt 2):281–293. doi: 10.1046/j.1469-7580.1999.19520281.x

Electron microscopic stereological study of collagen fibrils in bovine articular cartilage: volume and surface densities are best obtained indirectly (from length densities and diameters) using isotropic uniform random sampling

TEEMU K LÅNGSJÖ 1 , MIKA HYTTINEN 1 , ALPO PELTTARI 3 , KARI KIRALY 2 ,4 , JARI AROKOSKI 5 , HEIKKI J HELMINEN 1 ,✉
PMCID: PMC1467991  PMID: 10529063

Abstract

Results obtained by the indirect zonal isotropic uniform random (IUR) estimation were compared with those obtained by the direct point and interception counting methods on vertical (VS) or IUR sections in a stereological study of bovine articular cartilage collagen fibrils at the ultrastructural level. Besides comparisons between the direct and indirect estimations (direct IUR vs indirect IUR estimations) and between different sampling methods (VS vs IUR sampling), simultaneous comparison of the 2 issues took place (direct VS vs indirect IUR estimation). Using the direct VS method, articular cartilage superficial zone collagen volume fraction (Vv 41%) was 67% and fibril surface density (Sv 0.030 nm2/nm3) 15% higher (P<0.05) than values obtained by the indirect IUR method (Vv 25% and Sv 0.026 nm2/nm3). The same was observed when the direct IUR method was used: collagen volume fraction (Vv 40%) was 63% and fibril surface density (Sv 0.032 nm2/nm3) 21% higher (P<0.05) than those obtained by the indirect IUR technique. Similarly, in the deep zone of articular cartilage direct VS and direct IUR methods gave 50 and 55% higher (P<0.05) collagen fibril volume fractions (Vv 43 and 44% vs 29%) and the direct IUR method 25% higher (P<0.05) fibril surface density values (Sv 0.025 vs 0.020 nm2/nm3) than the indirect IUR estimation. On theoretical grounds, scrutiny calculations, as well as earlier reports, it is concluded that the direct VS and direct IUR methods systematically overestimated the Vv and Sv of collagen fibrils. This bias was due to the overprojection which derives from the high section thickness in relation to collagen fibril diameter. On the other hand, factors that during estimation tend to underestimate Vv and Sv, such as profile overlapping and truncation (‘fuzzy’ profiles), seemed to cause less bias. As length density (Lv) and collagen fibril diameter are minimally biased by the high relative section thickness, the indirect IUR method, based on utilisation of these estimates, is here regarded as representing a ‘gold standard’. The sensitivity of these 3 methods was also tested with cartilage from an in vitro loading experiment which caused tissue compression. In the superficial zone of articular cartilage Vv and Sv of collagen fibrils increased (P<0.05). This difference in the stereological estimates was only detected by the indirect IUR estimation but not by the direct VS or direct IUR methods. This indicated that the indirect IUR estimation was more sensitive than the direct VS or direct IUR estimations. On the basis of these observations, the indirect zonal IUR estimation can be regarded as the technique of choice in the electron microscopic stereology of cartilage collagen.

Keywords: Stereology, vertical section sampling, overprojection, truncation, isector

Full Text

The Full Text of this article is available as a PDF (3.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Afzelius B. A. Section staining for electron microscopy using tannic acid as a mordant: a simple method for visualization of glycogen and collagen. Microsc Res Tech. 1992 Mar 1;21(1):65–72. doi: 10.1002/jemt.1070210110. [DOI] [PubMed] [Google Scholar]
  2. Arokoski J. P., Hyttinen M. M., Lapveteläinen T., Takács P., Kosztáczky B., Módis L., Kovanen V., Helminen H. Decreased birefringence of the superficial zone collagen network in the canine knee (stifle) articular cartilage after long distance running training, detected by quantitative polarised light microscopy. Ann Rheum Dis. 1996 Apr;55(4):253–264. doi: 10.1136/ard.55.4.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aspden R. M., Hukins D. W. Collagen organization in articular cartilage, determined by X-ray diffraction, and its relationship to tissue function. Proc R Soc Lond B Biol Sci. 1981 Jul 14;212(1188):299–304. doi: 10.1098/rspb.1981.0040. [DOI] [PubMed] [Google Scholar]
  4. Baddeley A. J., Gundersen H. J., Cruz-Orive L. M. Estimation of surface area from vertical sections. J Microsc. 1986 Jun;142(Pt 3):259–276. doi: 10.1111/j.1365-2818.1986.tb04282.x. [DOI] [PubMed] [Google Scholar]
  5. Clarke I. C. Articular cartilage: a review and scanning electron microscope study. II. The territorial fibrillar architecture. J Anat. 1974 Nov;118(Pt 2):261–280. [PMC free article] [PubMed] [Google Scholar]
  6. Cruz-Orive L. M., Weibel E. R. Recent stereological methods for cell biology: a brief survey. Am J Physiol. 1990 Apr;258(4 Pt 1):L148–L156. doi: 10.1152/ajplung.1990.258.4.L148. [DOI] [PubMed] [Google Scholar]
  7. Curtin W. A., Reville W. J. Ultrastructural observations on fibril profiles in normal and degenerative human articular cartilage. Clin Orthop Relat Res. 1995 Apr;(313):224–230. [PubMed] [Google Scholar]
  8. Gundersen H. J., Bagger P., Bendtsen T. F., Evans S. M., Korbo L., Marcussen N., Møller A., Nielsen K., Nyengaard J. R., Pakkenberg B. The new stereological tools: disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS. 1988 Oct;96(10):857–881. doi: 10.1111/j.1699-0463.1988.tb00954.x. [DOI] [PubMed] [Google Scholar]
  9. Gundersen H. J., Bendtsen T. F., Korbo L., Marcussen N., Møller A., Nielsen K., Nyengaard J. R., Pakkenberg B., Sørensen F. B., Vesterby A. Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS. 1988 May;96(5):379–394. doi: 10.1111/j.1699-0463.1988.tb05320.x. [DOI] [PubMed] [Google Scholar]
  10. Gundersen H. J. Estimators of the number of objects per area unbiased by edge effects. Microsc Acta. 1978 Nov;81(2):107–117. [PubMed] [Google Scholar]
  11. Gundersen H. J., Jensen E. B. The efficiency of systematic sampling in stereology and its prediction. J Microsc. 1987 Sep;147(Pt 3):229–263. doi: 10.1111/j.1365-2818.1987.tb02837.x. [DOI] [PubMed] [Google Scholar]
  12. Hedlund H., Mengarelli-Widholm S., Reinholt F. P., Svensson O. Stereologic studies on collagen in bovine articular cartilage. APMIS. 1993 Feb;101(2):133–140. doi: 10.1111/j.1699-0463.1993.tb00092.x. [DOI] [PubMed] [Google Scholar]
  13. Hwang W. S., Li B., Jin L. H., Ngo K., Schachar N. S., Hughes G. N. Collagen fibril structure of normal, aging, and osteoarthritic cartilage. J Pathol. 1992 Aug;167(4):425–433. doi: 10.1002/path.1711670413. [DOI] [PubMed] [Google Scholar]
  14. Jurvelin J., Sämänen A. M., Arokoski J., Helminen H. J., Kiviranta I., Tammi M. Biomechanical properties of the canine knee articular cartilage as related to matrix proteoglycans and collagen. Eng Med. 1988 Oct;17(4):157–162. doi: 10.1243/emed_jour_1988_017_042_02. [DOI] [PubMed] [Google Scholar]
  15. Jørgen H., Gundersen G. Estimation of tubule or cylinder LV, SV and VV on thick sections. J Microsc. 1979 Dec;117(3):333–345. doi: 10.1111/j.1365-2818.1979.tb04690.x. [DOI] [PubMed] [Google Scholar]
  16. Király K., Hyttinen M. M., Parkkinen J. J., Arokoski J. A., Lapveteläinen T., Törrönen K., Kiviranta I., Helminen H. J. Articular cartilage collagen birefringence is altered concurrent with changes in proteoglycan synthesis during dynamic in vitro loading. Anat Rec. 1998 May;251(1):28–36. doi: 10.1002/(SICI)1097-0185(199805)251:1<28::AID-AR6>3.0.CO;2-A. [DOI] [PubMed] [Google Scholar]
  17. Mayhew T. M. The new stereological methods for interpreting functional morphology from slices of cells and organs. Exp Physiol. 1991 Sep;76(5):639–665. doi: 10.1113/expphysiol.1991.sp003533. [DOI] [PubMed] [Google Scholar]
  18. Minns R. J., Steven F. S. The collagen fibril organization in human articular cartilage. J Anat. 1977 Apr;123(Pt 2):437–457. [PMC free article] [PubMed] [Google Scholar]
  19. Noonan K. J., Hunziker E. B., Nessler J., Buckwalter J. A. Changes in cell, matrix compartment, and fibrillar collagen volumes between growth-plate zones. J Orthop Res. 1998 Jul;16(4):500–508. doi: 10.1002/jor.1100160416. [DOI] [PubMed] [Google Scholar]
  20. Panula H. E., Hyttinen M. M., Arokoski J. P., Långsjö T. K., Pelttari A., Kiviranta I., Helminen H. J. Articular cartilage superficial zone collagen birefringence reduced and cartilage thickness increased before surface fibrillation in experimental osteoarthritis. Ann Rheum Dis. 1998 Apr;57(4):237–245. doi: 10.1136/ard.57.4.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Parkkinen J. J., Lammi M. J., Karjalainen S., Laakkonen J., Hyvärinen E., Tiihonen A., Helminen H. J., Tammi M. A mechanical apparatus with microprocessor controlled stress profile for cyclic compression of cultured articular cartilage explants. J Biomech. 1989;22(11-12):1285–1291. doi: 10.1016/0021-9290(89)90232-7. [DOI] [PubMed] [Google Scholar]
  22. Paukkonen K., Helminen H. J. Decrease of proteoglycan granule number but increase of their size in articular cartilage of young rabbits after physical exercise and immobilization by splinting. Anat Rec. 1987 Sep;219(1):45–52. doi: 10.1002/ar.1092190109. [DOI] [PubMed] [Google Scholar]
  23. Weibel E. R., Paumgartner D. Integrated stereological and biochemical studies on hepatocytic membranes. II. Correction of section thickness effect on volume and surface density estimates. J Cell Biol. 1978 May;77(2):584–597. doi: 10.1083/jcb.77.2.584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Weiss C., Rosenberg L., Helfet A. J. An ultrastructural study of normal young adult human articular cartilage. J Bone Joint Surg Am. 1968 Jun;50(4):663–674. doi: 10.2106/00004623-196850040-00002. [DOI] [PubMed] [Google Scholar]
  25. Zambrano N. Z., Montes G. S., Shigihara K. M., Sanchez E. M., Junqueira L. C. Collagen arrangement in cartilages. Acta Anat (Basel) 1982;113(1):26–38. doi: 10.1159/000145534. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Anatomy are provided here courtesy of Anatomical Society of Great Britain and Ireland

RESOURCES