A Stereological Method for the Quantitative Evaluation of Cartilage Repair Tissue

Objective To implement stereological principles to develop an easy applicable algorithm for unbiased and quantitative evaluation of cartilage repair. Design Design-unbiased sampling was performed by systematically sectioning the defect perpendicular to the joint surface in parallel planes providing 7 to 10 hematoxylin–eosin stained histological sections. Counting windows were systematically selected and converted into image files (40-50 per defect). The quantification was performed by two-step point counting: (1) calculation of defect volume and (2) quantitative analysis of tissue composition. Step 2 was performed by assigning each point to one of the following categories based on validated and easy distinguishable morphological characteristics: (1) hyaline cartilage (rounded cells in lacunae in hyaline matrix), (2) fibrocartilage (rounded cells in lacunae in fibrous matrix), (3) fibrous tissue (elongated cells in fibrous tissue), (4) bone, (5) scaffold material, and (6) others. The ability to discriminate between the tissue types was determined using conventional or polarized light microscopy, and the interobserver variability was evaluated. Results We describe the application of the stereological method. In the example, we assessed the defect repair tissue volume to be 4.4 mm3 (CE = 0.01). The tissue fractions were subsequently evaluated. Polarized light illumination of the slides improved discrimination between hyaline cartilage and fibrocartilage and increased the interobserver agreement compared with conventional transmitted light. Conclusion We have applied a design-unbiased method for quantitative evaluation of cartilage repair, and we propose this algorithm as a natural supplement to existing descriptive semiquantitative scoring systems. We also propose that polarized light is effective for discrimination between hyaline cartilage and fibrocartilage.

[1]  H J Gundersen,et al.  The efficiency of systematic sampling in stereology and its prediction * , 1987, Journal of microscopy.

[2]  N. Südkamp,et al.  Results after microfracture of full-thickness chondral defects in different compartments in the knee. , 2006, Osteoarthritis and cartilage.

[3]  S W O'Driscoll,et al.  The Repair of Major Osteochondral Defects in Joint Surfaces by Neochondrogenesis with Autogenous Osteoperiosteal Grafts Stimulated by Continuous Passive Motion: An Experimental Investigation in the Rabbit , 1986, Clinical orthopaedics and related research.

[4]  J R Nyengaard,et al.  The Conneulor: unbiased estimation of connectivity using physical disectors under projection. , 1993, Bone.

[5]  James A Armitage,et al.  A design-based method for estimating glomerular number in the developing kidney. , 2011, American journal of physiology. Renal physiology.

[6]  M. Lind,et al.  A novel nano-structured porous polycaprolactone scaffold improves hyaline cartilage repair in a rabbit model compared to a collagen type I/III scaffold: in vitro and in vivo studies , 2012, Knee Surgery, Sports Traumatology, Arthroscopy.

[7]  C. Bünger,et al.  Dermatan sulphate in methoxy polyethylene glycol-polylactide-co-glycolic acid scaffolds upregulates fibronectin gene expression but has no effect on in vivo osteochondral repair , 2012, International Orthopaedics.

[8]  J R Nyengaard,et al.  Cartilage thickness in the hip joint measured by MRI and stereology--a methodological study. , 2007, Osteoarthritis and cartilage.

[9]  M. Hyttinen,et al.  Quantitative Analysis of Collagen Network Structure and Fibril Dimensions in Cartilage Repair with Autologous Chondrocyte Transplantation , 2010, Cells Tissues Organs.

[10]  H. Gundersen,et al.  Marrow space star volume in the iliac crest decreases in osteoporotic patients after continuous treatment with fluoride, calcium, and vitamin D2 for five years. , 1991, Bone.

[11]  B. Pakkenberg,et al.  Stereological Estimation of Epidermal Volumes and Dermo-Epidermal Surface Area in Normal Skin , 2011, Dermatology.

[12]  J. Nyengaard,et al.  Bone Formation and Resorption Are Both Increased in Experimental Autoimmune Arthritis , 2012, PloS one.

[13]  N. Südkamp,et al.  Is microfracture of chondral defects in the knee associated with different results in patients aged 40 years or younger? , 2006, Arthroscopy : the journal of arthroscopic & related surgery : official publication of the Arthroscopy Association of North America and the International Arthroscopy Association.

[14]  C. Bünger,et al.  ISSLS Prize Winner: Positron Emission Tomography and Magnetic Resonance Imaging for Monitoring Interbody Fusion With Equine Bone Protein Extract, Recombinant Human Bone Morphogenetic Protein-2, and Autograft , 2008, Spine.

[15]  B. Pakkenberg,et al.  Regulatory developmental neurotoxicity testing: a model study focussing on conventional neuropathology endpoints and other perspectives. , 2005, Environmental toxicology and pharmacology.

[16]  Bente Pakkenberg,et al.  2D and 3D assessment of neuropathology in rat brain after prenatal exposure to methylazoxymethanol, a model for developmental neurotoxicty. , 2005, Reproductive toxicology.

[17]  M. Spector,et al.  Chondral Defects in Animal Models: Effects of Selected Repair Procedures in Canines , 2001, Clinical orthopaedics and related research.

[18]  J. Nyengaard,et al.  Cartilage Thickness in the Hip Measured by MRI and Stereology Before and After Periacetabular Osteotomy , 2010, Clinical orthopaedics and related research.

[19]  J. Baas.  Adjuvant therapies of bone graft around non-cemented experimental orthopedic implants stereological methods and experiments in dogs , 2008, Acta orthopaedica. Supplementum.

[20]  Joseph M. Mansour,et al.  Mesenchymal Cell-Based Repair of Large Full Thickness Defects of Articular Cartilage , 1994 .

[21]  V. Goldberg,et al.  A semiquantitative scale for histologic grading of articular cartilage repair. , 1992, Acta anatomica.

[22]  D. Saris,et al.  Evaluation of histological scoring systems for tissue-engineered, repaired and osteoarthritic cartilage. , 2010, Osteoarthritis and cartilage.

[23]  R. Marx,et al.  Validation of a simple histological-histochemical cartilage scoring system. , 2001, Tissue engineering.

[24]  A. Barbero,et al.  Visual histological grading system for the evaluation of in vitro-generated neocartilage. , 2006, Tissue engineering.

[25]  L. Engebretsen,et al.  Intraarticular location predicts cartilage filling and subchondral bone changes in a chondral defect , 2010, Acta orthopaedica.

[26]  J. Nyengaard,et al.  Mitochondrial plasticity of the hippocampus in a genetic rat model of depression after antidepressant treatment , 2013, Synapse.

[27]  J. Nyengaard,et al.  Left Ventricular Morphology of the Giraffe Heart Examined by Stereological Methods , 2013, Anatomical record.

[28]  Peter R. Mouton,et al.  Principles and Practices of Unbiased Stereology: An Introduction for Bioscientists , 2002 .

[29]  P. Bullough,et al.  Histological Assessment of Cartilage Repair: A Report by the Histology Endpoint Committee of the International Cartilage Repair Society (ICRS) , 2003, The Journal of bone and joint surgery. American volume.

[30]  Lars Engebretsen,et al.  Autologous chondrocyte implantation compared with microfracture in the knee. A randomized trial. , 2004, The Journal of bone and joint surgery. American volume.

[31]  C. Sledge,et al.  Effect of Cultured Autologous Chondrocytes on Repair of Chondral Defects in a Canine Model* , 1997, The Journal of bone and joint surgery. American volume.

[32]  P. Luciw,et al.  Stereological analysis of bacterial load and lung lesions in nonhuman primates (rhesus macaques) experimentally infected with Mycobacterium tuberculosis. , 2011, American journal of physiology. Lung cellular and molecular physiology.

[33]  C. Hoemann,et al.  Stereological analysis of subchondral angiogenesis induced by chitosan and coagulation factors in microdrilled articular cartilage defects. , 2013, Osteoarthritis and cartilage.

[34]  R. Kandel,et al.  A New Histology Scoring System for the Assessment of the Quality of Human Cartilage Repair: ICRS II , 2010, The American journal of sports medicine.

[35]  Sally Roberts,et al.  Autologous chondrocyte implantation for cartilage repair: monitoring its success by magnetic resonance imaging and histology , 2002, Arthritis research & therapy.

[36]  E R Weibel,et al.  A simple tool for stereological assessment of digital images: the STEPanizer , 2011, Journal of microscopy.

[37]  A. Gomoll,et al.  Increased Failure Rate of Autologous Chondrocyte Implantation after Previous Treatment with Marrow Stimulation Techniques , 2009, The American journal of sports medicine.

[38]  W. Stanish,et al.  Subchondral pre-solidified chitosan/blood implants elicit reproducible early osteochondral wound-repair responses including neutrophil and stromal cell chemotaxis, bone resorption and repair, enhanced repair tissue integration and delayed matrix deposition , 2013, BMC Musculoskeletal Disorders.

[39]  Matthias Ochs,et al.  Quantitative microscopy of the lung: a problem-based approach. Part 1: basic principles of lung stereology. , 2013, American journal of physiology. Lung cellular and molecular physiology.

[40]  A. Grodzinsky,et al.  Effects of a cultured autologous chondrocyte‐seeded type II collagen scaffold on the healing of a chondral defect in a canine model , 2003, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[41]  R. Østerby,et al.  Optimizing sampling efficiency of stereological studies in biology: or ‘Do more less well!‘ , 1981, Journal of microscopy.