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International Cartilage Repair Society (ICRS) Recommended Guidelines for Histological Endpoints for Cartilage Repair Studies in Animal Models and Clinical Trials.

Hoemann C, Kandel R, Roberts S, Saris DB, Creemers L, Mainil-Varlet P, Méthot S, Hollander AP, Buschmann MD - Cartilage (2011)

Bottom Line: Methods were compiled from a literature review, and illustrative data were added.In animal models, treatments are usually administered to acute defects created in healthy tissues, and the entire joint can be analyzed at multiple postoperative time points.Standardized histology methods could improve statistical analyses, help interpret and validate noninvasive imaging outcomes, and permit cross-comparison between studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Institute of Biomedical Engineering, École Polytechnique, Montréal, Quebec, Canada.

ABSTRACT
Cartilage repair strategies aim to resurface a lesion with osteochondral tissue resembling native cartilage, but a variety of repair tissues are usually observed. Histology is an important structural outcome that could serve as an interim measure of efficacy in randomized controlled clinical studies. The purpose of this article is to propose guidelines for standardized histoprocessing and unbiased evaluation of animal tissues and human biopsies. Methods were compiled from a literature review, and illustrative data were added. In animal models, treatments are usually administered to acute defects created in healthy tissues, and the entire joint can be analyzed at multiple postoperative time points. In human clinical therapy, treatments are applied to developed lesions, and biopsies are obtained, usually from a subset of patients, at a specific time point. In striving to standardize evaluation of structural endpoints in cartilage repair studies, 5 variables should be controlled: 1) location of biopsy/sample section, 2) timing of biopsy/sample recovery, 3) histoprocessing, 4) staining, and 5) blinded evaluation with a proper control group. Histological scores, quantitative histomorphometry of repair tissue thickness, percentage of tissue staining for collagens and glycosaminoglycan, polarized light microscopy for collagen fibril organization, and subchondral bone integration/structure are all relevant outcome measures that can be collected and used to assess the efficacy of novel therapeutics. Standardized histology methods could improve statistical analyses, help interpret and validate noninvasive imaging outcomes, and permit cross-comparison between studies. Currently, there are no suitable substitutes for histology in evaluating repair tissue quality and cartilaginous character.

No MeSH data available.


Related in: MedlinePlus

Example of standardized histoprocessing to evaluate a human biopsy (A-D, from cadaveric knee medial femoral condyle) or sheep hyaline repair cartilage 6-month repair after treatment with microfracture and chitosan-GP/blood implant (E-H).43 Sections were stained for Safranin O, immunostained for collagen type II and collagen type I, and observed by polarized light microscopy (PLM). SZ = superficial zone; DZ = deep zone; AC = articular cartilage. Note the abnormal vascular invasion and mineralization (*) in this particular human biopsy above the tidemark (horizontal arrow, A-D), which is frequently observed in osteoarthritis.21
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fig4-1947603510397535: Example of standardized histoprocessing to evaluate a human biopsy (A-D, from cadaveric knee medial femoral condyle) or sheep hyaline repair cartilage 6-month repair after treatment with microfracture and chitosan-GP/blood implant (E-H).43 Sections were stained for Safranin O, immunostained for collagen type II and collagen type I, and observed by polarized light microscopy (PLM). SZ = superficial zone; DZ = deep zone; AC = articular cartilage. Note the abnormal vascular invasion and mineralization (*) in this particular human biopsy above the tidemark (horizontal arrow, A-D), which is frequently observed in osteoarthritis.21

Mentions: To analyze cartilage-bone integration and subchondral bone structure, repair biopsies should include bone and should be decalcified intact. Decalcification can be accomplished using acid or EDTA. Acids such 0.5 N HCl43 or 50% formic acid with 68 g/L sodium formate (pH ~3)42,105 are one way to remove calcium. Sufficient decalcification can be obtained in 0.5 N HCl at 4 °C after approximately 30 hours for 2-mm-diameter biopsies, 10 days for rabbit distal femurs, and 4 to 6 weeks for large animal osteochondral samples. Acid decalcification is compatible with immunostaining for both collagen type II and collagen type I in humans and sheep (Fig. 4) as well as rabbits if the samples are cryosectioned.107 However, the low pH can hydrolyze some antigens of interest and destroy their immunoreactivity. For this reason, some laboratories prefer to decalcify samples in EDTA, which chelates calcium at neutral pH, is less damaging to proteins, and preserves enzymatic staining (i.e., alkaline phosphatase in osteoblasts, or tartrate-resistant acid phosphatase in osteoclasts47). The time required for EDTA decalcification will be longer than acid solutions, although this can be somewhat shortened by gently agitating the specimen during the decalcification process and changing solutions frequently. Trace fixative (10% w/v EDTA/0.1% w/v paraformaldehyde, pH 7.2, or 0.5 N HCl/0.1% glutaraldehyde)52 can be included in decalcification solutions, especially during long decalcification procedures. Commercially available decalcification solutions are another alternative, but these tend to be more expensive and utilize the same types of methodologies. As each method has its strengths and drawbacks, it is recommended that the laboratory choose a method of decalcification, optimize it, and then determine how long the tissue can remain in the solution without affecting its immunoreactivity. Decalcification can be monitored and checks carried out to ensure the process is complete by using x-rays (i.e., faxitron of human biopsies62), ammonium oxalate calcium precipitation tests of the decalcification solution (animal specimens only),44,108 or cautiously puncturing into the subchondral bone using a 26-gauge needle.


International Cartilage Repair Society (ICRS) Recommended Guidelines for Histological Endpoints for Cartilage Repair Studies in Animal Models and Clinical Trials.

Hoemann C, Kandel R, Roberts S, Saris DB, Creemers L, Mainil-Varlet P, Méthot S, Hollander AP, Buschmann MD - Cartilage (2011)

Example of standardized histoprocessing to evaluate a human biopsy (A-D, from cadaveric knee medial femoral condyle) or sheep hyaline repair cartilage 6-month repair after treatment with microfracture and chitosan-GP/blood implant (E-H).43 Sections were stained for Safranin O, immunostained for collagen type II and collagen type I, and observed by polarized light microscopy (PLM). SZ = superficial zone; DZ = deep zone; AC = articular cartilage. Note the abnormal vascular invasion and mineralization (*) in this particular human biopsy above the tidemark (horizontal arrow, A-D), which is frequently observed in osteoarthritis.21
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4300784&req=5

fig4-1947603510397535: Example of standardized histoprocessing to evaluate a human biopsy (A-D, from cadaveric knee medial femoral condyle) or sheep hyaline repair cartilage 6-month repair after treatment with microfracture and chitosan-GP/blood implant (E-H).43 Sections were stained for Safranin O, immunostained for collagen type II and collagen type I, and observed by polarized light microscopy (PLM). SZ = superficial zone; DZ = deep zone; AC = articular cartilage. Note the abnormal vascular invasion and mineralization (*) in this particular human biopsy above the tidemark (horizontal arrow, A-D), which is frequently observed in osteoarthritis.21
Mentions: To analyze cartilage-bone integration and subchondral bone structure, repair biopsies should include bone and should be decalcified intact. Decalcification can be accomplished using acid or EDTA. Acids such 0.5 N HCl43 or 50% formic acid with 68 g/L sodium formate (pH ~3)42,105 are one way to remove calcium. Sufficient decalcification can be obtained in 0.5 N HCl at 4 °C after approximately 30 hours for 2-mm-diameter biopsies, 10 days for rabbit distal femurs, and 4 to 6 weeks for large animal osteochondral samples. Acid decalcification is compatible with immunostaining for both collagen type II and collagen type I in humans and sheep (Fig. 4) as well as rabbits if the samples are cryosectioned.107 However, the low pH can hydrolyze some antigens of interest and destroy their immunoreactivity. For this reason, some laboratories prefer to decalcify samples in EDTA, which chelates calcium at neutral pH, is less damaging to proteins, and preserves enzymatic staining (i.e., alkaline phosphatase in osteoblasts, or tartrate-resistant acid phosphatase in osteoclasts47). The time required for EDTA decalcification will be longer than acid solutions, although this can be somewhat shortened by gently agitating the specimen during the decalcification process and changing solutions frequently. Trace fixative (10% w/v EDTA/0.1% w/v paraformaldehyde, pH 7.2, or 0.5 N HCl/0.1% glutaraldehyde)52 can be included in decalcification solutions, especially during long decalcification procedures. Commercially available decalcification solutions are another alternative, but these tend to be more expensive and utilize the same types of methodologies. As each method has its strengths and drawbacks, it is recommended that the laboratory choose a method of decalcification, optimize it, and then determine how long the tissue can remain in the solution without affecting its immunoreactivity. Decalcification can be monitored and checks carried out to ensure the process is complete by using x-rays (i.e., faxitron of human biopsies62), ammonium oxalate calcium precipitation tests of the decalcification solution (animal specimens only),44,108 or cautiously puncturing into the subchondral bone using a 26-gauge needle.

Bottom Line: Methods were compiled from a literature review, and illustrative data were added.In animal models, treatments are usually administered to acute defects created in healthy tissues, and the entire joint can be analyzed at multiple postoperative time points.Standardized histology methods could improve statistical analyses, help interpret and validate noninvasive imaging outcomes, and permit cross-comparison between studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Institute of Biomedical Engineering, École Polytechnique, Montréal, Quebec, Canada.

ABSTRACT
Cartilage repair strategies aim to resurface a lesion with osteochondral tissue resembling native cartilage, but a variety of repair tissues are usually observed. Histology is an important structural outcome that could serve as an interim measure of efficacy in randomized controlled clinical studies. The purpose of this article is to propose guidelines for standardized histoprocessing and unbiased evaluation of animal tissues and human biopsies. Methods were compiled from a literature review, and illustrative data were added. In animal models, treatments are usually administered to acute defects created in healthy tissues, and the entire joint can be analyzed at multiple postoperative time points. In human clinical therapy, treatments are applied to developed lesions, and biopsies are obtained, usually from a subset of patients, at a specific time point. In striving to standardize evaluation of structural endpoints in cartilage repair studies, 5 variables should be controlled: 1) location of biopsy/sample section, 2) timing of biopsy/sample recovery, 3) histoprocessing, 4) staining, and 5) blinded evaluation with a proper control group. Histological scores, quantitative histomorphometry of repair tissue thickness, percentage of tissue staining for collagens and glycosaminoglycan, polarized light microscopy for collagen fibril organization, and subchondral bone integration/structure are all relevant outcome measures that can be collected and used to assess the efficacy of novel therapeutics. Standardized histology methods could improve statistical analyses, help interpret and validate noninvasive imaging outcomes, and permit cross-comparison between studies. Currently, there are no suitable substitutes for histology in evaluating repair tissue quality and cartilaginous character.

No MeSH data available.


Related in: MedlinePlus