2 Gross Morphology, Histology, and Immunohistochemistry

Articular Cartilage



Figure 7.8 Photodocumentationofgrossmorphologyfrombothtopandsideviews

of a bovine self-assembled construct along a ruler (bottom of image) allows for

dimensional information to be obtained.



be taken with a stand-mounted digital camera for all pertinent axes of

any tissue or tissue-engineered construct prior to mechanical testing

or using visualization techniques such as India ink. Thickness, width,

or diameter can be extracted from such images if an in-plane ruler is

included in the picture (Figure 7.8).

7.2.2 India Ink

Piercing the cartilage surface with a needle dipped in India ink produces a “split” that follows the direction of collagen fibrils. This technique provides a simple way of visualizing fibril orientation at the

cartilage surface, that is, via “split lines” (Figure 7.9) (Meachim 1972). A

diluted solution of India ink can also be pipetted onto cartilage, and the

excess washed off with PBS to increase the contrast of fibrillations or

other surface defects.

546



Experimental Protocols for Generation and Evaluation of Articular Cartilage



Figure 7.9 Split lines being produced in human cadaveric knee cartilage using a

sharpenedawldippedinIndiaink.(FromBelow,S.etal.,Arthroscopy18(6):613617,2002.Withpermission.)



A. Materials needed





Equipment

1. 22-gaugeneedle,sewingneedle,orawl









Reagents

1. Indiaink

2. PBS

Note: Split lines may be difficult to detect in very young cartilage.

B. Procedure











1. DilutetheIndiaink1:2withPBS.

2. DiptheneedleorawlintothedilutedIndiainksolution.

3. Lightlypokethecartilagesurfacewiththeneedle.



7.2.3 Histology

Common stains and their chemistry used for articular cartilage are

described in Section 5.2. The general procedure for preparing frozen sections differs from that for fixed paraffin-embedded sections.

However, subsequent staining is similar. Fixation involves chemically

cross-linking the components of the cells and tissue to preserve morphology. Fixatives for paraffin embedding include neutral buffered

547



Articular Cartilage



formalin, paraformaldehyde, and Bouin’s fixative. Frozen sections can

be produced with or without prior fixation. After embedding the sample

and sectioning, unfixed sections should be fixed prior to staining. The

use of a microtome or cryotome for sample sectioning is not described

here. It should be noted that sectioning requires a significant amount of

“art” to produce quality sections.

Positive and negative controls should be stained along with the samples of interest. Different tissues (native articular cartilage, tissueengineered articular cartilage, or fibrocartilage) will take up stains

differently based on tissue composition. If possible, slides should be

fixed, deparaffinized, and stained in a chemical fume hood.

7.2.3.1 Fixing Cryosectioned Slides

Cryosectioned slides should be fixed before staining if not prior to

embedding. Slides prepared from frozen sections should be placed

on a slide warmer for 10-20 minutes prior to fixation. Fixing directly

from the freezer or refrigerator can cause samples to wash off the

slide. For immunohistochemistry, slides may be fixed in cold acetone, as per the immunohistochemistry protocol for frozen sections

(Section 7.2.3.2).

A. Materials needed

Equipment

1. Glass slides

2. Slide holder and bath

3. Slide warmer





Reagents

1. 10%neutralbufferedformalin

Note: If possible, slides should be fixed in a chemical fume hood.



548



Experimental Protocols for Generation and Evaluation of Articular Cartilage



B. Procedure











1. Forallhistologystaining,fixslidesin10%neutralbufferedformalinfor10minutespriortostaining.Slidesshouldgodirectly

from the slide warmer into formalin.

2. Afterfixing,rinseindeionizedwater.

3. Slidesarereadyforfurtherstaining.



7.2.3.2   Clearing Paraffin Slides for Staining

Before staining or immunohistochemistry, paraffin-embedded slides

need to be cleared of paraffin and rehydrated. Following clearing,

paraffin-processed slides can be used for multiple staining protocols.

A. Materials needed

Equipment

1. Glass slides

2. Slide holder and bath







Reagents

1. Paraffinclearingagent,suchasSafeclear,Citri-solv,orxylene

2. Ethanol solutions, 70%, 80%, 95%, and 100%, prepared in

deionized or ultrapure water

Note: Slides should be cleared of paraffin in a chemical fume hood.

While xylene effectively removes paraffin, safer alternatives are

available (e.g., Safeclear and Citri-solv).

B. Procedure







1. Placeslidesinparaffinclearingagentfor5minutes.

2. Repeat step 1 with a fresh container of clearing agent. Clearing

agentfromthesecondstepcanbereusedforthefirstclearing

step for other slides.



549



Articular Cartilage















3.

4.

5.

6.

7.



Placeslidesin100%ethanolfor2minutes.

Placeslidesin95%ethanolfor2minutes.

Placeslidesin80%ethanolfor2minutes.

Placeslidesin70%ethanolfor2minutes.

Slidesarereadyforfurtherstaining.



7.2.3.3 Safranin O with Fast Green Counterstain

In articular cartilage, Safranin O dye stains sulfated glycosaminoglycans (sGAGs) red, while the background counterstains green by the

Fast Green, and cell nuclei stain a dark purple (Figure 7.10).

A. Materials needed

Equipment

1. Glass slides

2. Slide holder and bath

3. Coverslips

Reagents

1. Weigart’s hematoxylin kit (Sigma HT1079), prepared fresh

before use

2. FastGreen,0.001%,preparedindeionizedwater









(a)



500 µm



(b)



500 µm



Figure 7.10 Safranin O- and Fast Green-stained (b) and picrosirius red-stained

(a) paraffin-embedded section of neonatal canine distal fibula growth plate (10×).

(CourtesyofDr.NataliaVapniarsky.)



550



Experimental Protocols for Generation and Evaluation of Articular Cartilage













3. SafraninO,0.1%,preparedindeionizedwater

4. Aceticacid,1%,preparedindeionizedwater

5. Ethanolsolutions,70%,80%,90%,95%,and100%,prepared

in deionized or ultrapure water

6. Permountmountingmedium

Note: Safranin O and Fast Green solutions are stable at room temperature for several weeks or longer. Acid solutions should be prepared

by adding the concentrated acid solution to the water while mixing.

B. Procedure

























1.

2.

3.

4.

5.

6.

7.

8.



PlacefixedslidesinWeigart’shematoxylinfor1minute.

Washindeionizedwateruntilnomoredyewashesoff.

PlaceslidesinFastGreentocounterstainfor3minutes.

Washindeionizedwateruntilnomoredyewashesoff.

Placein1%aceticacidfor15minutes.

PlacedirectlyfromtheaceticacidintoSafraninOfor1minute.

Washindeionizedwateruntilnomoredyewashesoff.

Dehydrate through ascending ethanol in steps of 70%, 80%,

90%,95%,and100%;incubateslides2minutespersolution.

9. Dryslidesfor5minutes.

10. MountslidesusingPermountmediumandapplycoverslip.Allow

todryforseveralhourstoovernightbeforeimaging.



7.2.3.4 Picrosirius Red

In articular cartilage, picrosirius red stains all collagens red and the

background yellow. Stained slides can be viewed using polarized light

microscopy to detect collagen fiber alignment. Fibers will appear as

brighter regions or “streaks” against a dark background.

A. Materials needed

Equipment

1. Glass slides

551



Articular Cartilage



2. Slide holder and bath

3. Coverslips

Reagents

1. Picrosiriusred,0.1%,preparedinsaturatedaqueouspicricacid

(1.3%)

2. Ethanolsolutions,70%,80%,90%,95%,and100%,prepared

in deionized or ultrapure water

3. Permountmountingmedium











Note: Picrosirius red solution is stable at room temperature for several weeks or longer. Dried picric acid is explosive.

B. Procedure









1. Placefixedslidesinpicrosiriusredsolutionfor1hour.

2. Washindeionizedwateruntilnomoredyewashesoff.

3. Dehydrate through ascending ethanol in steps of 70%, 80%,

90%,95%,and100%;incubateslides2minutespersolution.

4. Dryslidesfor5minutes.

5. MountslidesusingPermountmediumandapplycoverslip.Allow

todryforseveralhourstoovernightbeforeimaging.









7.2.3.5 Hematoxylin and Eosin

Hematoxylin and eosin (H&E) is one of the most popular staining methods in histology and pathology. In articular cartilage, H&E will stain

glycosaminoglycans dark purple, while nuclei stain blue. Cytoplasm

and collagen will stain pink to red (Figure 7.11).

A. Materials needed

Equipment

1. Glass slides

2. Slide holder and bath

3. Coverslips

552



Experimental Protocols for Generation and Evaluation of Articular Cartilage



1 mm



Figure 7.11 H&E-stained and paraffin-embedded section of 5-month-old canine

articularcartilage.(CourtesyofDr.NataliaVapniarsky.)















Reagents

1. Harrismodifiedhematoxylin,filteredbeforeuse

2. Eosin Y solution

3. Aceticacid,2%,preparedindeionizedwater

4. Ammonium hydroxide, 1% (NH4OH), prepared in deionized

water

5. Ethanolsolutions,70%,80%,90%,95%,and100%,prepared

in deionized or ultrapure water

6. Permountmountingmedium

Note: Both Harris modified hematoxylin and Eosin Y are available

commercially as premade solutions from multiple vendors.

553



Articular Cartilage



B. Procedure





















1. Place fixed slides in Harris modified hematoxylin solution for

3 minutes.

2. Washindeionizedwateruntilnomoredyewashesoff.

3. Dipslidesin2%aceticacideighttimes.

4. Dip slides in deionized water eight times.

5. Dipslidesin1%ammoniumhydroxidesolutioneighttimes.

6. Dehydrate through ascending ethanol in steps of 70%, 80%,

90%,and95%;incubateslides2minutespersolution.

7. PlaceslidesinEosinYsolutionfor1minute.

8. Dehydratebydippingslidesin95%ethanolthreetimes;repeat

with100%ethanol.

9. Dryslidesfor5minutes.

10. MountslidesusingPermountmediumandapplycoverslip.Allow

todryforseveralhourstoovernightbeforeimaging.



7.2.3.6 Alcian Blue

In articular cartilage, Alcian blue stains glycosaminoglycans (or mucins)

blue. This dye should be maintained at a pH of 1.0 to detect sGAGs, or

at a pH of 2.5 to detect other glycosaminoglycans. See Figure 7.5 for an

example of Alcian blue staining of a micromass culture.

A. Materials needed

Equipment

1. Glass slides

2. Slide holder and bath

3. Coverslips







554



Reagents

1. AlcianBlue8GX,0.05%,pH2.5,in3%aceticacid,orpH1.0in

0.1Mhydrochloricacid

2. Ethanolsolutions,70%,80%,90%,95%,and100%,prepared

in deionized or ultrapure water

3. Permountmountingmedium



Experimental Protocols for Generation and Evaluation of Articular Cartilage



Note: Alcian blue solution is stable at room temperature for several

weeks or longer.

B. Procedure













1. PlacefixedslidesinAlcianbluesolutionfor1hour.

2. Washindeionizedwateruntilnomoredyewashesoff.

3. Dehydrate through ascending ethanol in steps of 70%, 80%,

90%,95%,and100%;incubateslides2minutespersolution.

4. Dryslidesfor5minutes.

5. MountslidesusingPermountmediumandapplycoverslip.Allow

todryforseveralhourstoovernightbeforeimaging.



7.2.4 Immunohistochemistry

Similar to antibody detection methods employed for Western blot and

enzyme-linked immunosorbent assay (ELISA), described in Section

7.3.3, immunohistochemistry uses antibodies to detect specific antigens, commonly proteins, on slides of tissues or cells. This allows for

detection and localization information to be determined for a specific

antigen. For example, this is commonly used to determine if type

I collagen is present in tissue-engineered articular cartilage. Some

antigens may be more favorably detected by immunohistochemistry from frozen sections as opposed to paraffin-embedded sections.

Also, individual antibodies will likely need to be tested on positive

controls to determine optimum dilutions and staining conditions.

Species variability may require different antibody conditions to be

used per species tested due to differences in antibody binding and

substrate specificity.

Following fixation and embedding (especially with paraffin), insufficient signal may be seen with immunostaining, as epitopes may be

obscured. Formalin or other aldehyde fixation forms protein cross-links

that mask the antigenic sites in tissue specimens, thereby giving weak

or false negative staining for immunohistochemical detection of certain

555



Articular Cartilage



proteins. To overcome this limitation, several methods of “antigen

retrieval” have been developed. The choice of which specific method

to use is dependent on both the antibody used andthe epitope being

analyzed.

7.2.4.1 Antigen Retrieval

A citric acid buffer-EDTA-based solution is used to break the protein cross-links produced during fixation, unmasking the antigens

and epitopes and enhancing antibody staining intensity. This antigen retrieval protocol is relatively gentle and works well with articular cartilage tissue samples that have been paraffin embedded. Slides

should be deparaffinized (Section 7.2.3.2) before undertaking antigen

retrieval.

A. Materials needed

Equipment

1. Slide holder

2. Stainingdishorotherheat-resistantcontainer

3. Hotplate









Reagents

1. Citric acid buffer: 10 mM citric acid, 2 mM EDTA, and 0.05%

Tween-20,pH6.2







Note: Citric acid buffer can be stored at room temperature for up to

3 months or at 4°C for up to 6 months. Incubation time in the citric

acid buffer may need to be optimized depending on sample type,

but should be kept consistent within a group of samples to reduce

artifactual staining intensity differences.

B. Procedure





556



1. Rinse deparaffinized slides (see Section 7.2.3.2) in deionized

water.