2 Histology, Immunohistochemistry, and Other Ultrastructural Techniques

Articular Cartilage



Control

Collagenase

C-ABC

Ribose



Intensity



30



20



10



0



400



450 500 550

Wavelength (nm)



600



Figure 5.9 Optical techniques can be used to evaluate the mechanical properties

of articular cartilage. In this case, changes in signals from laser-induced

autofluorescence correspond to changes in mechanical stiffness, altered using

collagenase, chondroitinase ABC (C-ABC), and ribose. Collagenase and C-ABC

degrade collagen and glycosaminoglycans, respectively, in articular cartilage, leading

to reduced stiffness. Ribose cross-links cartilage extracellular matrices, leading to

increased stiffness. For example, when the stiffness of cartilage matrix is altered by

these agents, the peak intensity resulting from laser-induced autofluorescence shifts

to different wavelengths. The nondestructive nature of laser-induced autofluorescence

has potential for use in the evaluation of cartilage mechanical properties. (From Sun,

Y. et al., Tissue Eng Part C Methods 18(3): 215-226, 2012. With permission.)



to this subject, as well as for ultrastructural techniques such as scanning electron microscopy (SEM) and transmission electron microscopy

(TEM) (An and Martin 2003; Mescher 2009). These techniques provide

information over several scales, from showing the general directions of

collagen alignment at the millimeter scale (Figure 5.10a) down to how

the collagen molecule is organized around the cells on the nanometer

scale (Figure 5.10c).

Of interest here, instead, is the histology of repair and engineered

tissues, in which biochemical components may be of different ratios

when compared with native tissues, and for which native morphology is absent and, therefore, cannot serve to guide the assessor. Care

must be taken not to misinterpret or overstate results based on histological evaluation, and it is important to keep in mind that while a

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Millimeters



Methods for Evaluating Articular Cartilage Quality



Micrometers



(a)



(b)

TM



ITM



Nanometers



ITM



PM



PM



TM

ITM



(c)

2



TM



Figure 5.10 Techniques to evaluate the structure of articular cartilage can assess a

dimensional range from millimeters (a, showing split lines) to micrometers (b, showing

tissue morphology) to nanometers (c, TEM showing cell and collagen structures).

(a, image from Below, S. et al., Arthroscopy 18(6): 613-617, 2002. b, image courtesy

of Dr. Natalia Vapniarsky. c, image from Hunziker, E. B. et al., Microsc Res Tech

37(4): 271-284, 1997. With permission.)



few studies have quantitatively correlated biochemical content with

histology (Martin et al. 1999), quantitative assessment of engineered

tissues should, in general, be accompanied with caution because

stains are only specific to certain types of extracellular components

and will not discern subspecies from each other. For specificity,

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immunohistochemical methods are more reliable, although these,

too, can suffer from nonspecific binding. In general, histological,

immunohistochemistry, and ultrastructural results are qualitative

to semiquantitative, providing information such as the absence or

presence of extracellular components of interest and their relative

distribution. Ultrastructural methods can yield quantitative results,

however, such as collagen diameter and degree of alignment; both of

these are important contributors to articular cartilage biomechanical

properties. Since the solid fraction of articular cartilage is made up

of cells, collagen, and glycosaminoglycans, a discussion of some of

the methods available for their assessment is presented in the next

sections.

5.2.1 Cells

Nuclear stains such as hematoxylin allow cell number and distribution

to be assessed. Stages of the cell cycle can also be identified by proliferating cell nuclear antigen (PCNA) and by terminal deoxynucleotidyl

transferase (TDT)-mediated dUTP (2′-deoxyuridine 5′-triphosphate)

nick end labeling (TUNEL), to assess proliferation and apoptosis,

respectively. Figure 5.11 shows an example of how TUNEL staining

is used. There are also in situ hybridization techniques where cellular

mRNA is bound by antisense RNA probes to determine chondrocyte

gene expression.

Cell viability assays for cell suspensions and smears typically function

by differences in cell membrane permeability (e.g., trypan blue will not

enter cells with intact cell membranes) or metabolism. For example, the

acetomethoxy derivate of calcein, after entering the cell, is metabolized

to calcein after intracellular esterases in live cells remove the acetomethoxy group; calcein is impermeable and retained to yield a signal that

fluoresces green (Figure 5.12). Cytoskeletal stains (e.g., rhodamine phalloidin) are important for articular cartilage biology since cell shape and

cytoskeletal arrangement are strongly correlated with the chondrocyte

and chondrogenic phenotypes.

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Methods for Evaluating Articular Cartilage Quality



Mutant



Merged



TUNEL



DAPI



Control



(a)



(b)



(c)



(d)



(e)



(f )



Figure 5.11 TUNEL staining can show DNA fragmentation and cell apoptosis.

In this case, TUNEL was used to stain cells of the nucleus pulposus of control

(Foxa2iCre;HIF-1αf/+) (a, c, and e) and mutant (Foxa2iCre;HIF-1αf/f) (b, d, and f) mice,

respectively, at birth. DAPI-stained nuclei fluoresce in blue (a and b). TUNEL staining

can be linked to various fluorophores and is green in this case (c and d). The merged

image (e and f) shows that the mutant’s HIF-1α deletion resulted in cell death in the

nucleus pulposus, which led to decreased biomechanical properties of the mouse’s

intervertebral disc. The bar is 200 μm. (From Merceron, C. et al., PLoS One 9(10):

e110768, 2014. Used under a PLoS One Creative Commons License.)



Cell morphology can also be visualized using cytoplasmic stains or

electron microscopy (e.g., SEM and TEM). By using TEM, intracellular features (e.g., Golgi and mitochondria) can be assessed. Visualizing

intracellular ion concentration (e.g., using a plethora of Ca2+ dyes) is useful for studying chondrocyte signaling and response to chemical and

mechanical stimuli (Mizuno 2005; Zhang et al. 2006).

Finally, clusters of differentiation and other cell surface markers or receptors can be used in conjunction with flow cytometry or fluorescenceactivated cell sorting (FACS) in determining the cell phenotype. It is

worth noting, however, that no definitive set of surface markers have

been identified as specific to chondrocytes. This can be particularly problematic for therapies that use stem cells. Where stem cell manipulation

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Articular Cartilage



24 hrs



(b)



(c)



(d)



(e)



(f )



(g)



(h)



(i)



1 wk



(a)

Baseline



4 wks



Culture control



(j)

Low



High



Figure 5.12 Confocal microscopy can be used in combination with live or dead

staining to show how cells progressively die in human articular cartilage following

impact. Live cells stain green, while dead cells stain red. In this case, cell death

is seen immediately after a high level of impact disrupts the cartilage surface

(d, g, j). (c, f, i) A low level of impact results in an injury that cannot be observed

clinically since there are no aberrations on the cartilage surface. However, as time

progresses to 4 weeks (h-j), an increasing number of dead cells can be observed

down the middle column. (From Natoli, R. M. et al., Ann Biomed Eng 36(5): 780792, 2008. With permission.)



occurs in vitro, flow cytometry data are typically used to verify that

the differentiated cells no longer exhibit surface markers associated

with stem cells. Even then, it can remain unclear whether the differentiated cells are chondrocytes. For regenerative medicine approaches

where stem cells are expected to differentiate in vivo into chondrocytes,

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(× 1000)

200 250



96.6%



CD105

102



(b)



103



104



585/42 PE-A



102



(e)



103



104



585/42 PE-A



104



585/42 PE-A



102



(f )



103



102



(h)



105



SSC-A

100 150



CD44 and CD29 CTRL



105



104



530/30 FITC-A



50



SSC-A

100 150



(g)



103



105



CD105 CTRL



105



50



SSC-A

100 150

50



CD166 CTRL

102



104



SSC-A

100 150



0.7%



CD49e



105



(× 1000)

200 250



530/30 FITC-A



103



530/30 FITC-A



(× 1000)

200 250



104



(× 1000)

200 250



(d)



103



102



(c)



SSC-A

100 150

50



SSC-A

100 150

50



95.2%



CD29

102



95.6%



CD44



105



(× 1000)

200 250



530/30 FITC-A



CD166



105



(× 1000)

200 250



104



(× 1000)

200 250



(a)



103



50



102



50



SSC-A

100 150



(× 1000)

200 250

SSC-A

100 150



96.6%



50



50



SSC-A

100 150



(× 1000)

200 250



Methods for Evaluating Articular Cartilage Quality



103



104



530/30 FITC-A



CD49e CTRL



105



102



(i)



103



104



585/42 PE-A



105



Figure 5.13 Flow cytometry can also be used to analyze cell markers to identify

ones useful in isolating cells. In this case, cells were labed with putative stem cell

surface markers CD105 (a), CD166 (b), CD44 (c), CD29 (d), and CD49e (e). While

only 0.7% of the isolated chondrocytes express CD49e (circled) in this study, 99% of

the cells in the clonal cell lines, obtained by adhesion to fibronectin, expressed this

marker after expansion. Corresponding immunoglobulin control samples (f-i) are also

presented. (From Williams, R. et al., PLoS One 5(10): e13246, 2010. Used under a

PLoS One Creative Commons License.)



phenotypic assessment would likely require biopsies. In addition to

surface markers (Figure 5.13), flow cytometry can offer additional information, such as cell size and organelle density.

5.2.2 Collagen

Histologically, general collagen staining can take advantage of the protein’s fibril organization, while collagen typing occurs through immunohistochemical staining. For instance, sirius red, used in a saturated

solution of picric acid (also known as picrosirius red), stains collagen

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fibers pink to red as the dye binds to collagen helices (Figure 5.14). Due

to the aspect ratio of the dye molecule, trapping accentuates alignment,

especially under polarized light (Junqueira et al. 1979), where binding

degree can be assessed as lighter-to-heavier staining transitions from

yellow to green. Other stains, such as aniline blue, can be used to stain

collagen, and, while polarized light microscopy for collagen alignment can also be performed without staining, the effect for observing

alignment is not as dramatic as when sirius red is used. SEM and TEM

can also be used to visualize collagen fiber thickness and orientation,

although these, like histology, are destructive methods.

Second harmonic generation (SHG) microscopy is an optical method that

may, in the future, allow for nondestructive quantitative assessment of

collagen alignment. Currently, SHG can be combined with various mathematical methods to determine collagen direction on histological sections (Figure 5.15). Collagen alignment is structurally important in native

articular cartilage, but despite how simple it can be to visualize, alignment is seldom assessed or reported in engineered or repair cartilage.



(a)



500 µm



(b)



500 µm



Figure 5.14 The picrosirius red method uses sirius red to stain collagen, as the dye

becomes trapped among the collagen fibers (a, tendon). Collagen is often stained in

combination with other extracellular matrix components, using for example a Masson’s

trichrome stain. In this case, aniline blue stains the fibers blue (b). Other stains, such

as Light Green SF yellowish, Fast Green FCF, methyl blue, and water blue, are also

routinely used for this trichrome stain. (Courtesy of Dr. Natalia Vapniarsky.)



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Methods for Evaluating Articular Cartilage Quality



(a)



10 µm



(b)



Figure 5.15 SHG can be used to determine collagen organization. In this case,

collagen fibers from chicken cartilage have been visualized using SHG and gradient

techniques, then pseudocolored (a). By superimposing ellipses over the fibers, one

can quickly see the local direction of the fibers (b). The more eccentric (flatter) the

ellipses, the more aligned is the collagen. (From Lilledahl, M. B. et al. in Confocal

Laser Microscopy—Principles and Applications in Medicine, Biology, and the Food

Sciences, ed. N. Lagali, Rijeka, Croatia: InTech, 2013, chap. 9. With permission.)



In addition to alignment, collagen fibril diameter and cross-links are

both important for articular cartilage biomechanical properties. Fibril

diameter can be assessed using electron microscopy (Figure 5.16), but

there are currently no widely used ultrastructural assays to evaluate

cross-links for type II collagen. Optical methods such as Raman microscopy are useful for discerning autofluorescence signatures of molecules;

this and other methods, such as fluorescence lifetime imaging microscopy (Sun et al. 2012), should be adapted to image articular cartilage

matrix cross-links in a reliable manner. As with alignment, fibril diameter and the degree of collagen cross-linking are seldom assessed or

reported for cartilages generated in  vitro or as a result of therapeutic

interventions; this is despite their structural and functional importance.

Ironically, these characteristics are important for cartilage-to-cartilage

integration, and their understudied nature may have been a hindrance

to advancements in this area.

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Articular Cartilage



(d) 10



Frequency (%)



(a)



Minimum diameter



8

6

4

2

0



50



(e) 20



Frequency (%)



(b)



0



100 150 200

Diameter (nm)



250



300



Area (tilt corrected)



15

10

5

0

0



(f )



Fibril area contribution (%)



(c)



10



6



20

30

Area (103 nm2)



40



50



Area contribution



5

4

3

2

1

0



0



10



20

30

Area (103 nm2)



40



50



Figure 5.16 Collagen fiber thickness can be quantified using TEM. In this case,

the collagen in rat-tail tendon was examined. The TEM image (a) was thresholded

to generate a black-and-white image for analysis (b). An algorithm was then used to

separate close fibril boundaries (c). From there, fibril diameters (d), area distribution

(e), and relative area contribution of fibrils of different sizes (f) can be determined.

The scale bars are 200 nm. (From Starborg, T. et al., Nat Protoc 8(7): 1433-1448,

2013. With permission.)



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Methods for Evaluating Articular Cartilage Quality



5.2.3 Sulfated Glycosaminoglycans

Dyes commonly used for staining cartilage glycosaminoglycans take

advantage of the negative charge of molecules, and these include

Safranin O, toluidine blue, and Alcian blue (Figure 5.17). Safranin O is

a red to orange stain, often used with Fast Green (a neutral dye) as a

counterstain. Articular cartilage should first be protonated in an acidic



(a)



(b)



(c)



Figure 5.17 Glycosaminoglycans in articular cartilage can be stained using toluidine

blue (a), Alcian blue (b), and Safranin O (red, c).



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Articular Cartilage



solution before dye binding occurs. Toluidine blue is a metachromatic

dye that changes from blue to pink-purple when the sulfated groups

of a glycosaminoglycan molecule bring several dye molecules close

together. This color change is only visible under aqueous conditions,

and dried toluidine blue complexes will appear blue. Toluidine blue has

been used in cartilage histology with aqueous mounts to distinguish

sulfated glycosaminoglycans from other anionic species.

Alcian blue staining for cartilage is common because it can be used with

periodic acid-Schiff (PAS) reagent to specifically stain for cartilage extracellular matrices of various dissociation constant (pKa) values. The pKa

of sulfate is lower than that of the carboxyl or phosphate groups, allowing for the sulfate to remain ionized at a lower pH than carboxyl or phosphate groups. For instance, at a pH of 2.5, both the sulfate and carboxyl

groups are ionized and will allow Alcian blue to bind to glycosaminoglycans. However, at a pH of 1.0, only the sulfate group will be ionized

for staining. The PAS technique may be applied to other cationic dyes

besides Alcian blue to stain carboxyl and sulfate groups at a pH of 2.5,

while only sulfated glycosaminoglycans will stain at a pH of 1.0.

The ability for Alcian blue to form insoluble complexes with glycosaminoglycans allows it to be combined with Verhoeff-Van Gieson methods

(e.g., in Movat’s pentachrome) to visualize glycosaminoglycan distribution in relationship to other extracellular matrix components. Soluble

Alcian blue complexes have also been used for quantitative assays

(Terry et al. 2000; Frazier et al. 2008).

Articular cartilage glycosaminoglycan assays have relied heavily on

the anionic nature of these molecules, and caution must be exercised

to ensure that glycosaminoglycans, and not other anionic matrices, are

measured. As shown with Alcian blue in solutions of pH greater than

1.0, cationic dyes can also be used to bind to acidic, nonsulfated molecules that are not cartilage specific. Furthermore, these cationic dyes,

whether used qualitatively or quantitatively, do not give information on

which kind of glycosaminoglycan is present (e.g., chondroitin sulfate vs.

418