What measuring tools are best for the minimally invasive, non-

destructive assessment of cartilage quality in vivo?

K.J.P. van Assen

1 Introduction
1.1 Articular Cartilage
Articular cartilage is a thin layer of connective tissue that covers the end of articulating bones in arthrodial joints
[1]. It is able to absorb and dissipate joint loads, while
providing an almost frictionless bearing surface [2]. The two
principal matrix components that are responsible for these
unique biomechanical properties are proteoglycans and
collagen fibrils. Proteoglycans consist of a core protein to
which glycosaiminoglycans (GAGs) are covalently linked.
The strandlike GAGs, are large negatively charged
polysaccharides that stick out from the core protein. The
GAGs and proteoglycans can associate to form even larger
polymeric complexes in the extracellular matrix [3].
Molecules of aggrecan, for example, assemble with
hyaluronan in cartilage matrix to form aggregates that are
as big as a bacterium, as depicted in Figure 1.1. The
negative charge of all the GAGs draws water into the tissue
and thereby generates an osmotic swelling pressure. This
pressure is counteracted by the tense collagen meshwork
[4]. A typical collagen molecule is namely featured by its
long, stiff, triple-stranded helical structure [3]. Al-
Figure 1.1 An aggrecan aggregate from fetal

terations in any of these cartilage constituents can lead to bovine cartilage [3].
impaired joint function or osteoarthritis (OA) [5].
1.2 Osteoarthritis
Osteoarthritis is a disabling disease associated with joint pain and restricted mobility, especially in the elderly. Post-
traumatic osteoarthritis (PTOA) however affects people of all ages and is initiated by joint trauma, for example
cartilage, meniscus and ligament tears (which are mainly caused by excessive loading such as accidental falls and
sport injuries) [6]. Injuries of the anterior cruciate ligament (ACL) for example are very common and can lead to
increased forces on the cartilage, causing early degeneration [7].

1.3 Assessing Cartilage Quality

Cartilage has limited self-healing properties because of its avascular and aneural nature. Therefore, assessment
methods capable of monitoring the subtle changes, when degeneration is still potentially reversible, are needed. An
early technique for accurately identifying structural and compositional changes in cartilage was assessment by
histological staining. These assessments are performed through the use of quantitative scoring systems. This score
requires the observer to rate different aspects of the tissue. Although this method has been effective for overall
matrix grading, there are several drawbacks to its use: time consumption, variability in tissue staining, subjective
analysis (leading to large interobserver variability) and the inability to translate to in vivo applications [8]. But most
importantly, it requires tissue harvesting, which may induce cartilage pathology [2]. Because of this last well-
recognised limitation, various non-destructive assessment techniques have been developed, including
roentgenography, computed tomography (CT) and magnetic resonance imaging (MRI). Unfortunately, these
techniques provide little detailed information on a cellular level with limited sensitivity to early OA changes [2] [4],
1
and thereby these techniques can only detect OA when biomolecular changes have already taken place (changes in
GAGs and collagen) [9] Moreover, the resolution of MRI is problematic, since cartilage is typically less than 2–3 mm
thick and the evaluation would rely heavily on the interpretation of a few pixels. In addition, its high cost, relatively
long imaging time, large size of equipment, and limited availability limit its widespread clinical use [1].
Another technique relies on osteoarthritis-specific biomarkers in body fluids. These biomarkers would allow
assessment of the disease but their levels can significantly vary owing to physical activity or food consumption, so
the interpretation of results often leads to incorrect conclusions. [4] Hence, arthroscopy currently remains the gold
standard for non-destructive OA assessment. These assessments currently rely on visual evaluation and manual
palpation of the cartilage surface with a metallic hook during arthroscopy [6] [2]. Thereby, a surgeon can
qualitatively evaluate cartilage stiffness during arthroscopic procedures by means of manual indentation of the
cartilage with this blunt arthroscopic hook [10] [11] (see Figure 1.2). However, existing underlying damage may go
undetected as the surface appears intact [5].
Thus, since this method are highly
subjective and limited to surface
evaluations, better and more reliable
alternatives for quantitative evaluation
of cartilage are inevitable [10]. Various
alternatives have been proposed in the
past years and there has been
considerable interest in developing
handheld devices that aim to acquire
more detailed and objective
information about cartilage quality
intra-operatively, particularly as these
technologies might be capable of
detecting early degeneration Figure 1.2 Blunt probe for palpating the articular surface during before visible surface
changes occur arthroscopic procedures [11]. [11]. The aim of this study is to review these alternatives, regarding the
following research question: ”What measuring tool is best for the minimally invasive, non-destructive assessment of
cartilage quality in situ.”
2 Methods
Search and Selection
A systematic literature search in the PubMed and Embase databases was conducted on 11-5-2020. Relevant
synonyms for the search terms considering ”instrument”, ”assessment”, ”degeneration” and ”cartilage” were used
for the search string, as depicted in Table 1.

Table 1 Search string as used for the systematic review on PubMed and Embase

1
instrument[Title/Abstract] OR probe[Title/Abstract] OR tool[Title/Abstract] OR device[Title/Abstract] OR
handpiece[Title/Abstract] OR handheld[Title/Abstract]

2
assessment[Title/Abstract] OR grading[Title/Abstract] OR measurement[Title/Abstract] OR
detection[Title/Abstract] OR evaluation[Title/Abstract] OR analysis[Title/Abstract] OR
characterization[Title/Abstract] OR determination[Title/Abstract] OR validation[Title/Abstract])

3
degeneration[Title/Abstract] OR osteoarthritis[Title/Abstract] OR cartilage defect[Title/Abstract] OR
osteochondral defect[Title/Abstract] OR osteochondral lesion[Title/Abstract] OR
deterioration[Title/Abstract] OR cartilage lesion[Title/Abstract] OR quality[tiab]

2
 4
cartilage[Title/Abstract] OR cartilaginous[Title/Abstract] OR chondral[tiab] OR osteochondral[tiab] OR
cartilage[MeSH]

After removal of duplicates, title and abstract screening was performed according to predetermined inclusion and
exclusion criteria. These criteria were formulated based on the research question. A study was included if its goal is
to evaluate a technique or tool that measures/assesses the quality of cartilage. Moreover, the described technique
should potentially be non-destructive, minimally invasive and applicable for in situ measurements of articular
cartilage. Articles making use of language other than English were excluded as well as articles that studied non-
articular cartilage (e.g. intervertebral discs). Furthermore, articles in which the described technique is not validated
(e.g. with histology or mechanical testing) were also excluded. Lastly, studies that focus solely on arthroscopic
evaluations/assessments were also excluded, as this technique is based on visual and tactile assessment and has
thus a suboptimal reliability.

Critical Appraisal
The techniques found in the remaining articles were then divided into spectroscopic techniques, microscopic
techniques and other alternatives for assessment of articular cartilage. These techniques were critically appraised
for their relevance. Therefore, per technique, the following data have been collected: (1) The number of articles (2)
The testing environment (3) The specimen the technique has been used on (4) Whether the technique is already
available as being minimally invasive or not and (5) Whether the technique has been validated or not. Using this
information, a selection of the most relevant techniques could be made. In Sections 3.2-3.4 a short description is
given of the most relevant techniques and their applications considering articular cartilage are summarized. In
Section 4 the advantages and disadvantages of each technique are enlisted for comparison.
3 Results
3.1 Results of the Systematic Search
Search and Selection
After removal of duplicates, the search yielded 1063 unique articles. These articles were then screened on title and
abstract according to predetermined inclusion and exclusion criteria named above. After screening all the articles
on title and abstract, 90 articles remained. These were then screened on full-text, yielding 65 articles. An additional
15 articles were included, thus yielding a total of 80 articles. A flowchart of the complete systematic search and
inclusion- and exclusion criteria can be found in Figure 3.1.

3
Figure 3.1 An overview of the complete systematic search and inclusion- and exclusion criteria.

3.2 Spectroscopic Techniques

Spectroscopy is the study of the interaction of matter and electromagnetic radiation. A continuum of different types
of electromagnetic radiation (each associated with a particular energy range) makes up the electromagnetic
spectrum (Figure 3.2) [13]. The electromagnetic radiation interacts with material in the form of scattering,
reflection, absorption and transmission. These interactions depend on the physical and chemical properties of the
specific material [14] [15]. Different methods of spectroscopy are available for different regions of electromagnetic
radiation [14].

4
Figure 3.2 The electromagnetic spectrum. Electromagnetic radiation with the highest energy (the highest
frequency and the shortest wavelength) is located on the left, whereas electromagnetic radiation with the lowest
energy (the lowest frequency and the longest wavelength) is located on the right [13].

The input of energy into a molecule will cause it to either translate, rotate or vibrate [16]. The latter is dealt with in
vibrational spectroscopy. Although some techniques differ in several aspects, their basic physical origin is the same:
the principle is based on the vibration of the bonds between two atoms. Each stretching and bending vibration of a
given bond occurs with a characteristic frequency. When a molecule is bombarded with radiation of a frequency
(often described by wavenumber, which is the number of waves in 1 cm) that exactly matches the frequency of one
of its bonds, the molecule absorbs the energy. This feature is used in both Infrared Spectroscopy and Optical
Reflectance Spectroscopy which will be discussed in Sections 3.2.1 and 3.2.4 If the frequency of the incoming
radiation however does not match any frequency of the bonds in the molecule, the light will either be transmitted
(most of the times) or scattered. The latter is used in Raman spectroscopy, which will be dealt with in Section 3.2.2.

3.2.1 Near-Infrared Spectroscopy

As already mentioned above, when the frequency of a bond within a molecule exactly matches that of the radiation,
the molecule absorbs energy. By directing light onto a diffraction rate, light is split into several beams (of different
frequency). These beams are then directed at the sample of interest. By individually examining each wavenumber,
a spectrum can be formed as the one in Figure 3.3 [13]. Then, by determining the wavenumbers of the energy
absorbed, one can ascertain what kind of bonds a compound has. For example, the stretching vibration of a C=O
bond absorbs energy with wavenumber 1700 cm−1, whereas the stretching vibration of an O-H bond absorbs energy
with wavenumber 3400 cm−1. In the near-infrared region (λ=770-2500nm) mostly the overtones of vibrations
(water, CH-, NH-, SH- and OH-groups) can be observed. Therefore organic substances show a strong signal, which
led to multiple applications of NIRS in many fields during the last decades. Since absorptions in the NIR spectral
range arise mainly from OH, CH, NH and SH bonds, which characterize the fundamental molecular make-up of
biological materials, NIR spectroscopy is capable of detecting both micro- and macroscopic changes in articular
cartilage structure and composition [19]. Due to its relative high penetrations depth of 1–10mm into organic
samples it gives a good insight into the composition of material. It is possible to measure water content,

5
Figure 3.3 An infrared spectrum shows the percent transmission of radiation versus the wavenumber of the
radiation. The (C=O) absorbs at 1705 cm−1 and the (O–H) stretch absorbs at 3450 cm−1 [13].

oxygen–hemoglobin, glucose, lipids, proteins and other substances in tissues [14]. Cartilage undergoes complex
changes in matrix composition (water, proteoglycans, collagen) during the initial stage of cartilage degeneration.
Spahn et al [20] discovered that these complex deviations from the normal matrix composition are correlated with
changes in near-infrared absorption and that therefore near-infrared spectroscopy (NIRS) might be useful in the
diagnosis of arthritis [14].

Several studies have shown that NIR spectroscopy is capable of separating cartilage samples into different groups
relative to the severity of degeneration: the NIR spectra correlate significantly with the Mankin score (Afara et al,
2012) and GAG content (Afara et al 2014) of rat knee cartilage. The spectral data also correlate significantly with the
structural integrity, cellularity and matrix straining components of the Mankin score (Afara et al 2017). An overview
of these studies and their validations can be found in Table 12. NIR spectroscopic data have also been compared to
MIR spectroscopic data obtained from the identical tissue regions of juvenile bovine knee joints for collagen and
proteoglycan content (Palukuru et al, 2016).

Near Infrared Fiber Optics

Recent advances have been made for coupling NIR spectroscopy into an arthroscopic probe by using fiber-optics,
since they are compatible with the small dimensions of the arthroscope. Thereby, the routine arthroscopy could be
extended with spectroscopy. An example of such a near infrared-fiber optical probe as described by Prakash et al
can be seen in Figure 3.4. An overview of studies that have used such NIR fiber optic probes and their validation is
given in Table 12.
Spectral absorbances correlated with MIR measurements and data from mechanical testing of bovine cartilage
(Hanifi et al, 2017). NIRS also showed significant correlations with three out of five subscores (symptoms, sports
and quality of life) of the Knee Injury and Osteoarthritis Outcome Score (KOOS).
Spahn et al investigated the complex deviations from the normal matrix composition (water, proteoglycan and
collagen content) and their correlation with changes in NIR absorption: ICRS grade, histology, increase of water, and
softening during initial cartilage were found to significantly correlate with NIR absorption considering knee cartilage
of female sheep. Spahn et al also examined the interobserver variance of NIR spectroscopy-based arthroscopic
cartilage evaluation in comparison with International Cartilage Repair Society (ICRS) arthroscopic grading.
The most common technique in relating complex NIR spectra and reference parameters such as cartilage
biomechanical properties is currenlty partial least squares (PLS) [6]. However, Sarin et al investigated the potential
of neural networks for analysis of cartilage spectral data. They demonstrated the potential of arthroscopic NIRS to
simultaneously monitor progressive degeneration of cartilage and subchondral bone in vivo in Shetland ponies
undergoing different experimental cartilage repair procedures (Sarin et al, 2018) and for in vivo evaluation of
articular cartilage thickness, proteoglycan (PG) content, and collagen orientation angle (Sarin et al, 2019). As
references, articular cartilage biomechanical properties and subchondral bone microstructure and density were
determined via indentation testing and computed tomography (Sarin et al, 2018) and cartilage PG content and

6
collagen orientation angle were determined with digital densitometry (DD) and polarized light microscopy (PLM)
Table 3 Overview of the studies on near infrared spectroscopy.

7
Figure 3.4 (A) A conventional arthroscopic hook used by orthopedic surgeons. (B) a custom near infrared optical
probe by Prakash et al. (C) A schematic diagram of the probe tip and the arrangement of the optical
fibers. [5]

(Sarin et al, 2019) respectively. Prakash et al have shown that by employing hybrid statistical regression models,
they can reliably predict biomechanical properties from NIR spectra during knee arthroscopy. They have
demonstrated the relationship between absorption spectra and cartilage biomechanical properties and thickness.

3.2.2 Raman Spectroscopy

As already mentioned, energy is absorbed when the frequency of one of the bonds in a molecule exactly matches
that of the incoming radiation. If the frequency of the incoming radiation however does not match any frequency of
the bonds in the molecule, the light will either be transmitted (most of the times) or scattered. With scattering, light
is absorbed and re-emitted in all directions. Light can be scattered in two ways: elastically, commonly known as
Rayleigh scattering or inelastically, known as Raman scattering. In the last case, some of the energy is absorbed by
the molecular vibrations, causing a small portion of light to be re-emitted at a different frequency than the incident
light. This causes a difference in energy that is unique to each molecule and can thus act as their fingerprint [16].
When combined with a microscope, Raman spectral images can be acquired, providing diffraction-limited spatial
information about the relative spatial distribution of biochemical constituents [70] as seen in Figure 3.5.
Albro et al have shown that Raman spectroscopic imaging measurements can be used as a valuable tool for
quantitatively measuring the distribution and organization of ECM constituents in native and engineered cartilage
tissue specimens. The acquired Raman spectroscopic profiles namely exhibited strong agreement with profiles
independently acquired via direct biochemical assaying of spatial tissue sections.
Kumar et al have investigated the correlation between vibrational spectral differences and the grade of osteoarthritis
in human cartilage, classified according to the ICRS and OARSI grading systems. Their study shows that Raman
spectroscopy can classify the different stage of osteoarthritic cartilage and can provide details on biochemical
changes. They conclude that the applied technique can be extended to in vivo diagnosis with the help of a
miniaturized Raman fiber probe which can be integrated within a clinical arthroscope.
Unal et al developed a new method to assess hydration status of human knee cartilage non-destructively using

8
Figure 3.5 (A) Raw spectra of native and engineered cartilage. Raman spectroscopic image (univariate analysis) of
ECM heterogeneities in 56-day cultured large engineered cartilage tissue construct for (B) the full cross-section (C)
a localized peripheral region. (D) Raman spectroscopic image of natural depth dependent ECM heterogeneities in
native articular cartilage [70].

Table 4 Overview of the studies on Raman spectroscopy.

Raman spectroscopy, and showed the association of Raman-based water and organic content measurement with
mechanical properties of cartilage. They further compared Raman-based water measurement to gravimetric and
magnetic resonance imaging based water measurement. There was a strong association between gravimetric and
RS-based water measurement. Moreover, they found that gravimetric and RS-based water contents were

9
significantly correlated with permeability and aggregate modulus. They also conclude that Raman spectroscopy has
the potential to be used clinically to monitor cartilage quality non-invasively or minimally invasively with Raman
probe during arthroscopy procedures.

Fiber-optic Raman Spectroscopy

Esmonde-White et al have therefore developed a
custom-made pen-like fiber-optic Raman probe as
seen in Figure 3.6. Articular joint surfaces of knee
cadaveric human tissue and tissue phantoms were
examined using this custom-designed Raman fiber
optic probe. The obtained spectra were compared
with reference spectra of cartilage, subchondral bone
and cancellous bone collected using Raman
microspectroscopy. This study provides a basis for
future clinical or animal model studies. The results of
the abovenamed studies can be found in
Table 4. Figure 3.6 The Raman fiber optic probe used is a custom-
built pen-like probe. The pen-like probe has collection
3.2.3 Fourier fibers adjacent to excitation fibers in the outer ring and
troscopy Transform Infrared Spec- collection fibers in the inner ring. [23]
To overcome the time consuming acquiring of spectra
mentioned with near infrared spectroscopy, a
scanning method has been developed that covers the complete wavelength region at once. This method is named
Fourier Transform (FT) spectroscopy. The principle of FT infrared spectroscopy is based on an Michelson
interferometer. Within this interferometer, the IR beam is directed at a beam splitter: the split beam then travels to
a fixed and moving mirror respectively. The beam is then recombined (causing interference) and finally directed
through the sample, onto a detector. This detector produces a raw signal of light intensity as a function of mirror
Table 5 Overview of the studies on Fourier transform infrared spectroscopy.

position. These raw signals are mathematically Fourier transformed into the classical IR plot (light intensity vs
wavenumber).

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