Arthroscopic repair surgery is commonly performed to treat joint injuries. However, its potential for successful repair is substantially limited by the weakness of current arthroscopic diagnostic techniques. These include qualitative visual evaluation of joint via arthroscope and tissue palpation with a metallic hook. Only in the last decade has NIRS been introduced with promising results for quantitative evalua-tion of knee tissues: articular cartilage [6, 18, 19, 30, 31, 33–35, 37, 40, 41, 43, 44, 47–49], subchondral bone [32], and meniscus [99, 117]. NIRS has been applied in mul-tiple ex vivo studies using human [39, 41] and animal samples, including bovine [33–35, 39, 40, 42, 43, 118], rat [31, 32, 37, 44], and sheep [30], thus providing valuable information for the clinical application of the technique. Bovine cartilage is widely used in testing the capability of NIRS for evaluation of cartilage integrity; this is because bovine cartilage thickness is similar to human’s. In clinical sense, equine would be a more appropriate animal model due to financial implications of cartilage lesions, for example, in racing horses.
Near infrared spectra has been shown to correlate with cartilage thickness [31, 33, 41], biomechanical properties [18, 30, 34, 41, 43], water content [30, 35], collagen content [45], and PG content [40, 41]. In addition to these functional and compo-sitional properties, NIR spectra has been correlated with severity of cartilage de-fects determined via conventional scoring systems including Mankin histological score [30, 31, 37, 44], ICRS score [18, 19, 47–49], and KOOS-score [6]. Additionally, investigations on wavelength-specific light propagation [42] and contribution of un-derlying bone [39] from NIR reflectance spectra have been conducted. However, no study has investigated the feasibility of NIRS for characterization of different cartilage layers or collagen orientation.
The research on OA-related diseases, such as PTOA, has mainly focused on the role of articular cartilage; however, several studies have suggested the underlying bone to have a substantial role on the progression of the disease [119,120]. Therefore, accurate and reliable evaluation of the underlying tissue (i.e., subchondral bone plate and subchondral trabecular bone) would be of high clinical significance. Afara et al [32] showed that the NIR spectral region (4000 - 12500 cm−1) correlates with subchondral bone mineral density (BMD) and bone volume fraction (BV) in a rat model; however, the thickness of rat cartilage is substantially thinner than that of equine or human cartilage [50]. In addition, McGoverinet al[39] showed that NIR spectra measured from the direction of cartilage surface also includes contributions from the underlying bone, thus suggesting that the technique could be feasible for assessing bone integrity through relatively thick cartilage.
In addition to the NIR spectral region, VIS and MIR have been applied for
car-tilage evaluation [121, 122]. With respect to penetration of light into biological tis-sues [35], light in the MIR region is restricted to the superficial layer of cartilage and is, thus, mainly applied in laboratory environment for histological imaging.
Several studies have adopted VIS region for evaluation of tissues [41, 121] as the VIS light penetrates deeper into soft tissues. Thus, spectral measurements in this region would include contributions from multiple tissues. Furthermore, to separate tissue contributions, a detailed understanding of light penetration into soft tissues is required.
Interestingly, most laboratory studies have utilized PCR or PLSR to investigate the relationships between NIR spectra and the reference parameters. The analy-ses have mainly focused on the comparison of spectral regions or spectral shapes, with very few studies utilizing sophisticated variable selection techniques [114,123].
Additionally, shallow neural networks could provide substantial benefit over these conventional multivariate techniques [124, 125].
Arthroscopic NIRS was firstly introduced by Spahn et al[47] for evaluation of OA by prediction of ICRS scores. Otherin vivo studies by Spahn et al [19, 48, 49], Hofmannet al[6], and Martickeet al[18] have shown arthroscopic predicted ICRS scores to have good reliability when compared to ICRS scores based on MRI and X-ray images. However, these conventional imaging modalities are suboptimal for scoring cartilage defects. Furthermore, these studies are based on ineffective and simplistic determination of the ratio between two spectral peaks and focus on eval-uation of cartilage lesion severity. More recent ex vivostudies have adopted novel multivariate approaches to determine cartilage properties via NIRS [44]. These more sophisticated approaches would enable the evaluation of the cartilage surrounding the defect and, therefore, to evaluate the extent of the post-traumatic degeneration around the original lesion.
As no cure currently exists for OA or PTOA, several cartilage repair and regen-eration techniques are being developed and optimized. However, current arthro-scopic diagnostics measures are subjective and unreliable and thus not feasible for delineation of injured area to be repaired or monitoring tissue healing after repair surgery. Therefore, several studies have investigated the potential of NIRS to moni-tor engineered cartilage with positive outcomes [21, 38, 45, 46].
Overall, NIRS has shown promise for evaluation of cartilage based onex vivo findings in animal models. However, adaptation of recently introduced advanced analysis algorithms forin vivo environment should be investigated to demonstrate the clinical potential of the technique. Additionally, as NIRS provides quantitative information on cartilage composition, the technique would benefit from combina-tion with a high resolucombina-tion imaging modality, such as OCT, for a holistic optical diagnosis of cartilage integrity during arthroscopic surgery.
4 AIMS OF THIS THESIS
Cartilage defects, ruptured menisci or ligaments may be treated via arthroscopic intervention. The current highly-subjective arthroscopic evaluation of the severity and extent of joint or cartilage injury is performed based on visual evaluation and by palpating tissue surface with a metallic hook. The diagnostics would benefit from introduction of novel quantitative arthroscopic techniques which could enable, for the first time, the surgeons to objectively evaluate the extent of compromised tissue and, thus, improve patient care. Therefore, the aims of this thesis were:
• To investigate the reproducibility of scoring cartilage lesions based on con-ventional arthroscopic visualization, ultrasound, and OCT, and develop an automated algorithm to score cartilage lesion severity based on OCT imaging
• To investigate the relationship between NIR absorption spectra and functional properties of articular cartilage
• To investigate the combined potential of OCT and NIRS in the evaluation of cartilage composition and structure
• To design, manufacture, and test a novel arthrosopic NIRS fiber probe
• To investigate the potential of arthroscopic NIRS for evaluation of cartilage and subchondral bone properties in equine jointsin vivo
5 MATERIALS AND METHODS
The samples for studiesI-IIIwere acquired from a slaughterhouse in Utrecht, Nether-lands and, thus, no ethical permissions were required. The permission to conduct study IV was obtained from the local Ethics Committee for Animal Experiments in compliance with the Institutional Guidelines on the Use of Laboratory Animals, Utrecht, Netherlands (Permission: DEC 2014.III.11.098). Due to the large number of spectral and reference measurements required for the studies, multiple measure-ments were acquired per joint to ensure reliable model training. A summary of the samples used in this thesis is presented in Table 5.1.
Five equine fetlock joints were utilized in studiesItoIII. In studyIV, anin vivo experiment was conducted in Utrecht University, in which two artificial defects were created on both knees of Shetland ponies (N= 7), and filled with four experimental repairs (fibrin glue,GelMA cap,GelMA, andreinforced GelMA) [126]. After 12 months follow-up, the ponies were sacrificed and their knees examined in arthroscopy to in-vestigate the health of osteochondral tissues surrounding the repair sites. Samples including the repair site and interjacent tissue were extracted and subjected for ref-erence analyses. Additionally, similar samples were extracted from control ponies (N= 3).
Table 5.1:Summary of materials and methods utilized in studiesI-IV Study Joint Number of Measurement Methods
joints points
I Equine fetlock N= 5 n= 43 Arthroscope, OCT, ultrasound, Artscan 200, mechanical testing II Equine fetlock N= 5 n= 869 in vitroNIRS,
mechanical testing III Equine fetlock N= 5 n= 530 in vitroNIRS, OCT,
quantitative microscopy IV Equine stifle N= 20 n= 236 in vivoNIRS,µCT,
mechanical testing
5.1 NEAR INFRARED SPECTROSCOPY
The NIR spectral measurements in studies II-III were collected from the same equine samples. The reflectance spectroscopy system consisted of a light source (Avalight-HAL-S, Avantes BW, Apeldoorn, Netherlands), a spectrometer (AvaSpec-ULS2048XL, Avantes BW), and a reflectance fiber optic probe. The wavelength re-gion and resolution of the spectrometer were 200–1160 nm and 0.4 nm, respectively.
The probe contained seven (d= 600µm) honeycomb-oriented fibers, with the central fiber collecting the light back to the spectrometer and six emitting the light into the sample (Figure 5.1). For analyses, the spectral region of 0.70–1.15µm was utilized.
In studyIV, a novel reusable fiber optic probe was designed forin vivo appli-cations. The probe (d = 3.25 mm) resembles the shape of an arthroscopic hook to enable perpendicular contact with sample surface in narrow joint cavities. Probe window (d = 2 mm) includes a total of 114 fibers, of which 100 emit the light into the sample and 14 collect the reflected and scattered light back to spectrometer sys-tem. In addition, collecting fibers were organized in two lines (7 in each) and aligned with spectrometer slit to minimize light intensity loss. The probe was manufactured from stainless steel with the casing protecting the fibers made from medical grade plastic. This durable and robust probe is sterilizable in autoclave at 122 ◦C. The spectroscopic measurement system consisted of two spectrometers (AvaSpec-2048 and Avalight-NIR256-2.5-HSC, Avantes BW), a light source (AvaLight-HAL-S-Mini, Avantes BW), and the novel fiber optic probe (Figure 5.1). The spectrometers cov-ered the wavelength regions 0.40–1.10µm and 1.00–2.50µm with resolutions of 0.7 nm and 6.0 nm, respectively.
Both systems were calibrated prior to every measurement session with dark ref-erence and reflectance standard (Spectralon, SRS-99, Labsphere Inc., North Sutton, USA) to account for variation in the ambient light intensity and to calculate the absorption spectrum. The integration times of spectrometers were adjusted to max-imize signal-to-noise ratio (SNR) without saturating the signal. Furthermore, the measurements were always performed in perpendicular contact between the sam-ple and the fiber optic probe. In studiesIIandIII, the sample preservation was en-sured by keeping the non-measurement locations covered with phosphate-buffered saline (PBS) moisted clothes. Furthermore, the measurement locations were con-stantly sprayed with PBS. In studyIV, the measurement locations were submerged in Ringer solution in bothin vivoarthroscopic andin vitrolaboratory measurements.
In the laboratory, the spectral measurements were performed three times to in-vestigate the coefficient of variation (CV):
CV=
where SD is the standard deviation of the absorbance spectra, and X the average spectrum. The average spectrum of each sample was calculated for multivariate modeling. In arthroscopic measurements (study IV), 15 consecutive spectra, each average of ten acquisitions, were recorded. Out of these spectra, seven spectra (with the largest area between a linear fit and measured spectrum in spectral region 0.42–
0.75µm) were excluded from all measurement locations.
Savitzky-Golay second derivative preprocessing and smoothing was utilized in studyII, and studies III and IV, respectively. In studies IIand III, a third order
smoothing window of 25 nm length was used when predicting cartilage functional properties, composition, and structure. In studyIV, the smoothing window lengths for the two spectrometers were 14 nm and 84 nm when predicting cartilage proper-ties and 26 and 84 nm when predicting subchondral bone properproper-ties.
Figure 5.1: The laboratory NIRS fiber probe utilized in studiesII-III(A) and the novel, robust, and reusable arthroscopic NIRS probe (B and C) utilized in Shetland pony arthroscopies (studyIV). In subfigure C, the illuminating fibers (n=100) and the collecting fibers (n=14, dark fibers) are observable.