2.1.

Specimen Preparation—Day 0

Fresh hind legs were obtained from 2- to 5-month-old pigs, weighing about 40 kg from the Primary Industries abattoir in Singapore. Each hind leg was dissected above the knee joint within 3 h of slaughter. Using a trephine, 100 full-depth cartilage explants of 4 mm in diameter and 2.5 mm in depth were extracted from the menisci-covered tibial region.¹⁶ These explants were randomly divided into four equal groups: the control (unimpacted healthy cartilage) and three groups that were subjected to unconfined single impact loads of 15, 20, and 25 MPa (Refs. 16 to 18). All explants were immediately immersed in isotonic Dulbecco modified Eagle medium (DMEM, 280 mOsm, pH 7.4) (Gibco, Basel, Switzerland) supplemented with 1-g/L L-glutamine, 10-mg/mL streptomycin sulfate, 10,000-units/mL penicillin G sodium, and 10% (v/v) fetal bovine serum¹⁹ (Sigma-Aldrich, St. Louis, Missouri). They were incubated at 37°C and 5% CO₂ in a humidity-controlled incubator for up to 7 days. The cell culture medium was changed every 3 days.

2.2.

Single Impact-Unconfined Compression—Day 0

Three groups of explants were subjected to a single impact-unconfined compression. A stainless steel 10-mm-diam compression plate and a dental cement pot (base-liquid and powder, Dentsply, Tianjin, China) was attached to the single column testing system (Instron, model 3345, Norwood, Massachusetts). Each moist explant from the impact group was placed on a sterile dental cement pot directly beneath the compression plate. A compression preload of 10 N was applied to ensure that the entire surface of the cartilage was in close contact with the compression plate. The impact groups were subjected to the corresponding peak stresses (15, 20, or 25 MPa) at a displacement rate of 2 mm/s using a uniaxial 5 × 10³ N load cell. After each impact, the explant was removed from the dental cement pot, immersed in fresh cell culture medium, and incubated. Control and impacted explants were randomly selected for cell viability assessment, histology, and Raman spectroscopy on day 7.

2.3.

Cell Viability Test—Day 7

On day 7, explants (n = 5 for each impact group) were removed from the cell culture medium and incubated for 30 min in 2 ml of medium of 0.1% fluorescein diacetate, and 0.05% propidium iodide (Sigma-Aldrich, St. Louis, Missouri). A confocal fluorescence microscope (Fluroview FV300; Olympus) with an excitation wavelength of 488 nm was used to image the cells on the cartilage surface, called chondrocytes. To visualize live cells, a narrow-bandpass filter centered at 550 nm (green) with an FWHM of 20 nm was used. To visualize dead cells, a narrow-bandpass filter centered at 650 nm (red) with an FWHM of 20 nm was used. A 20× objective lens was used to obtain images of the center of each explant. Pixel area fractions of viable and nonviable cells were calculated using Image J (Version1.4; NIH, Bethesda, Maryland). From these images, the percentage cell viability, given as P _L / (P _L + P _D), where P _L is the number of green pixels representing live cells, and P _D is the number of red pixels representing dead cells, was calculated. Their values were normalized and thereafter compared between the control and each impacted group.

2.4.

Histology Protocol—Day 7

On day 7, explants (n = 15 for each impact group) were fixed in 10% buffered formalin on a shaker for 2 weeks and then decalcified in 30% formic acid for another 2 weeks. Thereafter, the explants were dehydrated and cleared in ethanol and toluene for 16 h. The explants were then embedded in paraffin blocks and 8-μm cartilage slices were obtained using a microtome (RM2255, Leica Microsystems, Nussloch, Germany). Next, the sections were deparaffinized and stained using hematoxylin and eosin and Safranin-O/Fast Green to visualize the osteochondral structure, cell distribution, and proteoglycan or GAG contents. The histological appearance of the cartilage was finally evaluated by five independently trained investigators blinded against the control and impact groups to eliminate any bias. A modified Mankin scoring system was employed to grade the extent of cartilage disruption ^{22, 23, 24} (Table 1).

Fig. 7

Comparison of the mean perpendicular polarized Raman spectra from 800 to 1200 cm⁻¹ among control and impact groups. The singlet amide III Raman peak at 1268 cm⁻¹ is observed for the control group, while the doublet amide III peaks at 1264 and 1274 cm⁻¹ are found for all impact groups. The amide III Raman bands have been vertically shifted for better visualization.

Table 1

Modified Mankin scores grading system (Refs. 22, 23, 24).

2.5.

Raman Spectrometry Test—Day 7

We compared linearly polarized (i.e., perpendicular and parallel polarized) Raman spectra of porcine explants (n = 10 for control and 15-MPa impact groups; n = 5 for 20- and 25-MPa impact groups) with the nonpolarized Raman spectra of the same explant on coverslips (vfm coverslips, CellPath Ltd., United Kingdom) using a confocal Raman spectrometer system (inVia, Renishaw, United Kingdom) coupled with a microscope (DMI 5000M, Leica) in a backscattering geometry.²⁵ The parallel- or perpendicular-polarized micro-Raman measurements were performed by changing the direction of a linear polarizer (analyzer) placed in front of the CCD detector to be parallel or perpendicular with respect to the polarization direction of the linear polarizer placed in front of the 785-nm laser. The 785-nm laser beam (maximum output of 100 mW, Renishaw, United Kingdom) was focused onto explant with a beam size of ∼1 μm via a microscope objective [40×, numerical aperture (NA) 0.9, Leica, Germany]. The tissue Raman spectra were detected by a NIR-enhanced CCD detector (Peltier cooled at −70°C, Renishaw, United Kingdom) through a Czerny-Turner-type spectrograph (f = 250 mm) equipped with a holographic grating (1800 gr/mm). The spectral resolution of the micro-Raman system is ∼4 cm⁻¹. The nonpolarized and polarized (i.e., parallel and perpendicular) micro-Raman spectra can be acquired in tandem on the same samples by our confocal micro-Raman system. Each Raman spectra was collected with an integration time of 5 s and a 785-nm laser illumination power of ∼1 mW on the samples.

2.6.

Raman Data Processing

Each raw Raman spectrum was smoothed using the third-order Savitzky–Golay smoothing filter, and the fifth-order polynomial ^{26, 27, 28} was found to be optimal for fitting the autofluorescence background in the noise-smoothed spectrum. This polynomial was subtracted from the smoothed raw spectrum to yield the tissue Raman spectrum alone.²⁷ The multiple-component model was used to deconvolute selected Raman bands. To facilitate a computer fit to the data, the component Raman spectral intensity I(x) was assumed to be Gaussian²⁹:

2.7.

Statistical Analysis

A Mann-Whitney test was performed to compare the modified Mankin score from each impact group against the control group. In addition, a one-way anlysis of variance (ANOVA) was performed followed by post hoc Bonferroni tests for comparison of modified Mankin scores, normalized cell viability scores and biochemical changes (e.g., pyranose intensity 1126 cm⁻¹, proline to hydroxyproline band areas ratio 856 cm⁻¹/875 cm⁻¹, amide III band's red shift and FWHM of amide I band 1664 cm⁻¹). Differences were considered significant at the p < 0.05 level. An unpaired two-sided Student's t test was also used to compare changes between the FWHM of the parallel polarized amide I band and the FWHM of the perpendicular polarized amide I band within each impact and control group. Again, differences were considered significant at p < 0.05. To separate biochemical changes of impacted cartilage explants from control explants, the decision lines were selected using user-defined separate functions to obtain the optimal diagnostic specificity and sensitivity. The specificity was measured as the number of explants correctly identified as the normal unimpacted cartilage, while the sensitivity was measured as the number explants correctly identified as impacted cartilage.

3.1.

Cell Viability

Figures 1a and 2 show the cell viability results for the control and impact groups. No significant cell death (p > 0.05, one-way ANOVA and post hoc Bonferroni test) was noted for the 15-MPa impact group (n = 5) when compared to the control (n = 5). However, significant chondrocytes death (p < 0.05, one-way ANOVA and post hoc Bonferroni test) was noted for the 20- (n = 5) and 25-MPa (n = 5) impact groups. These results are consistent with previous chondrocytes viability findings¹⁸ that the threshold for cell death was between 15 and 20 MPa.

Fig. 1

(a) Comparison of normalized cell viability profile among the control (n = 5) and impacted cartilage explants (n = 5 for each group); (b) comparison of modified Mankin score profile of control (n = 15) and impacted cartilage explants (n = 15 for each group); (c) comparison of mean parallel polarized Raman intensity at 1126 cm⁻¹ among the control (n = 10) and impacted 15- (n = 10), 20- (n = 5), and 25-MPa (n = 5) cartilage explants, where the decision line (I ₁₁₂₆ = 53) yields a sensitivity of 90% and specificity of 90% to separate the control from the impact groups; and (d) comparison of the Raman band area ratio of proline (856 cm⁻¹) to hydroxyproline (875 cm⁻¹) from the parallel polarized Raman spectra among control and impacted cartilage explants, where the decision line (I ₈₅₆/I ₈₇₇ = 1.85) yields a sensitivity of 88% and specificity of 95% to separate the control from the impact groups. Note that the symbol * denotes statistically significant difference detected between the impact group and the control group, where p < 0.05. The y axis error bars indicate ± 1 standard deviation (SD).

Fig. 2

Confocal images of the cartilage surfaces of control and impacted groups after 7 days. Green colors (pixels) in the first column represent live cells; red colors (pixels) in the second column represent dead cells [see Fig. 1a for normalized cell viability profiles]. (Color online only.)

3.2.

Histological Findings

Figure 3 shows the example of the histological slides for the 15-MPa impacted explants, revealing surface irregularities with fibrillation and small clefts limited to the superficial zone. Slight reductions of PG staining were also observed but no tidemark (boundary between calcified and uncalcified cartilage) disruptions were found. The mean modified Mankin score for the 15-MPa impacted explants was 4.48 ± 1.57 points [Fig. 1b]. This score is classified as a mild osteoarthritic stage, grade I.²⁴ Statistically, this score is not significantly higher (p > 0.05) than modified Mankin score for the control group, 3.64 ± 1.02 points (n = 15). Hence, the modified Mankin score revealed no substantial structural changes between the control and the 15-MPa impact group. On the other hand, histological results for the 20- and 25-MPa impact groups (Fig. 3) revealed extended clefts from the superficial to the calcified zone. In these high-impact groups, tidemarks were disrupted and severe reduction in PG staining in deep regions was noted. Some explants were also found to have a complete disorganization of structure. The 20- and 25-MPa impacted groups explants were graded with mean modified Mankin scores of 8.16 ± 1.83 and 8.42 ± 2.34 points (n = 15), respectively [Fig. 1b]. These scores were classified as grade 4, a severe osteoarthritic stage,²⁴ which is significantly higher (p < 0.05) than the control group. Hence, the modified Mankin score revealed significant structural changes in the 20- and 25-MPa impact groups.

Fig. 3

Histology slides of the sectioned cartilage explants stained using: (a) hemotoxylin and eosin and (b) Safranin-O/Fast Green. Slight reduction in proteoglycan (PG) staining with no matrix disruptions at 15-MPa impacted explants. Obvious reductions in proteoglycans staining and matrix disruptions are observed in 20- and 25-MPa impacted explants.

3.3.

Raman Spectroscopy Results for Cartilage

Multiple Raman spectra were measured from the control (n = 10), 15- (n = 10), 20- (n = 5), and 25-MPa (n = 5) impact groups, respectively. A maximum of four spectra on the same cartilage explants were obtained at various locations to reduce the chances of overheating,³⁰ and serious disturbances of the cellular structure. All cartilage explants were also kept moist with physiological saline solution during Raman measurements. Figure 4 shows the mean nonpolarized and polarized (parallel and perpendicular) Raman spectra of all impact and control groups, exhibiting similar Raman spectral shapes and primary bands positions. Raman peaks were observed at the following locations: 856, 875, 940, 1003, 1042, 1126, 1160, 1207, 1265–1279, 1450, 1560, and 1660 cm⁻¹. Table 2 outlines the tentative biochemical assignments of respective Raman peaks observed in porcine cartilage. ^{10, 13, 14} Exceptions were noted in the parallel polarized Raman band at 957 and 1063 cm⁻¹ (Fig. 5), where the phosphate shoulder peak at 957 cm⁻¹ was observed only in the impact groups but not in the control group, while the sulphate shoulder peak at 1063 cm⁻¹ was observed only in 15-MPa impact group. On the other hand, the mean perpendicular polarized Raman spectra [Fig. 4c] revealed a significant overlapping of Raman bands between 800 and 1200 cm⁻¹. Compared to the parallel polarized Raman spectra [Fig. 4b], Raman bands at 856, 875, 940, 957, and 1560 cm⁻¹ were not detected in the perpendicular polarized Raman spectra [Fig. 4c]. The perpendicular polarized Raman intensities for Raman bands at 1003 and 1042 cm⁻¹ [arrows in Fig. 4c] were found to be notably reduced by 3 to 4 times relative to the parallel polarized Raman intensity [arrows in Fig. 4b]. This indicates that the Raman bands at 1003 and 1042 cm⁻¹ exhibit parallel dichroism in cartilage tissue.

Fig. 4

Comparison of Raman spectra among control and impacted porcine cartilage explants at 15, 20, and 25 MPa, respectively, obtained a week after impact: (a) mean nonpolarized Raman spectra, (b) mean parallel polarized Raman spectra, and (c) mean perpendicular-polarized Raman spectra. A single-headed arrow indicates the band where the parallel dichroism is observed, while the double-headed arrow indicates the FWHM of amide I raman band.

Fig. 5

Comparison of mean parallel polarized Raman spectra in the region of 800 to 1200 cm⁻¹ among the control and impacted porcine cartilage explants at 15, 20, and 25 Mpa, respectively, obtained 7 days after impact. The single arrow indicates the pyranose ring group, where a significant decrease in intensity is observed after impact [see Fig. 1(c)].

Table 2

Raman peak positions and tentative assignments of major vibrational bands observed in normal and impacted articular cartilage explants using polarized Raman spectroscopy (Refs. 10, 13, 14).

√: Peak present in Raman spectra; * Peak only appears in impact groups.

3.4.

Pyranose Ring Raman Band

For the pyranose ring Raman band at 1126 cm⁻¹ (arrow in Fig. 5), its mean parallel polarized Raman intensity was observed to have a significant decrease for all impact groups [Fig. 1c]. Note that the Raman intensity measurements were found to be quite consistent (of less than 5% variations in Raman intensities among different locations on the cartilages) for each group. Significant differences (p < 0.0005) were obtained between the control and impact groups. The mean parallel polarized pyranose intensity of the control group can be separated from all impact groups with a 90% sensitivity and 90% specificity (when a decision line I ₁₁₂₆ = 53 is selected) [Fig. 1c]. In contrast, no decision line could be made to separate the control group from impact groups due to the insignificant modified Mankin scores between the control and 15-MPa impact groups [Fig. 1b]. For the mean nonpolarized pyranose intensity at 1126 cm⁻¹, no statistical difference was found between the control and each impact group (p > 0.05) [Fig. 6a].

Fig. 6

(a) Comparison of mean nonpolarized pyranose Raman band intensity at 1126 cm⁻¹ among control and impacted porcine explants, note that each impact group is not statistically different from the control group (p = 1); and (b) comparison of mean nonpolarized band area ratio of proline (856 cm⁻¹) to hydroxyproline (875 cm⁻¹) among control and impacted cartilage explants, where each impact group is not statistically different from the control group (p = 1). The y axis error bars indicate ± 1 SD.

3.5.

Raman Band Area Ratio of Proline to Hydroxyproline

Figure 1d shows the mean Raman band area ratio values of proline (856 cm⁻¹) to hydroxyproline (875 cm⁻¹) in the parallel polarized spectra, illustrating the significant decrease (p < 0.05) for all impact groups. In contrast to the modified Mankin scores [Fig. 1d], where no decision lines could be made, a separate line (I ₈₅₆/I ₈₇₅ = 1.85) can be determined to differentiate the mean proline:hydroxyproline band area ratios of the control from all the impact groups with a 95% sensitivity and 88% specificity. For the mean nonpolarized Raman spectra for proline: hydroxyproline band area ratios [Fig. 6b], there is no statistical difference between the control and the impact groups (p > 0.05).

3.6.

Amide III and Amide I Bands

As summarized in Table 3, a significant Raman shift of amide III peak position was noted in both the mean nonpolarized and parallel polarized Raman spectra. However, in the mean perpendicular polarized Raman spectra, the amide III Raman band appears to be doublet peaks at 1264 and 1274 cm⁻¹ for all the impact groups band, as compared to the singlet peak at 1268 cm⁻¹ for the control group (Fig. 7). For the amide I band at 1664 cm⁻¹, its FWHM for each impact group was significantly narrower (p < 0.05, unpaired two-sided Student's t test) in the parallel polarized spectra [double arrows in Fig. 4b] compared to the perpendicular polarized spectra Fig. 4c]. Note that the mean amide I FWHM values of Raman spectra for the control group were 60 ± 11 (n = 10), and 107± 13 (n = 10), respectively, in the parallel and perpendicular polarized spectra. For the 15-MPa group, the mean amide I FWHM values were 61 ±13 (n = 10) and 107 ± 20 (n = 10) in the parallel and perpendicular polarized spectra, respectively. For the 20-MPa impact group, the mean amide I FWHM values were 65 ±10 (n = 5) and 96 ±15 (n = 5) in the parallel polarized and perpendicular spectra, respectively. For the 25-MPa impact group, the mean amide I FWHM values were 63 ±10 (n = 5) and 113 ± 8 (n = 5) in the parallel and perpendicular polarized Raman spectra, respectively.

Table 3

Comparison of the Raman shift and the p value (one-way ANOVA with post hoc Bonferroni test) of the amide III peak positions obtained from the parallel polarized Raman spectra for control and impacted cartilage explants.

n = number of explants.

s = significant difference compared to control group, p < 0.05.