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Article

Detecting Early Changes in Cartilage Collagen and Proteoglycans Distribution Gradients in Mice Harboring the R992C Collagen II Mutant Using 2D Correlation Infrared Spectroscopy

by
Jolanta Fertala
,
Andrzej Steplewski
† and
Andrzej Fertala
*
Department of Orthopaedic Surgery, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA
*
Author to whom correspondence should be addressed.
†
Dr. A. Steplewski passed away unexpectedly during manuscript preparation.
Biophysica 2025, 5(3), 24; https://doi.org/10.3390/biophysica5030024 (registering DOI)
Submission received: 15 April 2025 / Revised: 9 June 2025 / Accepted: 19 June 2025 / Published: 22 June 2025
(This article belongs to the Collection Feature Papers in Biophysics)
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Abstract

:
Collagen II is a vital structural component in developing bones and mature cartilage. Mutations in this protein cause spondyloepiphyseal dysplasia, a disease characterized primarily by altered skeletal growth and manifesting with a range of phenotypes, from lethal to mild. This study examined transgenic mice harboring the R992C (p.R1124C) substitution in collagen II. Previous research demonstrated significant growth abnormalities and disorganized growth plate structure in these mice, and histological signs of osteoarthritic changes in the knee joints of 9-month-old mice with the R992C mutation. Our study focuses on detecting early structural changes in the articular cartilage that occur before histological signs become apparent. Through microscopic and spectroscopic analyses, we observed significant alterations in the distribution gradients of collagenous proteins and proteoglycans in the cartilage of R992C mutant mice. We propose that these early changes, eventually leading to articular cartilage degeneration in older mice, underscore the progressive nature of osteoarthritic changes linked to collagen II mutations. By identifying these early structural aberrations, our findings emphasize the importance of early detection of osteoarthritic changes, potentially facilitating timely, non-surgical interventions.

1. Introduction

Spondyloepiphyseal dysplasia (SED; OMIM 183900) is a rare genetic disorder caused by mutations in the COL2A1 gene, which encodes procollagen II. Its mature form, collagen II, is the main structural component of cartilage. Like other skeletal dysplasias arising from collagen protein mutations, SED presents a broad spectrum of skeletal abnormalities, ranging from mild to life-threatening [1,2]. A hallmark of SED is short stature, resulting from developmental disruptions in bone growth. Milder forms of SED often feature early-onset osteoarthritis (OA) [3].
The molecular bases underlying genotype–phenotype correlations in SED remain unclear. Researchers suggest that variations in the type and location of amino acid substitutions in collagen chains may determine the extent of collagen damage and skeletal abnormalities. A prevailing hypothesis is that mutations near the N-terminus lead to milder phenotypes. In contrast, mutations near the C-terminus result in more severe manifestations. For example, the R75C mutation has been associated with mildly reduced stature, chondrodysplasia, and early-onset OA. At the same time, the R789C substitution has been linked to severe SED [4,5].
Donahue et al. described a spontaneous R992C mutation in mice (p.R1124C), where homozygous littermates exhibited significant growth abnormalities [6]. Later, Holt et al. observed OA in the knee cartilage of 9-month-old heterozygous mice [7]. Our group generated transgenic mice with tetracycline (Tet)-dependent regulation of the R992C collagen II mutant. Focusing on the growth plate, we used this model to examine the effects of the collagen II mutation at various stages of skeletal development [8,9,10]. Our findings showed that the R992C mutation disrupts early bone development, leading to irreversible bone abnormalities, even after suppressing R992C expression in newborn mice [8].
In this study, we analyzed the articular cartilage of mice carrying the R992C mutation at 6 weeks and 6 months of age, stages where histology did not reveal OA changes [7]. We used Fourier-transform infrared (FTIR) spectroscopy to assess critical components, specifically collagenous proteins and proteoglycans, in the superficial layer of tibial articular cartilage. We then applied two-dimensional correlation FTIR spectroscopy (2DCS) to characterize the distribution gradients of these components, as previously described by us and others [11,12].
Our results reveal subtle alterations in essential structural components of the cartilage matrix, providing new insights into the pathogenesis of cartilage harboring collagen II mutations.

2. Materials and Methods

2.1. Mutation Nomenclature

Consistent with the literature, the R992C (mouse p.R1124C) amino acid substitution was named, considering the first glycine of the collagen triple-helix number one.

2.2. Transgenic Mice

According to the Guide for the Care and Use of Laboratory Animals guidelines, all mice received humane care. Procedures performed on animals were approved by Thomas Jefferson University’s Institutional Animal Care and Use Committee (Protocol#766E).
We analyzed articular tibial cartilage from transgenic mice harboring the R992C mutation in collagen II. These mice were initially generated for studies of growth plate aberrations, the results of which we have already described in detail [8,9,10]. Crucial features of our model include the following: (i) Besides the DNA construct for the R992C collagen II mutant, the expression of tetracycline (Tet) transactivator (tTA) facilitated Tet-dependent regulation of the expression of the construct. (ii) Cre recombinase, the expression of which was driven by a chondrocyte-specific Col2a1-cre promoter, enabled the chondrocyte-specific expression of the mutant. (iii) The simultaneous expression of all transgenes (i.e., for mutant collagen II, tTA, and Col2a1-cre) was needed to produce the exogenous R992C collagen II. (iv) The expression of the mutant construct was only possible in the absence of doxycycline (Dox, a synthetic Tet variant), while expression is inhibited by supplying Dox in the drinking water (Tet-off system). (v) Triple-transgenic mice are described as mutants (MTs). (vi) Because of the genetics, not all littermates were positive for all three transgenes needed to produce R992C collagen II. Thus, mice lacking at least one of the three required transgene DNA constructs (i.e., either for R992C mutant, tTA, or Col2a1-cre) are named wild type (WT); in these mice, exogenous R992C collagen II is not produced, but endogenous normal collagen II is [8,9]. In the study described here, WT mice lacked at least one of three required transgenes (see above), while MT mice harbored all of them. Considering that we utilized the Tet-off regulation system, there was no need to supply Dox to the mice to achieve both phenotypes.

2.3. Experimental Groups

Because OA changes were observed histologically only in 9-month-old mice harboring the R992C mutation in collagen II, we analyzed tibial cartilage from 6-week-old (6 w) and 6-month-old (6 m) mice to study possible early changes in the cartilaginous matrices [7]. For the 6 w/WT group, we analyzed 8 males and 7 females, and for the 6 w/MT group, we analyzed 3 males and 9 females. For the 6 m/WT group, we utilized 2 males and 15 females, while for the 6 m/MT group, we utilized 2 males and 6 females. As we indicated earlier, only 14.6% of transgenic mice carried all three transgenes needed to express the R992C mutant. This value closely reflected the theoretical value of 12.5% predicted as the percentage of possible triple-transgene offspring generated with the applied breeding protocol [9]. Consequently, the number of MT mice available for experiments was lower than that of the WT.

2.4. Histology of Articular Cartilage

Cartilage specimens were stained with hematoxylin and eosin (H&E, StatLab, McKinney, TX, USA) to visualize the general tissue morphology and cellularity. In addition, the tibia–femur complexes were stained with picrosirius red (Polysciences Inc., Warrington, PA, USA) to visualize collagen fibrillar deposits or safranin O (StatLab) for proteoglycans (Eclipse E600, Nikon Inc., Melville, NY, USA).

2.5. Microscopic Assays of Cartilage Cellularity and the Interterritorial Matrix

Because of the complex curvature of femoral articular surfaces, sectioning for histological assays often creates highly variable femoral cartilage planes. Thus, we focused on analyzing digital images of tibial articular cartilage characterized by more uniform geometry. Gray images of H&E-stained cartilage cross-sections were studied using image analysis software (NIS Elements version 3.22.14, Nikon Inc.). Multiple regions of interest (ROIs) from the lateral and medial sites encompassing areas demarcated by the cartilage surface and the tidemark were analyzed.
First, we analyzed the area occupied by the interterritorial matrix and expressed it as a percentage of the total ROI area. Next, we analyzed the circularity of chondrocytes, a parameter describing the cell roundness measured as the area-to-perimeter ratio. A circularity of “1” indicates perfect circles; all other shapes are characterized by a circularity smaller than “1”. Unlike the shape factor (described below), circularity excludes the local irregularities of a cell. Finally, we measured the shape factor, a parameter that defines the roughness of an object. For this parameter, a value of “1” represents an ideal, i.e., smooth, shape (e.g., an ideal circle or a square). Values smaller than “1” indicate different roughness degrees.
The percentage area means of the interterritorial matrix were tabularized according to the mutation status and the age group. For each mouse, the values from multiple ROIs were averaged. Consequently, the statistical analysis of differences among these groups was conducted using the ANOVA test (IBM SPSS Statistics for Windows version 26 software, IBM Corp., Armonk, NY, USA). In all assays, statistical significance was defined as p ≤ 0.05. The cell morphology parameters were presented as histograms (OriginPro version 2023, OriginLab Corporation, Northampton, MA, USA).

2.6. Microscopic Assays of Collagen and Proteoglycan Distribution

To measure the distribution gradients of collagenous proteins (COLs) and proteoglycans (PGs) in the articular cartilaginous matrix, we utilized digital images of consecutive cross-sections stained with picrosirius red for COLs or safranin O for PGs. Camera settings for taking pictures were kept constant for a specific staining type. Subsequently, we used NIS Elements software to generate pixel intensity profiles along the vectors drawn perpendicularly from the cartilage surface to the tidemark line. The intensity profiles were collected for each mouse along numerous vectors placed across the cartilage cross-sections. When drawing the vectors, we traced them through the interterritorial matrix areas to minimize the impact of chondrocyte-derived pixels.
Pixel intensity values (Y-axis) and their corresponding positions on the vector (X-axis) were tabularized. The slopes for each vector were obtained using the linear slope function (Excel, Microsoft 365, version 2408, Microsoft Corp., Redmond, WA, USA). Subsequently, the slope values were averaged for each mouse, and the averages were tabularized according to the mutation status and the age group.
The statistical analysis of differences among the slopes was performed using the ANOVA test. The statistical significance was defined as p ≤ 0.05. Finally, the results of the measurements were described and presented graphically (OriginPro version 2023).

2.7. Fourier-Transform Infrared Spectroscopy (FTIR)-Based COL and PG Assays

FTIR spectroscopy, utilized here, uses an infrared beam to analyze the chemical composition of biological samples [13]. Unlike light microscopy, FTIR spectroscopy of tissues does not require any labeling of the specimens, minimizing potential artifacts (Figure 1A). It offers quantitative and qualitative information about measured elements in the tissue spatial context [14,15].
The fundamental principle of this method is generating spectra from vibrations of molecular bonds exposed to the infrared beam. Since biological macromolecules, including COLs and PGs, have distinctive spectral signatures, it is possible to quantify them and map their position within the tissue [16,17].
We used FTIR spectroscopy to visualize the cartilage relative content of COLs and PGs (Figure 1B). The PG/collagen ratio was measured as the ratio of the sulfated PG-associated peak centered around the 1240 cm−1 wavenumber [ν] (arising from the symmetric stretching of the -SO3 groups) and the COL-specific peak (occurring due to CH2 wagging vibrations of proline side chains) centered around 1338 cm−1 ν [12,16].
For these assays, paraffin-embedded 5 μm thick tissue sections were deposited on MirrIR low-e microscope slides (Kevley Technologies, Chesterland, OH, USA). Then, an FTIR spectrometer (Spotlight 400, Perkin Elmer, Waltman, MA, USA) was used to analyze selected ROIs. The measurements were conducted in the reflectance mode, in the 4000 cm−1 ν to 748 cm−1 ν spectral range, at a pixel resolution of 6.25 μm, 8 scans per pixel, and a spectral resolution of 4 cm−1 ν. The background spectrum was obtained from the area not occupied by the tissue using the same scanning parameters as those described above. Spectrum Image software created co-added spectra from scanned ROIs (PerkinElmer, Inc.).

2.8. Two-Dimensional Correlation FTIR Spectroscopy (2DCS)

In brief, 2DCS is a technique used to extract information from a set of spectral data collected from a specimen exposed to an external perturbation. For example, a perturbation can be a gradual change in a solvent’s temperature, pH, or ionic strength. During these changes, the specimen’s FTIR spectra are recorded continuously. Because individual components of the analyzed specimen, e.g., components X and Z, may change their physicochemical properties differently with a changing external parameter, collected spectra reflect these changes. Consequently, variations in spectral signatures in components X and Z induced by, for example, temperature changes, are utilized to create 2D correlation spectra using cross-correlation analysis. The 2DCS method simplifies complex spectra consisting of many overlapping peaks, improves spectral resolution, and identifies the sequence of changes represented by the variations in spectral intensities [18,19].
Here, we collected FTIR spectra from consecutive layers of femoral articular cartilage from the MT and WT mice (see Figure 1C). The order of the layers was from the surface toward the tidemark. For each ROI, we analyzed 27 consecutive layers. Data from corresponding layers were averaged for each mouse group, generating 27 averaged spectra for 6 w/WT, 6 w/MT, 6 m/WT, and 6 m/MT groups.
In the context of the 2DCS principles, the “perturbance” we introduced was the change in the cartilage depth. Since COLs and PGs are the crucial components of the cartilaginous matrix, we measured the depth-dependent correlation of the contents of these components. Since the results of the 2DCS assays of the consecutive layers reflect the structural relation between spatial COL and PG arrangement, we could study potential changes in this relation due to the R992C mutation. These measurements were based on averaged spectral data from the WT or MT group’s corresponding cartilaginous regions and layers.
Then, 2D maps showing the synchronous and asynchronous correlation of the COL-specific (ν1) and PG-specific peaks (ν2) were generated using OriginPro software. Based on these analyses, we obtained the 2D correlation values from the COL-derived (1338 cm−1 ν) and PG-associated (1240 cm−1 ν) cross-peaks according to methods described elsewhere [11,12]. The PG-associated peak centered around 1240 cm−1 ν may have overlapped with signals from collagen amide III vibration, with significant mixing with CH2 wagging vibration from the glycine backbone and proline side chain [16]. Consequently, for the 2DCS assays, we also generated 2D maps that utilized the PG-associated peak, centered around 1060 cm−1 ν, arising from C-O stretching vibrations of the carbohydrate residues in PGs [20,21].
Please note that the synchronous plots show the degree of similarity between two distinct spectral intensity changes occurring at two independent wavenumbers (here, ν1, encompassing the COL-specific peak, and ν2, encompassing the PG-specific peak). The positive correlation cross-peaks indicate a simultaneous change in analyzed components in the same direction (i.e., increasing or decreasing together along the cartilage depth). In contrast, the negative synchronous 2D correlation of the cross-peaks indicates that while one spectral intensity increases, the other decreases simultaneously [22,23].
The interpretation of the asynchronous spectrum relies on the sign of the corresponding cross-peak in the synchronous spectrum. Unlike the synchronous correlation, which represents simultaneous changes, the asynchronous correlation reflects sequential changes in the signal intensities. Namely, the asynchronous cross-peaks overlap if two dynamic spectral intensities are out of phase. Consequently, if the signs of both synchronous and asynchronous peaks present in the exact coordinates (here, 1338/1240 cm−1 ν) are the same, the intensity change at wavenumber ν1 (here, the COL-specific peak) occurs before that at ν2 (here, the PG-specific peak). In contrast, when the signs of cross-peaks from the synchronous and asynchronous correlations are different, intensity variation in ν1 takes place after ν2 [22].

3. Results

3.1. Mice Expressing the R992C Mutation in Collagen II

Our group thoroughly described the morphological features of transgenic mice expressing exogenous R992C collagen II. In brief, mice harboring this mutation were smaller due to altered skeletal growth. Their tibial growth plates were disorganized with poor columnar arrangement of chondrocytes [8,9,10]. Osteoarthritic changes were not apparent in the articular cartilage observed microscopically in 6 m MT mice (Figure 2).

3.2. Microscopic Assays of the Interterritorial Matrix Area and Cellularity

Since the architecture of the COL- and PG-rich cartilaginous matrix impacts the number, spatial organization, and shape of resident chondrocytes, we studied crucial features of these cells dwelling in the matrix containing R992C collagen II.
First, we measured the interterritorial matrix area as the percentage of analyzed ROIs (Figure 3). A two-way ANOVA was conducted to examine the effects of the genotype (WT vs. MT) and age of the mice (6 w vs. 6 m) on the interterritorial matrix area. The interaction effect between genotype and age on the measured area was not statistically significant (F(1, 48) = 1.077, p = 0.305). An analysis of the main effect for genotypes was performed, which indicated that the main effect was statistically significant (F(1, 48) = 5.702, p = 0.021). However, the main effect of age was not statistically significant (F(1, 48) = 0.332, p = 0.567).
We have also analyzed the morphological parameters of chondrocytes in articular cartilage. As indicated in Figure 4, the overall distribution patterns of these parameters were similar in the corresponding WT and MT groups. The histograms also suggested that the cell counts in the analyzed areas in the MT mice appeared lower than in the WT group. This observation fits our results of the larger interterritorial matrix area in the MT group described above (Figure 3).

3.3. Microscopic Assays of PG and COL Distribution in Articular Cartilage

We measured the PG distribution along the articular surface–tidemark axis by measuring the pixel intensities of the safranin O-stained samples (Figure 2). Subsequently, we expressed the PG distribution patterns as slopes of the lines fitted to the pixel intensity distribution gradient (Figure 5). In these assays, the negative slope indicated a decrease in PGs from the surface to the tidemark. Inversely, the positive slopes implied an increase in PGs from the surface toward the tidemark.
A two-way ANOVA was conducted to examine the effects of the genotype (WT vs. MT) and age of the mice (6 w vs. 6 m) on the PG slopes. The interaction effect between genotype and age on the PG gradient was not statistically significant (F(1, 48) = 1.47, p = 0.231). Therefore, an analysis of the main effect for genotype was performed, which indicated that the main effect was statistically significant (F(1, 48) = 7.567, p = 0.008). However, the main effect of age was not significant (F(1, 48) = 3.633, p = 0.063).
Two-way ANOVA was conducted to examine the effects of the genotype (WT vs. MT) and age of the mice (6 w vs. 6 m) on the COL slopes. The interaction effect between genotype and age on the COL gradient was not statistically significant (F(1, 48) = 1.38, p = 0.245). Therefore, an analysis of the main effects for genotype and age was performed. There was neither a significant main effect of genotype (F(1, 48) = 1.713, p = 0.197) nor one of age (F(1, 48) = 1.419, p = 0.239).
Except for the positive COL and PG gradients observed in the 6 m/MT mice, COL and PG gradients for other groups were negative.

3.4. FTIR-Based COL and PG Assays

FTIR methodology was used to visualize PG and COL distribution. The PG/COL and COL/PG ratios were measured based on the sulfated PG-associated and the COL-associated peaks (Figure 1). The relative PG content was relatively high at the articular cartilage surface (SC) and the growth plate (GP). In contrast, the relative COL content was high in the spongy bone (SP) and subchondral bone (SB) areas.

3.5. 2DCS Measurements

We also measured the synchronous and asynchronous 2D correlations between changes occurring in COLs and PGs as a function of the articular cartilage depth (Figure 1). Figure 6 and Figure 7 show 2D maps of the synchronous and asynchronous correlations of the COL-specific (ν1) and PG-specific peaks (ν2). Based on these analyses, we obtained the 2D correlation values from the cross-peaks of a COL-derived peak (centered around 1338 cm−1 ν1) and PG-derived peak (centered around 1240 cm−1 ν2). In addition, 2D correlation values from the cross-peaks of a COL-derived peak, centered around 1338 cm−1 ν1, and PG-derived peak, centered around 1060 cm−1 ν2, were also calculated (Appendix A, Figure A1 and Figure A2).
Average synchronous correlation values were positive (Figure 6 and Figure 7). Thus, the depth-dependent changes in the COL-specific and PG-specific FTIR-derived signals co-occur in the same direction in all corresponding groups. In addition, analysis of the gradient slopes from histological and FTIR assays indicates that except for the 6 m/MT mice, in which COL and PG gradients were positive, in other groups, these gradients were negative (Figure 6 and Figure 7). These results suggest that except for the 6 m/MT group, where the COL and PG content was increasing along the cartilage surface–tidemark axes, the content of these molecules was decreasing in other groups.
In addition, the asynchronous 2D correlation assay indicates that in the 6 w/WT and 6 w/MT groups, the asynchronous correlation values were positive (Figure 6). This result suggests that the COL depth-dependent changes occurred before PG changes. In contrast, in the 6 m/WT group, the asynchronous correlation value was negative, indicating that the COL depth-dependent changes occurred after PG changes. The correlation values were positive in the 6 m/MT group, suggesting that the COL depth-dependent changes occurred before PG changes.
Similar results were obtained with the PG-associated peak at 1060 cm−1 ν2 (Figure A1 and Figure A2).

4. Discussion

Mutations in collagen II are associated with a range of skeletal growth aberrations [24]. The precise mechanisms by which single-amino-acid substitutions lead to clinical abnormalities are poorly understood. However, researchers have observed mutation-associated changes at multiple levels: molecular, collagen fibril, chondrocyte, and cartilage. At the molecular level, amino acid substitutions in collagen II alter the structure of individual collagen molecules, often reducing their thermostability [25]. Some mutations negatively affect collagen II fibril morphology and may decrease fibril number, weakening the cartilage [26]. Furthermore, malformed mutant collagen II molecules may accumulate in chondrocytes, frequently leading to cellular stress and apoptosis [27,28].
Since short stature is the primary feature of SED, researchers focus on how collagen II mutations impact growth plates [24]. Our group has studied mice with the R992C collagen II mutation, showing severe growth plate disruptions, including in chondrocytes and the ECM [9]. We also reported that the R992C mutation reduces the thermostability of mutant collagen II and triggers endoplasmic reticulum stress [9,25].
While our group has analyzed growth plate changes in transgenic mice with the R992C mutation, Holt et al. reported OA-like microscopic changes in the temporomandibular and knee joints of affected mice [7]. They noted that these changes appeared in the temporomandibular joints at 2 months and in the knee joints at 9 months. We hypothesized that subtle cartilage degeneration might begin before microscopic-level changes are evident. Consequently, we examined 6-week-old and 6-month-old mice expressing the R992C collagen II mutation.
The biomechanical and structural integrity of articular cartilage depends on the proper spatial organization of COLs and PGs [29,30]. Previous studies utilizing various assays, such as electron microscopy and biochemical techniques, have shown that the fibrillar architecture of collagen in articular cartilage is organized depth-dependently, with fibrils running parallel to the surface in the superficial zone and forming arch-like structures in deeper zones [31,32,33]. However, the depth-dependent quantities of COLs and PGs are less clear, partly due to the lack of standardized analytical models, varied methodologies, and differences in species, anatomical sites, and age [29,34,35,36].
Using microscopic and FTIR techniques, our study analyzed the effect of the R992C mutation on early changes in COL and PG distribution patterns. An initial indicator of matrix alterations was the observed decrease in chondrocyte numbers, resulting in a marked increase in the interterritorial matrix area in the articular cartilage of MT mice, similar to changes reported in their growth plates [8]. Although we did not investigate mechanisms behind reduced chondrocyte cellularity in MT mice in this study, our prior research demonstrated chondrocyte apoptosis linked to the R992C mutation [27]. Similarly, Liang et al. reported apoptosis-driven chondrocyte death in mice with the G970S (p.G1170S) collagen II mutation [37].
We found no significant differences in the morphological features of articular cartilage chondrocytes between 6-week-old and 6-month-old MT mice. Cell shape and circularity assays showed no significant differences between WT and MT groups. This suggests that chondrocyte quantification is a more informative indicator of collagen II mutation-related changes in articular cartilage than morphological characteristics alone. However, as Hunziker et al. have suggested, advanced microscopic techniques measuring 3D chondrocyte characteristics, such as volume density, matrix volume per chondrocyte, and cell surface area, may yield more insightful information on the relationship between collagen II mutations and cell morphology [31].
We further examined potential early alterations in the depth-dependent distribution of COLs and sulfated PGs. We measured the distribution gradients of these macromolecules in WT and MT groups using microscopic and spectroscopic methods. While most gradients were negative, we observed positive gradients for COLs and PGs in the 6-month-old MT group. Notably, these gradients were shallow, suggesting minor depth-dependent changes in COLs and sulfated PGs along the surface-to-tidemark axis. Negative PG gradients in all WT groups and the 6-week-old MT group were consistent with findings from Poole et al., who reported negative gradients for biglycan and decorin in normal human cartilage. In contrast, these gradients were positive in osteoarthritic cartilage samples [34].
Since positive gradients were only observed in the 6-month-old MT group, this may indicate progressive changes leading to gradient shifts. Histological studies showing OA changes in 9-month-old but not younger mice support this hypothesis [7]. The transition from decreasing COL and PG gradients in WT and 6-week-old MT groups to increasing gradients in 6-month-old MT mice may result from faster degradation of these molecules on the cartilage surface compared to in deeper zones, consistent with the degeneration patterns in patients with osteoarthritis [38,39].
Our results highlight early alterations in the complex distribution patterns of critical COL and PG molecules in articular cartilage with the R992C mutation in collagen II. The key to detecting these changes was using 2DCS to analyze depth-dependent COL and PG distributions. Combining this technology with histology and image analysis allowed us to detect mutation- and age-related abnormalities in articular cartilage containing the R992C mutation.
While conventional microscopic methods have been applied to study COLs and PGs in healthy and degenerative cartilage, they have limitations in accurately detecting these molecules. For example, picrosirius red staining, commonly used for collagen, may vary based on the collagen triple-helix structure, with denatured collagen binding less dye than its native form [40]. As collagen degradation and denaturation occur in OA progression, picrosirius staining may not be optimal for detecting subtle depth-dependent collagen changes in MT mice. Additionally, binding to non-collagenous proteins can interfere with dye specificity [41]. Similarly, though adequate for healthy cartilage, PG-specific stains like safranin O lose sensitivity in degenerative tissues [42].
Our findings on COL and PG gradient shifts provide new insights into the architecture of cartilage and the pathomechanisms associated with the R992C mutation in collagen II. Although early OA changes in MT mice stem from structural abnormalities in collagen, these mechanisms may also apply to OA arising from injury or inflammation. As OA changes in patients are often detected at late stages with significant cartilage loss, our results underscore the potential of new approaches for early OA detection. Non-destructive spectroscopic methods for monitoring cartilage changes in situ, for instance, could aid in identifying early cartilage erosion and inform preventive interventions [43,44,45].

5. Conclusions

Mutations in collagen II, the primary structural component of articular cartilage, disrupt normal skeletal growth and are often associated with dwarfism. These skeletal abnormalities are typically evident during both prenatal and postnatal development. In contrast, osteoarthritic changes become apparent only at later stages, once the articular cartilage exhibits advanced and extensive degeneration. Therapeutic interventions to halt cartilage damage are considerably more difficult at that point. Therefore, early detection of osteoarthritic changes presents a critical window for interventions to delay or prevent cartilage erosion.
In this study, we identified early abnormalities associated with two key structural components of cartilage affected by a collagen II mutation linked to osteoarthritis. Using Fourier-transform infrared (FTIR) spectroscopy, we detected significant disruptions in the spatial gradients of collagenous proteins and proteoglycans. Because these gradients are essential to maintaining the mechanical properties of articular cartilage, their alteration signals early structural compromise that can ultimately lead to cartilage erosion.
Our findings underscore the importance of the proper distribution of essential macromolecules along the cartilage surface–subchondral bone axis. From a clinical standpoint, this research highlights the potential of FTIR spectroscopy as a tool for detecting early osteoarthritic changes, before they progress to irreversible cartilage damage.

Author Contributions

Conceptualization, A.F. and J.F.; methodology, A.F.; validation, A.S., A.F. and J.F.; formal analysis, A.F.; investigation, J.F. and A.S.; resources, A.F.; data curation, J.F.; writing—original draft preparation, A.F.; writing—review and editing, J.F.; visualization, J.F.; supervision, A.F.; project administration, A.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no specific grants from public, commercial, or not-for-profit funding agencies.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors upon request.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
SEDSpondyloepiphyseal dysplasia
OAOsteoarthritis
TetTetracycline
FTIRFourier-transform infrared spectroscopy
tTATetracycline transactivator
Col2a1Collagen II gene
DoxDoxycycline
MTMutant
WTWild type
R992CArginine to cysteine mutation in collagen II amino acid position 992
6-w6-week-old mice
6-m6-month-old mice
H&EHematoxylin and eosin
ROIRegion of interest
COLCollagen
PGsProteoglycans
2DCSTwo-dimensional correlation FTIR spectroscopy
SCCartilage surface
GPGrowth plate
SPSpongy bone
SBSubchondral bone
G970SGlycine to serine substitution in collagen II at amino acid position 970. The number “1” position is the first glycine in the collagen II triple-helical domain.
p.G1170SGlycine to serine substitution in procollagen II at amino acid position 1170. The number “1” position is the first methionine in the procollagen II.
p.R1124CArginine to cysteine substitution in procollagen II at amino acid position 1124. The number “1” position is the first methionine in the procollagen II.

Appendix A

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Figure A1. 2DCS synchronous and asynchronous maps showing the correlation values of the COL and PG changes occurring along the articular cartilage depth in 6 w mice. Please note that crucial 2D correlation values at ν1/ν2 coordinates are indicated in each panel. These values are at the collagen-derived (ν1; centered around 1338 cm−1) and sulfated PG-associated (ν2; centered around 1060 cm−1) cross-peaks indicated by asterisks (*). A graphic presentation of the cartilage-depth-dependent COL and PG changes is also demonstrated. The arrows show the directions of the depth-dependent changes in COL (▼) and PG (▼) signals. A longer arrow indicates that the changes it represents (e.g., in COLs) occur before those in an analyzed partner (e.g., in PGs).
Figure A1. 2DCS synchronous and asynchronous maps showing the correlation values of the COL and PG changes occurring along the articular cartilage depth in 6 w mice. Please note that crucial 2D correlation values at ν1/ν2 coordinates are indicated in each panel. These values are at the collagen-derived (ν1; centered around 1338 cm−1) and sulfated PG-associated (ν2; centered around 1060 cm−1) cross-peaks indicated by asterisks (*). A graphic presentation of the cartilage-depth-dependent COL and PG changes is also demonstrated. The arrows show the directions of the depth-dependent changes in COL (▼) and PG (▼) signals. A longer arrow indicates that the changes it represents (e.g., in COLs) occur before those in an analyzed partner (e.g., in PGs).
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Figure A2. 2DCS synchronous and asynchronous maps showing the correlation values of the COL and PG changes occurring along the articular cartilage depth in 6 m mice. Please note that crucial 2D correlation values at ν1/ν2 coordinates are indicated in each panel. These values are at the collagen-derived (ν1; centered around 1338 cm−1) and sulfated PG-derived (ν2; centered around 1060 cm−1) cross-peaks indicated by asterisks (*). A graphic presentation of the cartilage-depth-dependent COL and PG changes is also demonstrated. The arrows show the directions of the depth-dependent changes in COL (▼) and PG (▼) signals. A longer arrow indicates that the changes it represents (e.g., in COLs) occur before those in an analyzed partner (e.g., in PGs).
Figure A2. 2DCS synchronous and asynchronous maps showing the correlation values of the COL and PG changes occurring along the articular cartilage depth in 6 m mice. Please note that crucial 2D correlation values at ν1/ν2 coordinates are indicated in each panel. These values are at the collagen-derived (ν1; centered around 1338 cm−1) and sulfated PG-derived (ν2; centered around 1060 cm−1) cross-peaks indicated by asterisks (*). A graphic presentation of the cartilage-depth-dependent COL and PG changes is also demonstrated. The arrows show the directions of the depth-dependent changes in COL (▼) and PG (▼) signals. A longer arrow indicates that the changes it represents (e.g., in COLs) occur before those in an analyzed partner (e.g., in PGs).
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Figure 1. FTIR-based assays of PG and COL distribution in articular cartilage. (A) An image of an unstained knee joint observed using an FTIR-associated light microscope. (B) Maps of the COL/PG and PG/COL ratios of a region indicated in panel A. Red/orange colors represent high ratios, while blue/green colors represent low ratios. (C) An H&E-stained joint showing the areas of articular cartilage used for 2DCS assays. (D) A representative raw spectrum from cartilage regions indicated in panel (C). Spectral peaks used in 2DCS assays are indicated. Symbols: Fe, femur; Tb, tibia; ME, meniscus; SC, superficial cartilage; GP, growth plate; SP and SB, spongy and subchondral bone; A, absorbance.
Figure 1. FTIR-based assays of PG and COL distribution in articular cartilage. (A) An image of an unstained knee joint observed using an FTIR-associated light microscope. (B) Maps of the COL/PG and PG/COL ratios of a region indicated in panel A. Red/orange colors represent high ratios, while blue/green colors represent low ratios. (C) An H&E-stained joint showing the areas of articular cartilage used for 2DCS assays. (D) A representative raw spectrum from cartilage regions indicated in panel (C). Spectral peaks used in 2DCS assays are indicated. Symbols: Fe, femur; Tb, tibia; ME, meniscus; SC, superficial cartilage; GP, growth plate; SP and SB, spongy and subchondral bone; A, absorbance.
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Figure 2. Cross-sections of the knee joint from a 6 m MT mouse stained with picrosirius red for COLs (A,C) and safranin O for PGs (B,D). Boxes in (A,B) indicate enlarged areas in corresponding (C,D) panels. Symbols: Fe, femur; Tb, tibia; AC, articular cartilage; GP, growth plate; BO, bone; ME, meniscus; SC, superficial cartilage; CC, calcified cartilage.
Figure 2. Cross-sections of the knee joint from a 6 m MT mouse stained with picrosirius red for COLs (A,C) and safranin O for PGs (B,D). Boxes in (A,B) indicate enlarged areas in corresponding (C,D) panels. Symbols: Fe, femur; Tb, tibia; AC, articular cartilage; GP, growth plate; BO, bone; ME, meniscus; SC, superficial cartilage; CC, calcified cartilage.
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Figure 3. Analysis of the area occupied by the acellular interterritorial matrix in tibial articular cartilage of analyzed mice (individual points). The interquartile range between the 25th and 75th percentiles determines each box. The lines within the boxes represent the medians, while the whiskers delineate the standard deviation values.
Figure 3. Analysis of the area occupied by the acellular interterritorial matrix in tibial articular cartilage of analyzed mice (individual points). The interquartile range between the 25th and 75th percentiles determines each box. The lines within the boxes represent the medians, while the whiskers delineate the standard deviation values.
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Figure 4. Histograms showing the circularity and shape distributions of chondrocytes observed in tibial articular cartilages. Each bar represents the count of cells within a specific range (bin) of parameter values.
Figure 4. Histograms showing the circularity and shape distributions of chondrocytes observed in tibial articular cartilages. Each bar represents the count of cells within a specific range (bin) of parameter values.
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Figure 5. Comparison of the COL and PG distribution slopes along the cartilage surface–tidemark axis in analyzed mice (individual points). The interquartile range between the 25th and 75th percentiles determines each box. The lines within the boxes represent the medians, while the whiskers delineate the standard deviation values.
Figure 5. Comparison of the COL and PG distribution slopes along the cartilage surface–tidemark axis in analyzed mice (individual points). The interquartile range between the 25th and 75th percentiles determines each box. The lines within the boxes represent the medians, while the whiskers delineate the standard deviation values.
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Figure 6. 2DCS synchronous and asynchronous maps showing the correlation values of the COL and PG changes occurring along the articular cartilage depth in 6 w mice. Please note that crucial 2D correlation values at ν1/ν2 coordinates are indicated in each panel. These values are at the collagen-derived (ν1; centered around 1338 cm−1) and sulfated PG-derived (ν2; centered around 1240 cm−1) cross-peaks indicated by asterisks (*). A graphic presentation of the cartilage-depth-dependent COL and PG changes is also demonstrated. The arrows show the directions of the depth-dependent changes in COL (▼) and PG (▼) signals. A longer arrow indicates that the changes it represents (e.g., in COLs) occur before those in an analyzed partner (e.g., in PGs).
Figure 6. 2DCS synchronous and asynchronous maps showing the correlation values of the COL and PG changes occurring along the articular cartilage depth in 6 w mice. Please note that crucial 2D correlation values at ν1/ν2 coordinates are indicated in each panel. These values are at the collagen-derived (ν1; centered around 1338 cm−1) and sulfated PG-derived (ν2; centered around 1240 cm−1) cross-peaks indicated by asterisks (*). A graphic presentation of the cartilage-depth-dependent COL and PG changes is also demonstrated. The arrows show the directions of the depth-dependent changes in COL (▼) and PG (▼) signals. A longer arrow indicates that the changes it represents (e.g., in COLs) occur before those in an analyzed partner (e.g., in PGs).
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Figure 7. 2DCS synchronous and asynchronous maps showing the correlation values of the COL and PG changes occurring along the articular cartilage depth in 6 m mice. Please note that crucial 2D correlation values at ν1/ν2 coordinates are indicated in each panel. These values are at the collagen-derived (ν1; centered around 1338 cm−1) and sulfated PG-derived (ν2; centered around 1240 cm−1) cross-peaks indicated by asterisks (*). A graphic presentation of the cartilage-depth-dependent COL and PG changes is also demonstrated. The arrows show the directions of the depth-dependent changes in COL (▼) and PG (▼) signals. A longer arrow indicates that the changes it represents (e.g., in COL) occur before those in an analyzed partner (e.g., in PGs).
Figure 7. 2DCS synchronous and asynchronous maps showing the correlation values of the COL and PG changes occurring along the articular cartilage depth in 6 m mice. Please note that crucial 2D correlation values at ν1/ν2 coordinates are indicated in each panel. These values are at the collagen-derived (ν1; centered around 1338 cm−1) and sulfated PG-derived (ν2; centered around 1240 cm−1) cross-peaks indicated by asterisks (*). A graphic presentation of the cartilage-depth-dependent COL and PG changes is also demonstrated. The arrows show the directions of the depth-dependent changes in COL (▼) and PG (▼) signals. A longer arrow indicates that the changes it represents (e.g., in COL) occur before those in an analyzed partner (e.g., in PGs).
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Fertala, J.; Steplewski, A.; Fertala, A. Detecting Early Changes in Cartilage Collagen and Proteoglycans Distribution Gradients in Mice Harboring the R992C Collagen II Mutant Using 2D Correlation Infrared Spectroscopy. Biophysica 2025, 5, 24. https://doi.org/10.3390/biophysica5030024

AMA Style

Fertala J, Steplewski A, Fertala A. Detecting Early Changes in Cartilage Collagen and Proteoglycans Distribution Gradients in Mice Harboring the R992C Collagen II Mutant Using 2D Correlation Infrared Spectroscopy. Biophysica. 2025; 5(3):24. https://doi.org/10.3390/biophysica5030024

Chicago/Turabian Style

Fertala, Jolanta, Andrzej Steplewski, and Andrzej Fertala. 2025. "Detecting Early Changes in Cartilage Collagen and Proteoglycans Distribution Gradients in Mice Harboring the R992C Collagen II Mutant Using 2D Correlation Infrared Spectroscopy" Biophysica 5, no. 3: 24. https://doi.org/10.3390/biophysica5030024

APA Style

Fertala, J., Steplewski, A., & Fertala, A. (2025). Detecting Early Changes in Cartilage Collagen and Proteoglycans Distribution Gradients in Mice Harboring the R992C Collagen II Mutant Using 2D Correlation Infrared Spectroscopy. Biophysica, 5(3), 24. https://doi.org/10.3390/biophysica5030024

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