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Journal of Anatomy logoLink to Journal of Anatomy
. 2024 Jun 24;245(5):740–750. doi: 10.1111/joa.14102

Assessment of post‐trauma microstructural alterations in the rabbit knee cartilage and subchondral bone

Amanveer Singh 1, Hannah Mantebea 1, Farid Badar 1, Syeda Batool 1, Austin Tetmeyer 1, Gabrielle Abdelmessih 2, Talia Sebastian 2, Michael Newton 3,4, Kevin Baker 3,5,6, Sarah Salem 1, Yang Xia 1,✉
PMCID: PMC11470779  PMID: 38924533

Abstract

Early diagnosis of post‐traumatic osteoarthritis (PTOA) is critical for designing better treatments before the degradation becomes irreversible. We utilized multimodal high‐resolution imaging to investigate early‐stage deterioration in articular cartilage and the subchondral bone plate from a sub‐critical impact to the knee joint, which initiates PTOA. The knee joints of 12 adult rabbits were mechanically impacted once on the femoral articular surface to initiate deterioration. At 2‐ and 14‐week post‐impact surgery, cartilage‐bone blocks were harvested from the impact region in the animals (N = 6 each). These blocks were assessed for deterioration using polarized light microscopy (PLM), microcomputed tomography (μCT), and biochemical analysis. Statistically significant changes were noted in the impact tissues across the calcified zone (CZ) at 14 weeks post‐impact: the optical retardation values in the CZ of impact cartilage had a drop of 29.0% at 14 weeks, while the calcium concentration in the CZ of impact cartilage also had a significant drop at 14 weeks. A significant reduction of 6.3% in bone mineral density (BMD) was noted in the subchondral bone plate of the impact samples at 14 weeks. At 2 weeks post‐impact, only minor, non‐significant changes were measured. Furthermore, the impact knees after 14 weeks had greater structural changes compared with the 2‐week impact knees, indicating progressive degradation over time. The findings of this study facilitated a connection between mineralization alterations and the early deterioration of knee cartilage after a mechanical injury. In a broader context, these findings can be beneficial in improving clinical strategies to manage joint injuries.

Keywords: articular cartilage, impact injury, PLM, post‐traumatic osteoarthritis, μCT

We utilized multimodal high‐resolution imaging to investigate early‐stage deterioration in knee articular cartilage and the subchondral bone from a sub‐critical impact to the femur. Statistically significant optical retardation changes were noted in the impact cartilage across the calcified zone (CZ) at 14 weeks post‐impact. A significant reduction in bone mineral density (BMD) was also observed in the subchondral bone plate of the impact samples at 14 weeks.

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1. INTRODUCTION

In synovial joints, such as the knee, articular cartilage (AC) is a layer of connective tissue that coats the end of bones and is responsible for smooth joint movement and protection against compressive forces. Across its thickness, AC can be sub‐divided into four organized zones based on the structural differences in the extracellular matrix (ECM) of each layer: superficial zone (SZ), transitional zone (TZ), radial zone (RZ), and calcified zone (CZ) (Ulrich‐Vinther et al., 2003; Xia et al., 2001). The SZ, TZ, and RZ are uncalcified tissues. The CZ is located in the deepest region of the AC bounded by the tidemark and the cement line bordering the subchondral bone (Badar & Xia, 2022). The mineralization in the CZ provides a gradual transition of mechanical properties from the soft AC to the hard subchondral bone, this transition is crucial for joint stability and functionality (Clark, 1990; Mansfield & Winlove, 2012). The morphological structure and the chemical composition of the CZ are yet to be fully understood. In a previous study, calcium content has been found to be the major component in the CZ, accounting for 65%–75% of the dry weight (Zizak et al., 2003).

Just beneath the CZ lies the subchondral bone, a vital structure that supports the AC and helps in the even and gradual distribution of the mechanical load. The subchondral bone is anatomically divided into two structures: Subchondral bone plate (SBP) and subchondral trabecular bone (STB) (Goldring & Goldring, 2010). SBP is a thin layer located directly under the CZ consisting of tiny channels containing blood vessels and nerves that link the STB and the CZ, the size and number of these channels can change with age, pressure, and injuries (Madry et al., 2010; Milz & Putz, 1994). Relative to SBP, STB is more porous and consists of blood vessels, sensory nerves, and bone marrow. STB demonstrates a strong structural anisotropy (Li et al., 2013; Suri & Walsh, 2012).

Osteoarthritis (OA) is a progressive joint disease that can be initiated by several factors, such as aging, gender, joint injury, joint misalignment, and physical stress (Li et al., 2013). The risk of post‐traumatic osteoarthritis (PTOA) rises by 75%, after a significant trauma to the joint (Buckwalter & Mankin, 1998). Trauma to a joint not only initiates cartilage degradation but also negatively affects the subchondral bone, starting osteoarthritis by changing the microstructure of the AC and the subchondral bone (Li et al., 2013; Lories & Luyten, 2011). Remodeling and mineralization changes in the CZ and SBP can affect the mechanical and biological stability of the joint. The progression of OA at the microstructural level is not yet fully understood, but it is believed that structural changes in the CZ and subchondral bone play a crucial role in OA pathogenesis. Studies have shown that distinct structural changes in the subchondral bone can happen at different stages of OA progression (Li et al., 2013).

Animal models are highly useful for studying the early phases of PTOA because a controlled traumatic injury can be initiated and the changes in the joint can be monitored using various instruments and techniques at a precise duration after the initiation (Pond & Nuki, 1973; Xia, 1998; Xia, 2007). In recent years lower cost and higher availability have made rabbit models a popular choice compared with larger animal models. Additionally, the rabbit model has benefits over smaller animals, which lack sufficient cartilage tissue for investigations involving multiple studies (Cohen‐Solal et al., 2013; Kuyinu et al., 2016). In this study, we used advanced high‐resolution imaging techniques including polarized light microscopy (PLM), microcomputed tomography (μCT), and inductively coupled plasma (ICP) analysis to investigate the microstructural changes in the rabbit knee joint. We intentionally applied a sub‐critical impact, to purposely cause minor damage to the joint. By understanding how the sub‐critical impact affects the mineralization in the CZ and SBP, we seek to understand the connection between minor mechanical trauma and mineral distribution, which could provide insight into how a minor trauma can affect joint health.

2. METHODS

2.1. Sample preparation

In this study, we used 12 mature female New Zealand White (NZW) rabbits with approval from the institutional review committee (IACUC: AL‐2021‐07). The rabbits had the surgery when they were 8–9 months old with each weighing approximately 3–4 kg. The impact procedure was carried out by the surgeon, using a custom‐built impact device (Leucht et al., 2012) that had a metal indenter tip of 3 mm diameter, to strike the right femoral medial condyle one time. The medial femoral condyle at the impact location was measured to have an average overall width of 7 mm. All animal's left femoral condyle served as the contralateral control for comparison and did not undergo any surgical operation. Pressure sensing films (Fujifilm Prescale, Sensor Products Inc., Madison, NJ) were used to calibrate the impact, to be around 30 MPa. After impact, the animals were watched and were permitted to engage in normal activities until reaching their designated euthanizing periods of 2‐ and 14‐week post‐impact surgery. These time points were based on the progressive nature of the degenerative diseases in various animal models. Cartilage‐bone blocks of 2 × 2 × 3 mm3 were extracted from the femoral medial condyle of both the impacted knee and the contralateral knee (matched with the impact location). For analysis, we had a total of 12 blocks (n = 6 impact, n = 6 non‐impact) from the 2‐week group and 12 blocks (n = 6 impact, n = 6 non‐impact) from the 14‐week group. The cartilage‐bone samples were analyzed using PLM and μCT. In addition to the blocks harvested from the impact location for PLM and μCT imaging, cartilage samples were also extracted at the same time from the adjacent anterior and posterior portions of the impact location to measure calcium content in the CZ using ICP analysis.

2.2. Polarized light microscopy protocols

The cartilage‐bone samples were preserved in 10% buffered formalin and transferred to an outside agency for paraffin‐embedded tissue histology (Western Michigan University, School of Medicine, Kalamazoo, MI, USA). The 6 μm‐thick thin sections from histology were imaged quantitatively by the PLM system (Xia et al., 2001), at an objective of 10×, resulting in a resolution of 1.0 μm/pixel. For each histological section, the PLM system produced quantitative optical retardation images with units of nanometers, shown in Figure 1. These images were used to carry out analysis of the birefringent properties of the cartilage tissue.

FIGURE 1.

FIGURE 1

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Representative set of 2D quantitative optical retardation PLM images along with their associated depth‐dependent profiles (in unit of nm) from rabbit knee articular cartilage at 1 μm/pixel resolution. The profiles also contain the standard deviations. (a) Shows the optical retardation image and its profile from a non‐impact sample at 14‐week timepoint, including the depth‐wise zonal divisions of the cartilage and the retardation scale. (b) Shows the optical retardation image and its profile from an impact sample at 14‐week timepoint.

2.3. Microcomputed tomography protocols

Skyscan1174 (Bruker, Kontich, Belgium) was used for all μCT experiments, where the same experimental settings were used for all cartilage‐bone blocks: 45 kV, 700 μA, 0.5° rotation step, 10 averages, 0.2 mm aluminum filter, and 1024 × 1024 data matrix. Cartilage‐bone blocks were placed on a water‐soaked gauze pad to minimize the sample drying. The images were acquired at a resolution of 6.7 μm and the total scanning time for each sample was approximately 54 min. The scans were then reconstructed using NRecon (Bruker, Kontich, Belgium), and the reconstructed images were analyzed for bone mineral density (BMD) using CT‐Analyzer (Bruker, Kontich, Belgium). BMD measurements were calibrated to grams of hydroxyapatite per cm3 (g/cm3) against the density scales of the measured attenuation coefficient of standard μCT phantoms.

2.4. Inductively coupled plasma protocols

A Perkin Elmer inductively coupled plasma optical emission spectrometer (ICP‐OES) Optima 7000 DV (Waltham, MA) was used to determine the calcium molar concentration in the samples. The specimens were prepared for the 40Ca ICP‐OES analysis, based on the previously described methodology (Lesperance et al., 1992). The preparation began with the removal of bone from each cartilage‐bone block. The cartilage specimens were then placed in a saline bath. The specimens were then weighed for their wet weight, after which they were placed in an oven at 90°F for 3 h, where the cartilage samples were then taken out of the oven and weighed for their dry weight. The resultant tissues were then dissolved in 100 μL of concentrated HNO3 overnight and later diluted to 10 mL with ultrapure water for calculating calcium concentration. For calibration, five standard solutions were also evaluated.

2.5. Image/data analysis

2D PLM images of the histological sections were analyzed using ImageJ (National Institutes of Health, Bethesda, MD). A 100‐pixel‐wide region of interest was applied to the 2D optical retardation PLM images to generate a 1D profile along the cartilage depth for each sample. Optical retardation data from the 1D profile was further analyzed using Microsoft Excel to compare non‐impact knee (NIK) and impact knee (IK) samples. To analyze optical retardation in different regions of the tissue, the depth of the cartilage was divided into four regions: superficial zone (SZ), transitional zone (TZ), radial zone (RZ), and calcified zone (CZ), using the established criteria (Lee & Xia, 2013; Xia et al., 2001).

Reconstructed data from the μCT scans was analyzed using CT‐analyzer software. The region of interest (SBP region) was analyzed for BMD measurements, from the start of the SBP region, in each sample, a total of 100 axial slices along the depth were analyzed for mean BMD value in g/cm3. The depth of the SBP region was approximately 0.67 mm, which was equally divided into two regions (region‐1 and region‐2) of 50 slices each, and mean BMD was measured from each region.

The statistical analyses were conducted on optical retardation, BMD, and ICP measurements using the commercial software KaleidaGraph (Reading, PA). The paired student's t‐test was performed on all the samples for comparison between data from different groups (2‐week NIK, 2‐week IK, 14‐week NIK, and 14‐week IK), where a p < 0.05 was considered significant.

3. RESULTS

Before the specimens were harvested, the knee joints were visually inspected. All non‐impact femoral condyles in the 2‐ and 14‐week specimens looked healthy and had uniform coloring across the cartilage surface. For the 2‐week impact joints, there was no significant visual cartilage degeneration. For the 14‐week impact joints, a few impact joints showed minimal discoloration in and around the area of impact, while the other joints had no visual change.

3.1. PLM and ICP analysis

Figure 1 shows representative quantitative optical retardation images of non‐impact (A) and impact (B) cartilage samples at a resolution of 1.0 μm/pixel, followed by their respective depth‐dependent profiles. All individual data points from all optical retardation depth‐dependent profiles (i.e., all specimens) were pooled and compared in Figure 2, between the non‐impact and impact samples at the 2‐week and 14‐week periods. In Figure 2, the depth‐dependent data points of quantitative optical retardation values in cartilage were divided into four zones, to examine the effects of the impact on cartilage at various depths. The diagonal line in the correlation plots in Figure 2 has a slope of 1, which indicates no change between the comparisons. Any increase in the comparison will be displayed by shift in data points above the diagonal line and any decrease will be displayed below the diagonal line. The average value of each zone for non‐impact and impact samples at 2 and 14 weeks are shown in Table 1.

FIGURE 2.

FIGURE 2

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All data points from depth‐dependent optical retardation profiles, correlating the non‐impact and impact specimens from (a) 2‐week group, where no major shifts were noted. Slopes for each zone at 2‐Week: SZ‐0.83, TZ‐0.94, RZ‐0.97, and CZ‐0.99. (b) 14‐week group, showing a shift in values in the superficial and the calcified zone. Slopes for each zone at 14‐week: SZ‐0.69, TZ‐0.89, RZ‐0.89, and CZ‐0.74. The diagonal dotted line has a slope of 1, meaning no change between the comparisons. Shift above the diagonal line means an increment after impact, and below the diagonal line means a decrement after impact.

TABLE 1.

Summaries of the optical retardation in PLM analysis, calcium content in ICP analysis, and BMD measurements in μCT analysis.

2‐week NIK versus 2‐week IK 14‐week NIK versus 14‐week IK 2‐week IK versus 14‐week IK 2‐week NIK versus 14‐week NIK
2‐week NIK 2‐week IK % diff. p‐value 14‐week NIK 14‐week IK % diff. p‐value % diff. p‐value % diff. p‐value
Ret(nm) SZ 2.83 ± 0.11 2.84 ± 0.1 +0.4% 0.9 3.27 ± 0.13 2.63 ± 0.09 −22.0% 0.1 −8.0% 0.6 +14.0% 0.2
Ret(nm) TZ 3.36 ± 0.06 3.52 ± 0.05 +5.0% 0.7 3.51 ± 0.06 3.48 ± 0.05 −0.9% 0.5 −1.0% 0.6 +4.0% 0.3
Ret(nm) RZ 8.40 ± 0.07 8.45 ± 0.06 +0.6% 0.8 8.36 ± 0.07 7.89 ± 0.05 −6.0% 0.4 −7.0% 0.3 −0.5% 0.6
Ret(nm) CZ 7.17 ± 0.06 7.23 ± 0.06 +0.8% 0.6 8.49 ± 0.07 6.35 ± 0.08 −29.0% 0.01 −13.0% 0.1 +16.0% 0.1
Calcium (mM) 0.389 ± 0.145 0.297 ± 0.158 −23.0% 0.5 0.455 ± 0.102 0.244 ± 0.059 −46.0% 0.02 −20.0% 0.7 +15.0% 0.5
BMD (g/cm3) Average SBP 1.119 ± 0.022 1.118 ± 0.027 −0.1% 0.9 1.144 ± 0.028 1.074 ± 0.021 −6.0% 0.01 −4.0% 0.09 +2.0% 0.5
BMD (g/cm3) Region‐1 1.064 ± 0.022 1.053 ± 0.033 −1.0% 0.8 1.068 ± 0.011 1.026 ± 0.027 −4.0% 0.2 −3.0% 0.5 +0.4% 0.8
BMD (g/cm3) Region‐2 1.174 ± 0.021 1.184 ± 0.021 +0.8% 0.6 1.221 ± 0.026 1.123 ± 0.013 −8.0% 0.03 −5.0% 0.03 +4.0% 0.3
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Note: The data are presented as mean ± standard error along with percentage differences and statistical p‐values from the Paired Student t‐Test, the bold values in the table represent statistical significance (p‐value < 0.05).

Figure 2 shows that the comparisons between non‐impact and impact samples at 2 weeks had no major shifts between the two groups since data points were approximately equally spread out across the diagonal for all four regions. In contrast, the comparison between non‐impact and impact samples at 14 weeks displayed skewed data points for two regions. Both the SZ and the CZ had skewed data points below the diagonal line, illustrating a reduction in the optical retardation measurements for the impact group. Most shift in the optical retardation values was observed for the CZ where most of the data points were below the diagonal line, shift in the CZ region was statistically significant. The relevant region for CZ is highlighted with an ellipse‐shaped boundary in the figure. The statistical analysis for the PLM zone‐wise data is shown in Figure 3.

FIGURE 3.

FIGURE 3

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Box plots comparing depth‐dependent quantitative analysis of optical retardation among non‐impact and impact samples at (a) 2‐week and (b) 14‐week post‐impact. The analysis depicts statistically significant changes at a 14‐week comparison in the calcified zone. Statistical significance is denoted as *(p‐value < 0.05).

Figure 4 shows the depth‐dependent optical retardation profiles averaged from all specimens, for both non‐impact and impact groups at the 2‐week and 14‐week periods. At the 2‐week timepoint, the depth‐dependent profiles between the non‐impact and the impact groups were almost similar. In contrast, at the 14‐week timepoint, the depth‐dependent profile for the impact group had lower values in SZ and CZ, with the CZ having the largest differences (both trends are reflected in the spreading of the individual data points in Figure 2). The zonal‐averaged optical retardation values at different time points are summarized in Table 1. While the zonal optical retardation values between the non‐impact and impact specimens showed no prominent differences at 2 weeks. At 14 weeks, the SZ showed a reduction of 22.0% in retardation values in the impact group, while the TZ and the RZ showed a minor drop of 0.9% and 6.0% respectively for impact samples. The CZ at 14 weeks showed a 29.0% decrease in retardation values in the impact group, this decrease was statistically significant (p < 0.05). These changes in the optical retardation values are displayed using the box plots in Figure 3.

FIGURE 4.

FIGURE 4

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Comparison of averaged depth‐dependent optical retardation profiles from all samples, between the non‐impact vs impact specimens, at (a) 2‐week and (b) 14‐week post‐impact.

Table 1 also summarizes the calcium concentration in the cartilage specimens measured by ICP analysis (calcium content is largely contributed from the CZ of the cartilage). When comparing the non‐impact and impact groups at 2 weeks, the calcium concentration was lower by 0.092 mM in the impact group compared with the non‐impact group. The comparison at 14 weeks displayed a drop of 0.211 mM in the impact group compared with the non‐impact group, this difference between the two groups was statistically significant (p < 0.05). This analysis was helpful to support the changes observed in the CZ optical retardation values for the impact group as well as the findings in the 14‐week BMD measurements from μCT.

3.2. μCT analysis on the subchondral bone

To investigate the changes in the mineralization in the SBP initiated by the impact event, we carried out BMD analysis on the cartilage‐bone blocks using μCT imaging. The average change in the SBP's BMD was found to be statistically insignificant for the non‐impact Vs impact groups at 2 weeks, with a decrease of 0.1% in the impact group. In comparison, at 14 weeks the BMD dropped in the impact group with a change of 6.3%, which was statistically significant (p < 0.05). These changes in the BMD are displayed using the box plots in Figure 5b.

FIGURE 5.

FIGURE 5

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(a) 3D representation of rabbit knee subchondral bone using μCT imaging. (b) Box plots display bone mineral density (BMD) data from non‐impact and impact subchondral bone plate (SBP) samples at 2‐week and 14‐week post‐impact, where the statistical significance is denoted as *(p‐value < 0.05). The data in these plots were taken from the entire SBP region. (c) Box plots display the region‐wise BMD data from the non‐impact and impact samples at 2‐week and 14‐week. The region‐1 data were measured from the upper half of the SBP and region‐2 data were measured from the lower half of the SBP as shown in (a). Statistical significance is denoted as *(p‐value < 0.05).

The division of the SBP region from which the BMD values were measured is shown in Figure 5a. Box plots in Figure 5c for non‐impact and impact groups with BMD measurements were created for the two regions of SBP. It can be seen that the 2‐week specimens in both regions showed little deviation indicating minor changes in the BMD measurements between the non‐impact and impact groups. For region‐1 BMD changed by 1.0% and for region‐2 a minor change of 0.8% was noted, where both differences were statistically insignificant. The 14‐week specimens for both regions showed higher deviation compared with the 2‐week specimens, both regions noted a drop in BMD values for the impact group. Region‐1 for the impact group saw a decrease in BMD by 4.0% and region‐2 noted a drop of 8.4% for the impact group, this difference was statistically significant (p < 0.05).

4. DISCUSSION

This study tactically used a surgically induced sub‐critical impact injury in a rabbit model to identify the early progressions of PTOA. The high‐resolution PLM and μCT examination aimed to track the microstructural changes in the AC and the SBP by quantitatively analyzing the optical retardation and BMD changes in the specimens. Under sub‐critical circumstances (Mantebea et al., 2023), the low pressure of 30 MPa was intentionally used to initiate PTOA with minor yet early alterations. The early changes from the sub‐critical impact were not expected to produce highly visible and extensive damage in the cartilage‐bone specimen. These sub‐critical low‐pressure impacts occur frequently in daily activities for people of all ages, and they are harder to recall or identify compared with a single catastrophic event. The assessment of cartilage‐bone block quality can be determined by important factors like microdamage in peri‐articular mineralized tissue such as CZ of the cartilage and the SBP (Burr, 2004). We aimed to find out if the early changes in the impact specimens could be successfully identified. Any early deterioration that is successfully identified may help in the future development of a therapeutic intervention that may stop or reverse tissue degeneration. This report's findings attest to the early PTOA's effective identification.

In this study, quantitative analysis of optical retardation measurements from PLM images displayed notable changes in birefringent properties of the cartilage after an impact injury (Alhadlaq et al., 2007). At the 14‐week post‐impact surgery, we observed a significant reduction in optical retardation values of impact samples compared with the non‐impact samples, particularly in the CZ. When analyzing the CZ region in Figures 2 and 3, notable differences emerged where the majority of the retardation values in the CZ from the impact samples showed a prominent decrease. This observation confirmed structural changes in the CZ from the applied impact. However, at 2 weeks post‐impact surgery, no significant reduction was observed in the impact cartilage. While minimal changes were observed at the 2‐week mark, changes were more pronounced and evident at 14 weeks, indicating a progressive nature of cartilage degradation over time. Further research with more time points can better explain the dynamics of these changes over time.

At the 14‐week timepoint, we observed a significant reduction in calcium concentration in the impact samples. The drop in the calcium concentration seen in the ICP analysis aligned with the optical retardation results of the PLM findings. Past studies indicated that early‐stage remodeling of CZ and SBP differs significantly from the later stages of PTOA. In the early stages, CZ and SBP are known to undergo osteopenia, characterized by a decrease in mineral density which can also result in subtle thinning of the CZ. Whereas at later stages remodeling of the CZ and SBP takes a different trajectory exhibiting signs of sclerosis during which an increase in mineral density is observed (Burr & Gallant, 2012; Goldring, 2012; Kaspiris et al., 2022; Wang et al., 2022). CZ is the region in the cartilage where the calcium is deposited during the mineralization process. So, a significant reduction in calcium concentration in the cartilage can be indicatively responsible for the altered optical retardation values observed in the CZ of impact samples at 14 weeks.

In a prior μMRI investigation of early‐stage PTOA in rabbit knees, structural degradation was noted in the SZ and the CZ (Mantebea et al., 2023; Singh et al., 2024). After a direct hit to the surface of the femur condyle, the SZ was expected to show early degenerative alterations due to its location at the surface of the tissue at the impact point. Also, the SZ is known to show early degenerative alterations with the onset of PTOA (Kajabi et al., 2020; Korhonen et al., 2002). With its high stiffness, CZ has been found to be one of the first zones affected by trauma (Keinan‐Adamsky et al., 2005). We were particularly interested in the changes we observed in the CZ in our animal model when a sub‐critical impact was applied intentionally. CZ is a critical region where mineralization occurs in the cartilage, changes in this zone can have serious implications on cartilage health (Fan et al., 2022; Revell et al., 1990; Wang et al., 2022). Optical retardation changes observed in the CZ in this study strongly indicated alterations in the deposition of minerals, mainly calcium (Zizak et al., 2003). Any changes in the mineralization can lead to compromised structure and functionality of the cartilage subsequently. The changes in the mineralization of the CZ can greatly affect the load‐bearing ability of the cartilage in diseases like OA (Zhang et al., 2012).

Subchondral bone plays a crucial role in supporting the AC and is also known to undergo structural changes with OA progression (Madry et al., 2010). In this study, we aimed to understand how the BMD is affected in the SBP after a single sub‐critical impact. At 2 weeks no significant differences (0.1%) in BMD were noted between the non‐impact and impact specimens. However, at 14 weeks a statistically significant drop of 6.3% in BMD was observed in the SBP of impact samples. This difference again suggested the progressive structural changes from a sub‐critical impact in the bone plate over time. The altered BMD in the SBP at 14 weeks could be indicative of the changes in mineralization pattern as a response to the microdamage from the impact. While subchondral sclerosis is known to be a sign of advanced OA, SBP goes through several microstructural changes during different stages of OA (Intema et al., 2010; Li et al., 2013). In various animal studies, subchondral bone loss has been reported at the early stages of OA pathogenesis. Also, deterioration of the SBP has been related to cartilage degradation in OA (Batiste et al., 2004; Intema et al., 2010; Meyer et al., 2008). Significant remodeling and subchondral bone loss in human OA have also been observed at early stages (Bettica et al., 2002).

Both halves of the SBP in our study showed changes in the BMD between non‐impact and impact samples at 14 weeks. In the impact samples, region‐1 showed a drop of 4.0% in BMD, and a drop of 8.4% was observed in region‐2. Higher change in region‐2 could be due to the varying mechanical stress in the two regions. The second region was closer to the STB which experiences more load during the weight‐bearing activities and can go under more pronounced structural changes in response to the impact. STB experiences different responses in OA progression as it has significantly different porosity, density, and stiffness than the SBP (Burr, 2004). The BMD measurements in SBP from μCT were in line with PLM findings of optical retardation values in the CZ of the cartilage.

In this study, along with noted changes between non‐impact and impact samples at 14‐week and impact samples at 2 and 14 weeks, we also observed changes in the optical retardation and mineral concentration values between contralateral non‐impact samples at 2 and 14 weeks, in Table 1. These variations could have been introduced by the alteration in gait and weight distribution by the rabbit to compensate for the injured knee (Mcgibbon & Krebs, 2002). The altered biomechanical loading could stimulate adaptive changes in the cartilage and SBP of the contralateral non‐impact samples.

4.1. Limitations

Twelve adult female NZW rabbits were used in our animal procedures. All left femoral heads remained unimpacted and each right femoral medial condyle was impacted once. We cannot completely rule out the possibility of surgical stress contributing to cartilage deterioration since our study lacks sham surgery on the NIK. Also, the non‐impact samples did not serve as true control comparisons due to potential adaptive mechanisms introduced in the NIK while compensating for the injured knee. This choice of using a contralateral control limb in place of a sham group or sham limb was made tactically to drastically lower the number of rabbits needed for our research and spare the animals from the excruciating pain and suffering associated with bilateral surgical procedures which was a requirement suggested by the institutional review committee. Any inconsistency in impactor or limb positioning or rigidity during impact could theoretically have led to differences in impact energy between animals, however, we took numerous precautions to limit variability and maximize consistency between impacts. We conducted this study using a validated impactor device (Mantebea et al., 2023) which utilized a spring‐loaded weight for consistent impact force and velocity, and surgical limbs were rigidly mounted to the impactor frame via an intra‐op k‐wire to maximize rigidity for consistent transmission of force from the impactor to the articular surface. Furthermore, multiple hardware interlocks allowed the impactor device to be rigidly affixed once positioned on the femoral surface, ensuring rigidity during the impact event. Availability of male rabbits was extremely low for the experiments that were conducted during the COVID‐19 pandemic hence we limited our investigation to using only female rabbits, and therefore we are unable to assess potential sex differences in response to sub‐critical impact injury. All rabbits were randomly assigned to different groups and were monitored carefully. No specific trends related to age or weight were observed during the progression of cartilage‐bone block degradation from the impact.

5. CONCLUSIONS

In this study, we investigated the effects of a sub‐critical impact on rabbit knee AC and SBP at 2 and 14 weeks post‐impact surgery. With the use of multiple techniques including PLM imaging, μCT imaging, and ICP analysis, we assessed the response of AC and SBP to a sub‐critical impact and obtained several significant findings. At 2 weeks the degradation was not prominent, however, at 14 weeks statistically significant changes were evident. PLM imaging analysis exhibited reduced optical retardation values in the CZ of impact AC, indicating early structural changes in cartilage. A decline in calcium concentration was noted for the 14‐week impact AC samples, implying mineralization changes in response to the impact. μCT analysis of SBP at 14 weeks showed BMD reduction in the impact samples compared with the non‐impact samples. The BMD changes observed in the impact samples suggest bone remodeling in response to the impact. The observed alterations in optical retardation, calcium concentration, and BMD are collectively suggestive of early responses in AC and SBP due to a sub‐critical impact. The findings of this study have the potential to improve the knowledge about the early‐stage progression of injury‐related joint degradation, which can be useful in informing timely diagnosis and intervention for preserving joint health.

AUTHOR CONTRIBUTIONS

Amanveer Singh: acquisition of data, data analysis, data interpretation, drafting of the manuscript, and design. Hannah Mantebea: acquisition of data and data interpretation. Farid Badar: acquisition of data, data interpretation, critical revision of the manuscript, and design. Syeda Batool: acquisition of data and data interpretation. Austin Tetmeyer: acquisition of data. Gabrielle Abdelmessih: acquisition of data. Talia Sebastian: acquisition of data and critical revision of the manuscript. Michael Newton: acquisition of data and critical revision of the manuscript. Kevin Baker: concept and design. Sarah Salem: acquisition of data. Yang Xia: concept, design, data interpretation, critical revision of the manuscript, and approval of the article.

ACKNOWLEDGMENTS

The surgical procedures on the rabbits by Dr. Jaewon Chang, MD, and Dr. Tyler Enders, DO (Beaumont Hospital, Royal Oak, MI 48073) are gratefully acknowledged. This research was funded by an R01 grant (AR 69047, PI: Xia) from the National Institutes of Health (NIH). All the authors state that there are no conflicts of interest.

Singh, A. , Mantebea, H. , Badar, F. , Batool, S. , Tetmeyer, A. , Abdelmessih, G. et al. (2024) Assessment of post‐trauma microstructural alterations in the rabbit knee cartilage and subchondral bone. Journal of Anatomy, 245, 740–750. Available from: 10.1111/joa.14102

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from Journal of Anatomy are provided here courtesy of Anatomical Society of Great Britain and Ireland

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