Early Unloading After ACL Rupture and Prior to Surgical Restabilization in Mice Slows Post-Traumatic Osteoarthritis Progression

Abstract

Purpose:

People who sustain joint injuries such as anterior cruciate ligament (ACL) rupture often go on to develop post-traumatic osteoarthritis (PTOA). ACL injuries are often treated with ACL reconstruction, but there is typically a gap of several weeks between injury and surgery. However, it is unclear how loading or unloading of the injured joint during the early postinjury period affects the progression of PTOA. The goal of this study was to determine how unloading between noninvasive ACL injury and surgical restabilization of the injured joint affects PTOA progression in mice.

Findings:

Mice were subjected to noninvasive ACL injury or no injury followed by 1 week of hindlimb unloading (HLU) or normal cage activity. After 1 week of HLU or cage activity, mice underwent restabilization surgery or no surgery. ACL injury resulted in considerable epiphyseal trabecular bone loss regardless of HLU or cage activity. HLU groups exhibited significantly reduced chondrophyte/osteophyte formation, OA scoring, and synovitis at day 42. Single-cell RNA sequencing revealed that 1 week of HLU resulted in more neutrophils and less monocytes-macrophages in the injured joint.

Conclusions:

This study establishes that 1 week of HLU after ACL injury effectively slowed PTOA progression, suggesting that the early inflammatory response and joint instability play a key role in PTOA initiation and progression, and neutrophils and monocytes-macrophages play roles in the modulation. However, subsequent joint restabilization surgery caused greater inflammatory protease activity in the joint and exacerbated the loss of epiphyseal trabecular bone but did not significantly diminish OA score or synovitis.

Keywords

ACL injury osteoarthritis hindlimb unloading osteophytes subchondral bone joint restabilization scRNA-seq

Background

The anterior cruciate ligament (ACL) is crucial for providing anterior-posterior (AP) and rotational stability to the knee joint. The ACL is one of the most commonly injured joint structures, particularly for sports involving jumping and cutting such as basketball, soccer, and skiing.¹ ACL injury is one of the most common joint injuries, with around 70 per 100,000 people suffering an ACL rupture each year in the United States.² This type of injury often leads to post-traumatic osteoarthritis (PTOA), which can affect patients’ pain, mobility, and quality of life.³ PTOA can be initiated by multiple types of joint injuries including ACL rupture, meniscal injury, direct damage to the articular cartilage, or intra-articular bone fracture.⁴ Traumatic joint injuries present a unique opportunity for prophylactic treatments aimed at preserving joint health, since unlike primary OA there is a known event that initiates PTOA. Because PTOA is initiated by a traumatic joint injury, often during sports or high impact activities, the population affected by PTOA is usually younger and healthier than that affected by idiopathic (primary) OA.⁵ After ACL injury, ACL reconstruction is often performed, in which surgeons replace the injured ligament with autograft or allograft tissue to restore the biomechanical function of the ACL and restabilize the injured joint. This type of surgery is typically recommended for patients who are seeking to return to sports.⁶ In our previous study, we found that surgical restabilization of the knee joint immediately after ACL injury in mice could slow PTOA progression.⁷ However, in human patients there is typically a gap of several weeks between ACL injury and reconstruction surgery. It is currently unclear to what extent loading or unloading the injured knee joint during the time between ACL injury and restabilization surgery would affect the progression of PTOA.

Several preclinical studies have shown that early joint unloading after injury has some beneficial effects on joint health. For example, in dogs, hindlimb immobilization for 11 weeks reduced levels of interleukin-1α, tissue inhibitor of metalloproteinases 1 (TIMP-1), and chondroitin sulfate in synovium.⁸ Similarly, following medial meniscus transection in rats, immobilization reduced cartilage loss and decreased osteophyte formation.⁹ Another study showed that hindlimb unloading (HLU) of rats following intra-articular injection of monosodium iodoacetate diminished cartilage degradation in the joint.¹⁰ In contrast, another study found that physiologic loading of the injured joint following ACL injury in mice preserved chondrocyte health and delayed PTOA progression.¹¹ In our previous studies, we found that 7 days of hindlimb unloading (HLU) in mice after noninvasive ACL injury was able to diminish protease activity and synovitis in the injured knee joints and mitigate early epiphyseal trabecular bone loss; 7 days of HLU also decreased long-term osteophyte formation and PTOA progression.^12,13 However, it is unclear if early unloading followed by surgical joint restabilization will have an additive effect in preserving joint health after ACL injury.

In this study, we sought to determine if 7 days of HLU following ACL injury and prior to surgical restabilization of the mouse knee joint would affect PTOA progression compared to 7 days of normal cage activity before surgery. We hypothesized that joint restabilization after 7 days of HLU injury would be the most effective at slowing the progression of PTOA due to diminished early inflammation and tissue damage in the joint prior to restabilization surgery.

Methods

Animals

A total of 103 C57BL/6J female mice from The Jackson Laboratory (12 weeks old at the time of injury) were used in this study; mice were randomly assigned to 5 experimental groups (Fig. 1): 20 mice were uninjured, were not subjected to HLU, and had no surgical procedures (Uninj-Unop); 20 mice were injured, were not subjected to HLU, and had no surgical procedures (Inj-Unop); 19 mice were injured followed by 7 days of HLU but had no surgical procedures (Inj-HLU); 21 mice were injured, returned to normal cage activity for 7 days, then had restabilization surgery (Inj-Restab); and 23 mice were injured followed by 7 days of HLU then had restabilization surgery (Inj-HLU-Restab). Half of the mice in each group were euthanized 21 days after injury (14 days after surgery) and the remaining mice were euthanized 42 days after injury (35 days after surgery) for analysis of OA progression. Mice euthanized at day 42 were imaged with in vivo near-infrared fluorescence reflectance imaging (FRI) to determine protease activity in the joint using a commercially available fluorescence activatable probe at 14, 28, and 42 days after injury. All procedures were approved by the UC Davis Institutional Animal Care and Use Committee.

Figure 1.

Study design showing animal number per group, terminal time points, and intervention for each group.

Noninvasive ACL Injury

Mice were subjected to noninvasive ACL injury by using a single tibial compression overload as we have previously described.^14
-16 Mice were anesthetized by isoflurane inhalation and were placed in a prone position in the tibial compression setup. For ACL injuries, the right hindlimb of the mouse was subjected to a single compressive overload (ElectroForce 3200, TA Instrument, New Castle, DE) at 1 mm/s until injury (~10-12 N).

Hindlimb Unloading

Immediately following noninvasive ACL injury, 42 mice were subjected to hindlimb unloading (HLU) via tail suspension as previously described.^12,17 Mice were single housed during HLU in customized cages designed for tail suspension (Techshot, Inc., Greenville, IN); absorbent paper was placed beneath a wire mesh on the bottom of the cage. A plastic loop with a metal chain was attached to the tail base using superglue while mice were still under anesthesia from ACL rupture. These loops can allow 360° rotation due to attaching to a swivel, and were passed through by a bar across the length of the cage. Mice were placed at approximately 30° head-down angle, with both hindlimbs unable to touch the floor of the cage. Mice could move freely in the cage by using their forelimbs, and were given food, water, hydrogels, and nesting materials (Suppl. Fig. 1). Mice were in HLU for 7 days after which they were anesthetized by isoflurane inhalation for removal of the metal loops and swivels from tails using acetone, and mice were placed back into group housing in normal mouse cages for the remainder of the study.

Restabilization Surgery

Restabilization surgery was performed following 7 days of HLU or normal cage activity using an extra-capsular method first described in rats by Kokubun et al.¹⁸; we applied this technique in mice in our previous study.⁷ For this restabilization surgery, a bone tunnel was drilled in the anterior aspect of the proximal tibia, and a suture was passed through the tunnel and around the posterior site of the distal femur beneath the fabellofemoral ligaments, then the suture was tightly tied on the lateral side of the joint. A single surgeon (CAL) performed all surgeries to ensure consistency.

Fluorescence Reflectance Imaging (FRI) of Protease Activity

To evaluate inflammatory protease activity in the knee joints, both hindlimbs were imaged using in vivo Fluorescence Reflectance Imaging (FRI) as previously described.^13,16,19 Mice were anesthetized using isoflurane and were administered 2 nmol/150 μL of ProSense 680 (Perkin-Elmer, Waltham, MA) 24 hours prior to imaging by retro-orbital injection. ProSense 680 is a fluorescent probe activated by cathepsin proteases such as Cathepsin B, L, and S and Plasmin which are associated with the inflammatory response.²⁰ Mice were imaged at 14, 28, and 42 days after injury. Fur was removed from the anterior aspect of both legs using depilatory cream to expose knee joints. 24 hours after injection, mice were imaged individually in a 22.6-cm field of view in the imaging system (IVIS Spectrum, Perkin-Elmer, Waltham, MA). The excitation and emission wavelength were set as 675 ± 10 nm and 720 ± 10 nm, respectively, with system-auto exposure time. Mice were placed in consistent positions to ensure the entire knee joints were exposed and facing toward the camera. Ankles were taped down to hold the legs in place, and images were captured by the IVIS Living Image software. After images were captured, a region of interest (ROI) circle with consistent size (radius 0.35 cm) was placed over each knee joint on a black and white image. Total radiant efficiency (TRE; [photons/s]/[μW/cm²]) was calculated within the ROI by the software to evaluate fluorescence intensity of the knee joint.

Single-Cell RNA Sequencing (scRNA-seq)

Following 7 days of normal cage activity or HLU after ACL injury, 4 mice were euthanized for scRNA-seq (n = 2 per loading condition). Knee joints were collected, and muscle and tendon tissues were thoroughly removed while leaving knee joints intact. The samples were then stored in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY) on ice and transferred to a safety cabinet or clean bench for further soft-tissue cleaning. Knee joints were isolated by separating femur and tibia at the epiphyseal growth plates. The isolated knee joints were vortexed with 1% Fetal bovine serum (FBS) (Gibco, Grand Island, NY) in 1x Phosphate buffered saline (PBS) (Gibco, Grand Island, NY) to shed bone marrow content. The joint was then rinsed with PBS and digested in 5 mL of Collagenase 1 solution, 3 mg/mL Collagenase 1 (Worthington, Lakewood, NJ) dissolved in DMEM/F12 (Gibco, Grand Island, NY) +100 μg/mL DNase 1 (Roche Diagnostics, Indianapolis, IN,) to further separate the distal femur and proximal tibia and collect everything into digest solution to shake at 37 ℃ for 60 minutes. The digestion supernatant was then collected, and the process was repeated with the digestion solution on the remaining samples to gather more cells. After that, 500 μL Ammonium-Chloride-Potassium (ACK) lysis solution (Gibco, Grand Island, NY) was added to the solution for 5 minutes on ice to remove red blood cells. Five folds of DMEM/F12 +10% was added to stop reaction and the solution was centrifuged for 10 minutes at 500 G at 4 ℃. Cells were strained with a 70- to 100-μm strainer and were stored for further analysis. Isolated cell populations were processed according to the Next GEM Cell Fixed RNA Sample Preparation Kit (10× Genomics) then stored at -80° C. Upon removal from storage, cells were thawed, centrifuged at 850 rcf for 5 minutes at room temperature, and resuspended in 500 μL Quenching Buffer. The quantity and quality of the cells were assessed by Acridine Orange and Propidium Iodide dye on a Cellometer Auto 2000 (Nexcelom Bioscience, Lawrence, MA). A range of 300K–500K cells per sample were hybridized overnight and pooled equally using the mouse transcriptome kit (10× Genomics, part number PN-1000496). In total, 13,200 cells were loaded onto the 10x Chromium X, and the library was constructed according to the Chromium Fixed RNA Profiling Reagent Kits manual (10× Genomics). The final library quantity was assessed by Qubit 2.0 (Thermo Fisher, Waltham, MA), and quality was assessed by TapeStation HSD1000 ScreenTape (Agilent Technologies Inc., Santa Clara, CA). Equimolar pooling of libraries was performed based on QC values and sequenced on an Illumina® NovaSeq X Plus (Illumina, San Diego, CA) with a read length configuration of 150 PE for 200M PE reads per sample (100M in each direction).

scRNAseq-Seq Data Analysis

Raw scRNA-seq data was processed with 10× Genomics Cell Ranger software (version 6.0.0; 10× Genomics, Pleasanton, CA) as was done previously.²¹ The resulting output was then exported into the R package Seurat where quality control steps and additional analysis were performed.²² Data filtering criteria included: cells with <500 detected genes per cell, genes that were expressed by fewer than 5 cells, had mitochondrial >15%, or had aberrantly high gene count, all of which were removed. After preprocessing, normalization, feature selection, and data scaling, the experimental groups were integrated to generate a merged Seurat object. Once data was integrated, cells were then grouped into clusters for cell type discovery using Seurat’s “FindNeighbors,” and “FindClusters” functions followed by the visualization of the produced clusters using the non-linear dimensional reduction via uniform manifold approximation and projection (UMAP). Cluster marker genes were determined using Seurat’s “FindAllMarkers” function. Next, Seurat’s “FeaturePlot,” “DotPlot,” “VlnPlot,” and “Heatmap” functions were used to graphically plot the cell type–specific gene expression measurements among the datasets. Differentially expressed genes among clusters were identified using “FindMarkers” function. For either Injured or Injured HLU samples, the cell type proportions were estimated as a ration of the number of cells in each cell cluster relative to the total number of cells sequenced.

Micro-Computed Tomography Analysis of Trabecular Bone and Osteophytes

Injured/operated and contralateral knee joints were analyzed with micro-computed tomography (μCT) to determine epiphyseal trabecular bone microstructure and osteophyte formation (SCANCO μCT 35, Brüttisellen, Switzerland). Scan parameters were X-ray tube potential = 55 kVp, intensity = 114 mA, 10 μm isotropic nominal voxel size, integration time = 900 ms. Bone tissue global threshold was set to 567.431 mg HA/cm³. The volume of interest for epiphyseal trabecular bone was designated using manually drawn contours on 2D slices and included all the trabecular bone enclosed by the distal femoral growth plate. Trabecular bone volume fraction (BV/TV), trabecular thickness (Tb. Th), and other microstructural outcomes were calculated by the manufacturer’s analysis tool.

Total osteophyte volume was determined by the same contouring method; the volume of interest included all heterotopic mineralized tissue in the joint, the patella, fabellae, and menisci. Mineralized tissue was measured on both the injured limb and the contralateral limb, and the difference in bone volume between injured and contralateral limbs was calculated to get the total mineralized osteophyte volume.

Histological Analysis of Osteoarthritis and Synovitis

Following μCT scanning, whole knee joints were decalcified, embedded in paraffin, and 7-μm-thick sagittal histological slides were cut at 20-μm intervals from the medial and lateral articulations. The first section was selected at the point where the anterior and posterior horns of the medial meniscus appear as triangles. Slides were stained with Safranin-O (Sigma-Aldrich Inc., ST. Louis, MO) and Fast Green (Sigma-Aldrich Inc., ST. Louis, MO). At least 1 slide from the medial and lateral aspect of each joint with no folding on the articular cartilage and intact “W” shape growth plate on the femur were evaluated to score articular cartilage degeneration and synovitis. A modified 10-point grading OARSI scoring system to assess PTOA severity has previously described.²³ Briefly, a score of 0 indicates intact cartilage with strong staining on the femoral condyle and tibia; a score of 1 indicates minor fibrillation without cartilage loss; a score of 2 indicates clefts extending below the superficial zone; a score of 3 indicates preliminary signs of cartilage thinning on the femoral condyle and tibia; scores 4-10 correspond to increasing areas of cartilage erosion from the tibia and femur (4: 0%-10%, 5: 10%-20%, 6: 20%-25%, 7: 25%-50%, 8: 50%-75%, 9: 75%-90%, 10: greater than 90%). Synovitis scoring was performed using a 6-point scoring system grading cell lining and cell density of synovium as previously described by Lewis et al.²⁴ and Krenn et al.^25,26 Three independent readers graded both OA severity and synovitis on a selected section of each joint. All readers were blinded to experimental groups. The final scores were calculated by averaging 3 readers’ scores for each joint compartment.

Statistical Analysis

All data are presented as mean ± SD. One-way analysis of variance (ANOVA) with Tukey-Kramer post-hoc test was performed to detect statistical differences between the 5 experimental groups for protease activity, μCT data, OA grading, synovitis scoring, and scRNA-seq at each time point. GraphPad Prism (GraphPad Prism version 9, GraphPad Software, La Jolla, CA) was used to analyze and plot the data, and significance was defined as P < 0.05.

Results

FRI Shows Surgical Knee Restabilization Induces More Protease Activity

FRI analysis of protease activity showed no differences between any experimental groups in normalized radiant efficiency at day 14 or day 42, but significantly greater protease activity (up to 4-fold increase) was observed in restabilized mice at day 28 regardless of whether they had HLU or normal cage activity post injury (Fig. 2).

Figure 2.

Fluorescence reflectance imaging (FRI) of inflammatory protease activity in mouse knee joints at day 14, day 28, and day 42 after injury. (A) Representative images of uninjured and injured mice knee joints. (B) Average radiant efficiency of the injured leg normalized by the contralateral leg. The data show no significant effects of injury or unloading at these time points, but greater protease activity at day 28 in groups that had restabilization surgery.

HLU Diminishes Osteophyte Formation but Does not Change Trabecular Bone Microstructure

Consistent with our previous studies, we found that noninvasive ACL injury led to loss of epiphyseal trabecular bone after injury, with partial recovery at later time points. In unoperated groups, 21 days after ACL injury, approximately 15% loss of epiphyseal trabecular bone was found regardless of whether mice were subjected to 7 days of HLU or normal cage activity. Restabilization surgery caused considerably greater loss of epiphyseal trabecular bone (−27% to 32% BV/TV) at the same timepoint in both HLU and cage activity groups (Fig. 3A). A similar trend was also found in trabecular thickness. Injury resulted in a slight loss in trabecular thickness, and more loss was observed in the surgical groups (Fig. 3C). At day 42, we observed partial recovery of trabecular bone volume in both surgery groups, but persistent deficits in bone volume were observed in all injured mice except the Inj-HLU group (Fig. 3B). No significant difference in trabecular thickness was found between groups at this time point (Fig. 3D).

Figure 3.

Micro-computed tomography analysis of trabecular bone microstructure in the distal femoral epiphysis. (A) ACL injury alone caused approximately 15%-20% trabecular bone loss in the injured leg at day 21, while restabilization surgery caused approximately 30% bone loss at the same time point regardless of whether mice were subjected to HLU or normal cage activity. (B) Bone loss was partially recovered after 42 days, and significantly more recovery was observed in the groups that were subjected to HLU. (C) Trabecular thickness also showed a significant decrease in injured legs and significant decrease in surgical groups at day 21. (D) No significant differences in trabecular thickness were observed between any groups at day 42. Only significant differences between injured limbs are shown on graphs; *P value < 0.05, **P value < 0.01, ***P value < 0.001, ****P value < 0.0001.

Osteophyte formation in Inj-Unop, Inj-HLU, Inj-Restab, and Inj-HLU-Restab mice was detected by day 21, with considerably greater osteophyte volume in all injured groups at day 42 (Fig. 4). However, osteophyte volume in both HLU groups was significantly less than in cage activity groups (−46% relative to Inj-Unop mice; Fig. 4B). Osteophyte volume in restabilized joints with normal cage activity was similar to Inj-Unop leg while the surgery mice with HLU showed considerably less osteophyte formation. No significant differences in osteophyte volume were observed in restabilized groups.

Figure 4.

Quantification of osteophyte formation using micro-CT analysis. (A) representative 3D images of knee joint from each experimental group at day 42 illustrating the greater osteophyte volume in Inj-Unop and Inj-Restab mice. (B) Quantitative data show that osteophytes were found in Inj-Unop and Inj-Restab mice at day 21, and considerably greater osteophyte volume were observed in these two groups at day 42 compared to uninjured and HLU groups.

HLU Significantly Decreases OA Progression and Synovitis Based on Histological Grading

Quantification of chondrophyte area on day 21 histological images showed that considerable chondro/osteophytes formed in Inj-Unop mice (Fig. 5A). Chondro/osteophyte formation measured in Inj-HLU, Inj-Restab, and Inj-HLU-Restab groups was significantly less than in Inj-Unop mice (−70%). Little-to-no chondro/osteophyte formation was observed in Uninj-Unop and Inj-HLU-Restab mice (Fig. 5B). At day 42, the trend was similar to day 21 except chondro/osteophyte formation in Inj-Restab mice increased considerably (+65% compared to day 21). Both HLU groups showed significantly less chondro/osteophyte area than Inj-Unop mice at day 42 (Fig. 5B).

Figure 5.

Measurement of chondrophyte/osteophyte area on histological images. (A) Representative contouring of chondrophyte/osteophyte area for Inj-Unop and Inj-HLU-Restab mice at 42 days after injury. The three contouring areas for quantifying chondrophyte area were the anterior femur (AF), anterior meniscus (AM), and posterior tibia (PT). (B) At day 21, greater chondrophyte osteophyte area was found in the Inj-Unop mice compared to other groups. At day 42, both Inj-HLU and Inj-HLU-Restab groups had significantly less chondrophyte/osteophyte area than Inj-Unop and Inj-Restab mice. *P value < 0.05, **P value < 0.01, ****P value < 0.0001.

At day 21, we observed minimal OA progression in the Inj-HLU-Restab group (mean OA score 2.11 ± 0.70). Mild-to-moderate OA were observed in Inj-HLU and Inj-Restab mice (mean OA score 4.33 ± 1.05 and 3.82 ± 1.84, respectively). Moderate-to-severe OA was observed in Inj-Unop mice (mean OA score 6.13 ± 1.47) at this time point. At day 42, we observed more severe OA progression in Inj-Unop (mean OA score 7.60 ± 1.81) and Inj-Restab (mean OA score 5.90 ± 1.96) mice compared to day 21. However, OA was less severe in the Inj-HLU group (mean OA score 3.03 ± 1.24), and OA progression in the Inj-HLU-Restab group was minimal at this time point (mean OA score 1.87 ± 0.99; Fig. 6B ).

Figure 6.

Cartilage degradation and synovitis grading on Safranin-O and Fast Green stained images. (A) Representative histological images from each experimental group at 42 days after injury. Red arrows indicate the cartilage degradation which is assessed with OA scoring. Blue arrows show the region for synovitis scoring. (B) At day 21, moderate cartilage degeneration was found in Inj-Unop mice and mild-to-moderate degeneration was found in Inj-HLU and Inj-Restab mice. There was little cartilage degeneration observed in both Uninj-Unop and Inj-HLU-Restab mice. At day 42, moderate-to-severe cartilage degeneration was found in Inj-Unop mice and moderate degeneration was observed in Inj-Restab mice. However, in Inj-HLU and Inj-HLU-Restab mice, only mild cartilage degeneration was observed by day 42. (C) At day 21, Inj-Unop mice had significantly greater synovitis than Inj-HLU mice (Inj-Unop: 4.23 ± 0.83; Inj-HLU: 2.59 ± 0.55; Inj-Restab: 3.53 ± 1.22; Inj-HLU-Restab: 2.89 ± 0.64). At day 42, significantly less synovitis was observed in Inj-HLU and Inj-HLU-Restab mice compared to Inj-Unop and Inj-Restab. *P value < 0.05, **P value < 0.01, ***P value < 0.001, ****P value < 0.0001.

At day 21, there were no significant differences in synovitis score in any of the injured groups (Inj-Unop: 4.23 ± 0.83; Inj-HLU: 2.59 ± 0.55; Inj-Restab: 3.53 ± 1.22; Inj-HLU-Restab: 2.89 ± 0.64), although all injured groups had increased synovitis scores compared to Uninj-Unop mice. Both HLU groups (Inj-HLU and Inj-HLU-Restab) showed lower synovitis scores (1.90 ± 0.98 and 1.93 ± 0.67, respectively) than Inj-Unop (3.67 ± 0.65) and Inj-Restab (3.65 ± 1.11) joints at day 42 (Fig. 6C). However, there were no significant differences between restabilized and unoperated HLU mice.

Characterization of Cell Changes in the Mouse Knee Joint After 1 Week of HLU With scRNA-seq

In a previous study, we characterized immune cell changes in the mouse knee joint following ACL injury. After injury, monocytes/macrophages significantly increase in the knee joints while neutrophils were still the most abundant immune cells at all timepoints.²⁷ Compared with these data, we examined cellular composition and transcriptional changes across all cell types present in the injured joint to determine the effects of unloading. We identified ten major cell clusters based on distinct gene expression profiles (Fig. 7A). Cluster 1 was identified as neutrophils, characterized by the markers S100a8, S100a9. Cluster 2 corresponded to proliferating cells, marked by Top2a and Mki67. Cluster 3 represented monocyte-macrophages (Mono-macs), expressing Cd14 and Cd79a. Cluster 4 was classified as B cells, with markers Cd19 and Cd79a. Cluster 5 corresponded to synovial fibroblast (SFB), expressing Prg4 and Htra1. Cluster 6 was identified as mesenchymal progenitor/fibroblasts (MP/FB), marked by Postn and Prrx1. Cluster 7 was identified as T cells and natural killer cells (T/NK), based on Nkg7 and Cd3e markers. Cluster 8 was designated as chondrocytes (Ch), based on Acan and Col2a1 expression. Cluster 9 was identified as osteoblast (OB), marked by Col1a1 and Col1a2. Lastly, Cluster 10 was enriched for endothelial (Endo) markers Pecam1 and Cd34 (Fig. 7B).

Figure 7.

Single-cell RNA-seq results from injured knee joints of mice after 1 week of HLU or normal cage activity. (A) Determined cell clusters shown with Uniform Manifold Approximation and Projection (UMAP). Cluster 1: neutrophils; Cluster 2: proliferative cells; Cluster 3: monocyte-macrophages; Cluster 4: B cells; Cluster 5: synovial fibroblast; Cluster 6: mesenchymal progenitor and fibroblast; Cluster 7: T cells and natural killer cells; Cluster 8: chondrocytes; Cluster 9: osteoblast; Cluster 10: endothelial cells. (B) Dot plot showing the expression of highlighted genes in various cell clusters. Dot size represents the percentage of cells expressing a specific gene and the intensity of color shows the average expression of each gene. (C) Comparison of the proportion of different types of immune cells in the joint with and without HLU. Knee joints from mice in HLU showed more neutrophils and less macrophages and B cells than joints from mice in normal cage activity. (D) Hindlimb unloading induced transcriptional changes characterized by some significant upregulation of transcription factors and genes. (E) Monocyte-macrophage (Mono-Mac) populations showed changes in upregulation of transcription factors and inflammatory signaling molecules. (F) Transcriptional changes in mesenchymal including upregulation of transcription factors, growth factors, and cytoskeletal adaptor proteins.

One week of unloading significant impacted immune cells such as neutrophils levels, which increased to ~209% of the levels of injured mice with normal cage activity. Conversely, monocyte-macrophages decreased to 68%, B cells decreased to 67%, and T cells and natural killer cells increased to 127% of the levels of mice with normal cage activity. Other types of cells also showed some changes such as SFBs and MP/FBs, which decreased to 24% and 23% of the cell proportion, respectively. Osteoblasts increased to 3,756% while endothelial cells decreased to 35% (Fig. 7C).

Next, we investigated the transcriptional changes induced by HLU to understand how it may slow PTOA progression at the single-cell level, with a focus on genes upregulated by unloading. Notably, immune cell populations such as neutrophils exhibited a pro-resolution phenotype, characterized by significant upregulation of transcription factors Fos, Fosl1, and Nr4a1, as well as genes including Fkbp5, Pdcd6, Rnf125, Rnf40, and Sik1 (Fig. 7D). Monocyte-macrophage (Mono-Mac) populations showed broader transcriptional changes, with upregulation of transcription factors such as Nfkbiz, Rela, and Stat3; cytokines including Il1b, Il6, and Osm; and inflammatory signaling molecules such as Il6ra, Tlr2, Trem1, Csf2ra, Csf2rb, Csf3r, Irak2, Irak3, and Jak2 (Fig. 7E). These changes suggest enhanced activation of an M1-like inflammatory phenotype in response to HLU.

We also examined transcriptional changes within MP/FB populations. Here, HLU was associated with upregulation of transcription factors including Zbtb16, Foxo1, Klf15, and Tsc22d3; growth factors such as Bmp5, Cytl1, and Serpina3n; and cytoskeletal adaptor proteins like Tns2 (Fig. 7F). These changes suggest enhanced progenitor potential and support chondrogenic and osteogenic differentiation under unloading conditions. Taken together, the HLU-specific transcriptional changes observed in neutrophils and monocyte-macrophage populations suggest that these cells may serve complementary roles in modulating inflammation and tissue remodeling, ultimately contributing to the attenuation of PTOA progression.

Discussion

The main goal of this study was to investigate the long-term effects of early joint unloading after ACL injury and prior to surgical joint restabilization on PTOA progression. Consistent with our initial hypothesis, we found that 7 days of joint unloading was able to slow down OA disease progression, diminish osteophyte formation and synovitis after ACL injury regardless of whether the injured knee joint underwent restabilization surgery or not, suggesting that early joint protection immediately after ACL injury is more critical than longer term mechanical instability in this mouse injury model. We also found that 7 days of HLU following ACL injury changed immune cell populations in the joint, with a greater number of neutrophils and reduced macrophages in unloading joints. Altogether, these data suggest that joint unloading (non-weightbearing) following ACL injury can modulate early processes that contribute to PTOA progression before the joint stability is restored surgically.

The knee restabilization method we utilized in mice is not directly comparable to ACL reconstruction in humans. Since knee joints of mice are extremely small, restabilizing the joint by recapitulating human ACL reconstruction is technically difficult. Even though some research has performed ACL reconstruction in mice^28,29 and rats,^30
-32 it is unclear how effective those knee surgeries were at restoring the mechanical stability of the knee joints. As a result, we used a knee surgery technique that is similar to the procedures used in small dog breeds to stabilize a knee joint after ACL rupture,³³ which we previously showed to restore much of the anterior-posterior biomechanical stability of the knee joint.

The timing of ACL reconstruction has been debated for many years, and there is still no consensus regarding whether early or delayed surgery is superior. Based on the reviews from Evans et al.³⁴ and Smith et al.,³⁵ even though there were no differences in long-term results between immediate (less than 3 weeks) and delayed (4-6 weeks or longer) ACL reconstruction, there are still several complications corresponding to both immediate and delayed ACL reconstruction. Some literature suggests that performing surgery at least 3 weeks after injury was able to decrease the risk of arthrofibrosis.^36,37 However, there are still several critical factors that may affect the recovery and long-term PTOA progression such as joint condition, muscle strength, and post-surgery rehabilitation etc.³⁴ In this study, we aimed to focus on investigating the effect of joint unloading during the acute inflammation of the knee on the ruptured ACL. This study can provide patients with delayed ACL reconstruction a beneficial strategy immediately after ACL injury.

Few clinical studies have investigated the effect of weightbearing or non-weightbearing between ACL injury and reconstruction on OA progression. Wellsandt et al.³⁸ found that within 1 month after ACL injury, longer T2 relaxation times were showed in some gait changes and higher physical activity indicating some cartilage degradation. In addition, longer periods of disuse can also lead to muscle atrophy and bone loss, which would further accelerate OA progression.³⁹ Patients treated with different activities and without ACL reconstruction showing that changing early activities and adding some knee rehabilitation might be able to decrease incidence of knee OA.⁴⁰ These limited data suggest that the early period following ACL injury can have some effect on joint heath and also affect long-term PTOA progression. However, more studies are needed to understand the effects of loading or unloading during the early phase post injury.

Based on our previous study, the short-term effect of joint unloading after ACL injury includes changes in the early phase (<7 days) inflammatory response. In this study, we used ProSense 680, which is a well-established probe targeting cathepsin proteases including Cathepsin B, L, and S and Plasmin. It can indicate the activity of lysosomal cathepsin proteases, activated macrophages, neutrophils, and eosinophils in the inflammatory response.^20,41 Using ProSense 680 and FRI imaging, we found that restabilization surgery caused considerably more protease-activated fluorescent signal than noninvasive ACL injury alone, but this greater protease activity was not associated with accelerated PTOA progression. These data suggest that surgical procedures themselves increased the FRI signal of the knee joint, likely due to wound healing in the skin and overlying soft tissue, which may obscure the reading of protease activity within the joint caused by ACL injury. In this way, FRI analysis and fluorescent activatable probes such as ProSense 680 may not be particularly useful for surgical models of PTOA. These models may require more direct and invasive approaches such as PCR to measure protease activity or other biomarkers within the joint. Further investigation of the relationship between early protease activity and long-term joint degeneration and the effect of early joint unloading may help establish treatable targets for preserving joint health after injury.

Even though some amount of unloading might be able to mitigate the inflammatory responses and PTOA progression in preclinical models, long-term unloading can cause OA like effects even in uninjured joints.⁴² In healthy joints, non-weightbearing or immobilization can cause cartilage degradation, but this can be partially recovered with reloading.⁴³ Joint immobilization in dogs contributed to thinning of articular cartilage, reduced proteoglycan content, and decreased proteoglycan synthesis.⁴⁴ In addition, in rats, both short-term (1 or 2 weeks) or long-term (2 or 4 weeks) non-weightbearing after ACL reconstruction caused cartilage degeneration.⁴⁵

There is still no consensus on the optimal treatment during the early phase after joint injury including joint loading or unloading. In human cases, physicians and physical therapists will often recommend loading as tolerated after ACL injury to restore muscle health and strength and to maintain joint range of motion, which are beneficial in surgical repairs and post-surgical outcomes. However, loading of the injured joint too early or with too much intensity may exacerbate inflammation and aggravate the injured joint tissues. Therefore, there is a potential tradeoff between preventing long-term joint degeneration and maintaining short-term muscle/bone structure and joint function. For example, some studies have indicated that long-term inactivity can also cause muscle atrophy and bone loss, which could accelerate OA progression³⁹ and increase risks of other injuries such as fractures.⁴⁶ Identifying optimal biomechanical treatments that can be applied following ACL injury is important for both efficient rehabilitation and recovery from ACL reconstruction and for maintaining long-term joint health.

In this study, we found the trabecular epiphyseal bone loss at 21 days following ACL rupture is about 20%, which is consistent with our previous studies.^7,14 It is likely that this bone loss is partially driven by mechanical unloading of the injured joint due to pain and/or inflammation in the joint leading to bone remodeling in the subchondral compartment.¹⁵ Mechanical unloading is one of the main biomechanical interventions in this study, but we did not observe more bone loss in unloaded mice than in mice that returned to normal cage activity following ACL injury, suggesting that this bone loss does not arise primarily due to mechanical unloading. In addition, we found that restabilization surgery itself caused more trabecular bone loss than ACL injury alone. These findings are in agreement with our previous study, which showed that restabilization surgery was not able to mitigate early epiphyseal bone loss, suggesting that this bone loss is not caused by mechanical factors, but by biological factors such as inflammation.⁷ However, at day 42, we found more bone recovery in Inj-HLU mice, suggesting that early joint unloading might be able to reduce inflammation and further facilitate the recovery of bone loss in the long term.

Anderson et al.⁴⁷ described the time course following traumatic joint injuries within 14 days, which includes 4-7 days of inflammation in the early phase, 2-3 days of catabolic and anabolic processes in the intermediate phase, and 6-8 days of healing, remodeling, and matrix formation in the late phase. During the early stage, some inflammatory cytokines and degradative enzymes such as interleukin and matrix metalloproteinases (MMPs) can initiate the degradation of articular cartilage and subchondral bone, which are early processes in the development of PTOA.⁴⁷ In the current study, we applied unloading to the joint during this early phase aiming to decrease inflammatory responses and protease activity,^13,48
-50 reduce degradation of joint tissues, and diminish some amount of “wear and tear” caused by joint instability. This study confirmed that unloading during the early phase has long-term benefits in diminishing osteophyte formation, preventing articular cartilage degradation, and mitigating synovitis.

Consistent with our previous studies, we found that HLU could significantly delay chondro/osteophytes formation for at least 42 days after injury. Our previous studies showed that chondrophytes formed quickly (<1 week after injury) and then progressively mineralized to osteophyte over the following few weeks. In the current study, at day 21, there was greater chondrophyte formation at the anterior femur, anterior horn of the medial meniscus, and posterior tibia in the Inj-Unop group than in the Inj-HLU, Inj-Restab, and Inj-HLU-Restab groups. At day 42, more osteophyte formation was observed in these same regions, indicating the transition from chondrophytes to osteophytes during these 3 weeks, which is also consistent with other studies.^51,52 Little chondro/osteophyte formation was observed in Inj-HLU and Inj-HLU-Restab mice at either day 21 and day 42, showing that HLU after ACL injury can effectively prevent or delay osteophyte formation in this injury model. Based on the proposed function of osteophytes, which is to provide some amount of restabilization to the knee joint,⁵³ early HLU might be able to help maintain mechanical stability of knee joint to sustain long-term joint health.

Interestingly, mice with 7 days of normal cage activity following ACL injury that then underwent restabilization surgery did not exhibit any considerable benefit of the surgery for slowing or preventing PTOA. This finding is somewhat consistent with human cases, in which still more than half of patients receiving ACL reconstruction have PTOA in 10-20 years.⁵⁴ Our results suggest that early joint unloading in this model likely provided more mechanical and biological benefit for OA prevention. The mechanical and biological changes in the first week following injury clearly can affect long-term PTOA progression.⁴⁷ This is important clinically because there is no consensus about how much patients should load or unload their knee joint between injury and ACL reconstruction surgery. As a result, one main goal of this study was to use mice to investigate the effect of early unloading or mobilization prior to restabilization surgery to provide some insight to this question. Altogether, we postulate that both early unloading and subsequent restabilization of the joint are important for decreasing inflammation and restoring mechanical function of the joint and ultimately slowing or preventing PTOA progression.

One unique strength of this study is that we used single-cell RNA sequencing to further understand immune cell changes associated with joint unloading following ACL injury. Even though our analysis only used 2 mice per experimental group to investigate the changes with HLU, these preliminary results were able to note several interesting differences between these conditions, and these data provide a foundation for further investigation on the mechanism of cellular changes within injured knee joints with different biomechanical conditions. Few studies have used scRNA-seq to understand the effect of HLU in other tissues such as cardiovascular system.⁵⁵ In our previous study, we used scRNA-seq to identify immune cell populations in the joint including neutrophils, monocytes, macrophages, B cells, T cells, and so on and found that monocyte and macrophage populations changed significantly after noninvasive ACL injury.²⁷ Our scRNA-seq analysis in the current study found more neutrophils and fewer macrophages and B cells in the knees of mice that were unloaded for 7 days compared to those that returned to normal cage activity. Other studies also showed there are significant changes in the immune system following HLU. For example, Masoudi et al.⁵⁶ found neutrophil-to-lymphocyte ratio (NLR) increased with HLU which is corresponding to increasing neutrophils observed in our study. Pro-B and pre-B cells in the bone marrow were also found decrease in HLU mice⁵⁷ which is consistent to our finding in B cell population. Less IgM was also observed in HLU mice compared to those in aged mice.⁵⁸ Altogether, studies conclude that hindlimb unloading greatly suppressed or delayed the function of the immune system in preclinical models.⁵⁹

The early inflammatory response has been shown in many studies to be one of the critical triggers of PTOA progression,^60
-62 therefore the changes we observed in the immune cell populations following injury may indicate key differences that are mechanisms of joint degeneration leading to PTOA. We also discovered transcriptional changes within MP/FB and OB populations which are in agreement with other research suggesting that HLU induces the modulation the gene expression in different types of mesenchymal cell as well as mRNAs and proteins for ECM deposition, maturation and remodeling which may play key roles in maintaining bone microstructure.⁶³ The modulations of mesenchymal cells and chondrogenic and osteogenic cells found in our study may also play critical roles in preserving joint health but need further justification.

One limitation of this study is that we did not evaluate any soft-tissue changes caused by mechanical unloading without ACL injury. Some studies showed that in human subjects, long periods of non-weightbearing can cause muscle atrophy and bone loss, which might also accelerate OA progression.^64,65 Similarly, in preclinical models, HLU can lead to muscle atrophy and weakness;^66,67 HLU can also induce muscle fiber type switch from type I to type II.^68,69 Muscle strength and muscle fiber type might play a critical role in PTOA progression.⁷⁰ Therefore, it may be important to minimize the unloading period to only occur during the early inflammatory phase following injury. In our current and previous studies, we subjected mice to only 1 week of HLU in order to minimize the muscle atrophy and muscle fiber type switch, however it is not clear if 7 days of unloading is the optimal time for preventing PTOA. Future studies can investigate different periods of HLU (e.g., 14 or 21 days) and incorporate the effect of muscle atrophy and muscle type changes in PTOA progression. Future studies could also include an uninjured unloading group to compare the effects of HLU alone on cartilage, muscle, and bone morphology within the knee joints.

Although incorporating single-cell RNA sequencing is one of the strengths of this study, our analysis is limited because we only used 2 mice per group at a single time point to acquire preliminary results of immune cell changes with HLU. Future studies could perform a more thorough investigation of immune cell populations at multiple time points to strengthen our preliminary findings. Another limitation of this study is that even though 1 week of joint unloading altered the population of immune cells in the joint and effectively slowed PTOA progression in a mouse model of ACL injury, it is difficult to translate this intervention to human subjects directly. In both humans and rodent models, the inflammatory response following ACL injury can be categorized in different phases. In humans, the inflammatory response is much more prolonged than rodent models, with increased inflammatory cytokines and synovial fluid markers. The responses can remain high for months to years contributing to sustaining cartilage and joint degeneration. These long-term effects also contribute to hard translation between human and rodent models.^71,72 Joint injuries cause a temporally dependent adaption response that includes an early phase characterized by cell death/apoptosis and inflammation, an Intermediate Phase with both catabolic and anabolic processes, and a Late Phase of repair, remodeling, and matrix formation. The surge of inflammatory cytokines and degradative enzymes such as cathepsin proteases and matrix metalloproteinases (MMPs) during the early phase can initiate degradation of articular cartilage and subchondral bone.⁴⁷ In humans, ACL injury often includes direct damage to the articular cartilage and subchondral bone, which leads to further cell death, subchondral bone damage, bone marrow edema, and accelerated matrix remodeling.^73,74 Inflammatory cytokines and biomarkers of cartilage and bone remodeling are increased in synovial fluid within 24-48 hours after knee injury.⁷⁵ These markers are associated with proteolysis of aggrecan, type II collagen, cartilage matrix, and subchondral bone changes that can lead to PTOA.⁷⁶

Based on our results, early joint unloading can significantly decrease the inflammatory response contributing to joint degradation. Although the tail suspension hindlimb unloading model in mice is not directly translatable to humans after joint injury, the general principle of unloading the injured joint during the early phase after injury can inform strategies for ACL injury patients such as decreasing weightbearing or fully unloading during the first few days or weeks following injury. More clinical studies are needed to understand the effect of different unloading conditions in PTOA progression in human subjects and the effective window of opportunity for early unloading.

In the current study, even though the improvement in PTOA progression was clear in a rodent model, the equivalent amount of time for patients to unload their knee joints is difficult to determine. The time course of the inflammatory responses after ACL injury is somewhat different between human and mouse (although not nearly as different as the time course of PTOA disease progression). If the overall time of inflammation in human is fully understood, we might be able to apply the unloading concept within the inflammation period. In addition, some patients might not be able to get ACL surgery right after this inflammation time. Most physicians and physical therapists will recommend some rehabilitation soon after injury to maintain muscle mass and strength and maintain joint range of motion.⁷⁷ How these activities would affect long-term PTOA progression is also critical and need to be further investigated.

In summary, this study found that early unloading of injured mice knee joints prior to restabilization surgery was an effective intervention for slowing PTOA progression after ACL injury. The results from this study also suggest that unloading knee joint in human subjects during the early stage following ACL injury may be a beneficial intervention to prevent long-term joint degradation. However, further studies are needed to further confirm that the changes in inflammation during the early stage following injury are key mechanisms that can be targeted to delay PTOA progression or diminish the severity of PTOA in the future. Developing a more thorough understanding of how biomechanical interventions can be used for slowing osteophyte formation and PTOA development can help translate these strategies into the entire time course of therapeutics in human ACL injury in order to improve millions of patients affected by this debilitating disease.

Supplemental Material

sj-jpg-1-car-10.1177_19476035251411505 – Supplemental material for Early Unloading After ACL Rupture and Prior to Surgical Restabilization in Mice Slows Post-Traumatic Osteoarthritis Progression

Supplemental material, sj-jpg-1-car-10.1177_19476035251411505 for Early Unloading After ACL Rupture and Prior to Surgical Restabilization in Mice Slows Post-Traumatic Osteoarthritis Progression by Yu-Yang Lin, I-Shin Ju, Cesar Morfin, Elias H. Jbeily, Aimy Sebastian, Cassandra A. Lee, Gabriela G. Loots and Blaine A. Christiansen in CARTILAGE

Footnotes

ORCID iDs

Yu-Yang Lin

Blaine A. Christiansen

Ethical Considerations

Not applicable

Consent for Publication

Not applicable

Author Contributions

Yu-Yang Lin (Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing—original draft, Writing—review & editing), I-Shin Ju (Investigation, Methodology), Cesar Morfin (Methodology, Data curation, Visualization), Elias Jbeily (Methodology), Aimy Sebastian (Validation, Writing—review & editing), Cassandra A. Lee (Investigation, Methodology, Writing-review & editing), Gabriela Loots (Supervision, Project administration, Writing-review & editing), and Blaine Christiansen (Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing—review & editing).

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases, part of the National Institutes of Health, under Award Number R01 AR075013. This work was in part performed under the auspices of the USDOE by Lawrence Livermore National Laboratory (DE-AC52-07NA27344).

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Supplemental Material

Supplementary material for this article is available on the Cartilage website at .

References

Shimokochi

Shultz

SJ.

Mechanisms of noncontact anterior cruciate ligament injury. J Athl Train. 2008;43(4):396-408. doi:10.4085/1062-6050-43.4.396.

Sanders

Maradit Kremers

Bryan

Larson

Dahm

Levy

, et al. Incidence of anterior cruciate ligament tears and reconstruction: a 21-year population-based study. Am J Sports Med. 2016;44(6):1502-7. doi:10.1177/0363546516629944.

Dilley

Bello

Roman

McKinley

Sankar

Post-traumatic osteoarthritis: a review of pathogenic mechanisms and novel targets for mitigation. Bone Rep. 2023;18101658. doi:10.1016/j.bonr.2023.101658.

Dirschl

Marsh

Buckwalter

Gelberman

Olson

Brown

, et al. Articular fractures. J Am Acad Orthop Surg. 2004;12(6):416-23.

Riordan

Little

Hunter

Pathogenesis of post-traumatic OA with a view to intervention. Best Pract Res Clin Rheumatol. 2014;28(1):17-30. doi:10.1016/j.berh.2014.02.001.

Mercurio

Cerciello

Corona

Guerra

Simonetta

Familiari

, et al. Factors associated with a successful return to performance after anterior cruciate ligament reconstruction: a multiparametric evaluation in soccer players. Orthop J Sports Med. 2024;12(10):23259671241275663. doi:10.1177/23259671241275663.

Lin

Jbeily

Tjandra

Pride

Lopez-Torres

Elmankabadi

, et al. Surgical restabilization reduces the progression of post-traumatic osteoarthritis initiated by ACL rupture in mice. Osteoarthritis Cartilage. 2024;32(8):909-20. doi:10.1016/j.joca.2024.04.013.

Haapala

Arokoski

Rönkkö

Agren

Kosma

Lohmander

, et al. Decline after immobilisation and recovery after remobilisation of synovial fluid IL1, TIMP, and chondroitin sulphate levels in young beagle dogs. Ann Rheum Dis. 2001;60(1):55-60. doi:10.1136/ard.60.1.55.

Tsai

Cooper

Hetzendorfer

Warren

Chang

Willett

NJ.

Effects of treadmill running and limb immobilization on knee cartilage degeneration and locomotor joint kinematics in rats following knee meniscal transection. Osteoarthritis Cartilage. 2019;27(12):1851-9. doi:10.1016/j.joca.2019.08.001.

10.

Takahashi

Matsuzaki

Kuroki

Hoso

Joint unloading inhibits articular cartilage degeneration in knee joints of a monosodium iodoacetate-induced rat model of osteoarthritis. Osteoarthritis Cartilage. 2019;27(7):1084-93. doi:10.1016/j.joca.2019.03.001.

11.

Kotelsky

Elahi

Nejat Yigit

Proctor

Mannava

Pröschel

, et al. Effect of knee joint loading on chondrocyte mechano-vulnerability and severity of post-traumatic osteoarthritis induced by ACL-injury in mice. Osteoarthr Cartil Open. 2022;4(1):100227. doi:10.1016/j.ocarto.2021.100227.

12.

Hsia

Jbeily

Mendez

Cunningham

Biris

Bang

, et al. Post-traumatic osteoarthritis progression is diminished by early mechanical unloading and anti-inflammatory treatment in mice. Osteoarthritis Cartilage. 2021;29(12):1709-19. doi:10.1016/j.joca.2021.09.014.

13.

Hsia

Emami

Tarke

Cunningham

Tjandra

Wong

, et al. Osteophytes and fracture calluses share developmental milestones and are diminished by unloading. J Orthop Res. 2018;36(2):699-710. doi:10.1002/jor.23779.

14.

Christiansen

Anderson

Lee

Williams

Yik

Haudenschild

DR.

Musculoskeletal changes following non-invasive knee injury using a novel mouse model of post-traumatic osteoarthritis. Osteoarthritis Cartilage. 2012;20(7):773-82. doi:10.1016/j.joca.2012.04.014.

15.

Christiansen

Emami

Fyhrie

Satkunananthan

Hardisty

MR.

Trabecular bone loss at a distant skeletal site following noninvasive knee injury in mice. J Biomech Eng. 2015;137(1):0110051-6. doi:10.1115/1.4028824.

16.

Lin

Christiansen

BA.

Non-invasive compression-induced anterior cruciate ligament (ACL) injury and in vivo imaging of protease activity in mice. J Vis Exp. 2023;199:10.3791/65249. doi:10.3791/65249.

17.

van Loon

JJWA

Beysens

. Generation and applications of extra-terrestrial environments on earth. New York: River Publishers; 2022.

18.

Kokubun

Kanemura

Murata

Moriyama

Morita

Jinno

, et al. Effect of changing the joint kinematics of knees with a ruptured anterior cruciate ligament on the molecular biological responses and spontaneous healing in a rat model. Am J Sports Med. 2016;44(11):2900-10. doi:10.1177/0363546516654687.

19.

Satkunananthan

Anderson

De Jesus

Haudenschild

Ripplinger

Christiansen

BA.

In vivo fluorescence reflectance imaging of protease activity in a mouse model of post-traumatic osteoarthritis. Osteoarthritis Cartilage. 2014;22(10):1461-9. doi:10.1016/j.joca.2014.07.011.

20.

Conus

Simon

HU.

Cathepsins: key modulators of cell death and inflammatory responses. Biochem Pharmacol. 2008;76(11):1374-82. doi:10.1016/j.bcp.2008.07.041.

21.

Morfin

Sebastian

Wilson

Amiri

Murugesh

Hum

, et al. Mef2c regulates bone mass through Sost-dependent and -independent mechanisms. Bone. 2024;179:116976. doi:10.1016/j.bone.2023.116976.

22.

Butler

Hoffman

Smibert

Papalexi

Satija

Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol. 2018;36(5):411-20. doi:10.1038/nbt.4096.

23.

Mendez

Sebastian

Murugesh

Hum

McCool

Hsia

, et al. LPS-induced inflammation prior to injury exacerbates the development of post-traumatic osteoarthritis in mice. J Bone Miner Res. 2020;35(11):2229-41. doi:10.1002/jbmr.4117.

24.

Lewis

Hembree

Furman

Tippets

Cattel

Huebner

, et al. Acute joint pathology and synovial inflammation is associated with increased intra-articular fracture severity in the mouse knee. Osteoarthritis Cartilage. 2011;19(7):864-73. doi:10.1016/j.joca.2011.04.011.

25.

Krenn

Morawietz

Häupl

Neidel

Petersen

König

Grading of chronic synovitis–a histopathological grading system for molecular and diagnostic pathology. Pathol Res Pract. 2002;198(5):317-25. doi:10.1078/0344-0338-5710261.

26.

Krenn

Morawietz

Burmester

Kinne

Mueller-Ladner

Muller

, et al. Synovitis score: discrimination between chronic low-grade and high-grade synovitis. Histopathology. 2006;49(4):358-64. doi:10.1111/j.1365-2559.2006.02508.x.

27.

Sebastian

Hum

McCool

Wilson

Murugesh

Martin

, et al. Single-cell RNA-Seq reveals changes in immune landscape in post-traumatic osteoarthritis. Front Immunol. 2022;13:938075. doi:10.3389/fimmu.2022.938075.

28.

Camp

Lebaschi

Cong

Album

Carballo

Deng

, et al. Timing of postoperative mechanical loading affects healing following anterior cruciate ligament reconstruction: analysis in a murine model. J Bone Joint Surg Am. 2017;99(16):1382-91. doi:10.2106/JBJS.17.00133.

29.

Nakagawa

Lebaschi

Wada

Green

SJE

Wang

Album

, et al. Duration of postoperative immobilization affects MMP activity at the healing graft-bone interface: evaluation in a mouse ACL reconstruction model. J Orthop Res. 2019;37(2):325-34. doi:10.1002/jor.24177.

30.

Cheuk

Chiu

Yung

Rolf

Chan

KM.

Tripeptide-copper complex GHK-Cu (II) transiently improved healing outcome in a rat model of ACL reconstruction. J Orthop Res. 2015;33(7):1024-33. doi:10.1002/jor.22831.

31.

Leong

Kabir

Arshi

Nazemi

McAllister

, et al. Athymic rat model for evaluation of engineered anterior cruciate ligament grafts. J Vis Exp. 2015;97:52797. doi:10.3791/52797.

32.

Maurice

Godineau

Pichard

El Hafci

Autret

Bensidhoum

, et al. Remnants-preserving ACL reconstruction using direct tendinous graft fixation: a new rat model. J Orthop Surg Res. 2022;17(1):7. doi:10.1186/s13018-021-02890-9.

33.

Canapp

Jr.

The canine stifle. Clin Tech Small Anim Pract. 2007;22(4):195-205. doi:10.1053/j.ctsap.2007.09.008.

34.

Evans

Shaginaw

Bartolozzi

Acl reconstruction—it’s all about timing. Int J Sports Phys Ther. 2014;9(2):268-73.

35.

Smith

Davies

Hing

CB.

Early versus delayed surgery for anterior cruciate ligament reconstruction: a systematic review and meta-analysis. Knee Surg Sports Traumatol Arthrosc. 2010;18(3):304-11. doi:10.1007/s00167-009-0965-z.

36.

Shelbourne

Patel

DV.

Timing of surgery in anterior cruciate ligament-injured knees. Knee Surg Sports Traumatol Arthrosc. 1995;3(3):148-56. doi:10.1007/BF01565474.

37.

Almekinders

Moore

Freedman

Taft

TN.

Post-operative problems following anterior cruciate ligament reconstruction. Knee Surg Sports Traumatol Arthrosc. 1995;3(2):78-82. doi:10.1007/BF01552379.

38.

Wellsandt

Kallman

Golightly

Podsiadlo

Dudley

Vas

, et al. Knee joint unloading and daily physical activity associate with cartilage T2 relaxation times 1 month after ACL injury. J Orthop Res. 2022;40(1):138-49. doi:10.1002/jor.25034.

39.

Lepley

Davi

Burland

Lepley

AS.

Muscle atrophy after ACL injury: implications for clinical practice. Sports Health. 2020;12(6):579-86. doi:10.1177/1941738120944256.

40.

Neuman

Englund

Kostogiannis

Fridén

Roos

Dahlberg

LE.

Prevalence of tibiofemoral osteoarthritis 15 years after nonoperative treatment of anterior cruciate ligament injury: a prospective cohort study. Am J Sports Med. 2008;36(9):1717-25. doi:10.1177/0363546508316770.

41.

Gounaris

Tung

Restaino

Maehr

Kohler

Joyce

, et al. Live imaging of cysteine-cathepsin activity reveals dynamics of focal inflammation, angiogenesis, and polyp growth. PLoS ONE. 2008;3(8):e2916. doi:10.1371/journal.pone.0002916.

42.

Zhou

Wei

Liu

Mao

Zhang

, et al. Cartilage matrix changes in contralateral mobile knees in a rabbit model of osteoarthritis induced by immobilization. BMC Musculoskelet Disord. 2015;16:224. doi:10.1186/s12891-015-0679-y.

43.

Takahashi

Matsuzaki

Kuroki

Hoso

Physiological reloading recovers histologically disuse atrophy of the articular cartilage and bone by hindlimb suspension in rat knee joint. Cartilage. 2021;13(2 Suppl):1530S-1539S. doi:10.1177/19476035211063857.

44.

Brandt

KD.

Response of joint structures to inactivity and to reloading after immobilization. Arthritis Rheum. 2003;49(2):267-71. doi:10.1002/art.11009.

45.

Kaneguchi

Okahara

Masuhara

Doi

Yamaoka

Ozawa

. The effects of short-term non-weightbearing and immobilization after anterior cruciate ligament reconstruction on articular cartilage: long-term observation after reloading and remobilization. Tissue Cell. 2025;92:102628. doi:10.1016/j.tice.2024.102628.

46.

Kondo

Nifuji

Takeda

Ezura

Rittling

Denhardt

, et al. Unloading induces osteoblastic cell suppression and osteoclastic cell activation to lead to bone loss via sympathetic nervous system. J Biol Chem. 2005;280(34):30192-200. doi:10.1074/jbc.M504179200.

47.

Anderson

Chubinskaya

Guilak

Martin

Oegema

Olson

, et al. Post-traumatic osteoarthritis: improved understanding and opportunities for early intervention. J Orthop Res. 2011;29(6):802-9. doi:10.1002/jor.21359.

48.

Blomgran

Ernerudh

Aspenberg

A possible link between loading, inflammation and healing: immune cell populations during tendon healing in the rat. Sci Rep. 2016;6:29824. doi:10.1038/srep29824.

49.

Kohno

Yamashita

Abe

Hirasaka

Oarada

Ohno

, et al. Unloading stress disturbs muscle regeneration through perturbed recruitment and function of macrophages. J Appl Physiol. 2012;112(10):1773-82. doi:10.1152/japplphysiol.00103.2012.

50.

Grenon

Jeanne

Aguado-Zuniga

Conte

Hughes-Fulford

Effects of gravitational mechanical unloading in endothelial cells: association between caveolins, inflammation and adhesion molecules. Sci Rep. 2013;3:1494. doi:10.1038/srep01494.

51.

Markhardt

Kijowski

The clinical significance of osteophytes in compartments of the knee joint with normal articular cartilage. AJR Am J Roentgenol. 2018;210(4):W164-W171. doi:10.2214/AJR.17.18664.

52.

Blom

van Lent

Holthuysen

van der Kraan

Roth

van Rooijen

, et al. Synovial lining macrophages mediate osteophyte formation during experimental osteoarthritis. Osteoarthritis Cartilage. 2004;12(8):627-35. doi:10.1016/j.joca.2004.03.003.

53.

Hsia

Anderson

Heffner

Lagmay

Zavodovskaya

Christiansen

BA.

Osteophyte formation after ACL rupture in mice is associated with joint restabilization and loss of range of motion. J Orthop Res. 2017;35(3):466-73. doi:10.1002/jor.23252.

54.

Cinque

Dornan

Chahla

Moatshe

LaPrade

RF.

High rates of osteoarthritis develop after anterior cruciate ligament surgery: an analysis of 4108 patients. Am J Sports Med. 2018;46(8):2011-9. doi:10.1177/0363546517730072.

55.

Pan

Wang

Tie

, et al. Single-cell RNA sequencing of the carotid artery and femoral artery of rats exposed to hindlimb unloading. Cell Mol Life Sci. 2025;82(1):50. doi:10.1007/s00018-024-05572-x.

56.

Masoudi

Kalani

Alavianmehr

Mosleh-Shirazi

Mortazavi

SMJ

Farjadian

. Immune cell dysregulation in hindlimb unloading mouse model: implications for space applications. J Biomed Phys Eng. Epub 2024 Dec 3. doi:10.31661/jbpe.v0i0.2406-1776.

57.

Lescale

Schenten

Djeghloul

Bennabi

Gaignier

Vandamme

, et al. Hind limb unloading, a model of spaceflight conditions, leads to decreased B lymphopoiesis similar to aging. FASEB J. 2015;29(2):455-63. doi:10.1096/fj.14-259770.

58.

Fonte

Jacob

Vanet

Ghislin

Frippiat

JP.

Hindlimb unloading, a physiological model of microgravity, modifies the murine bone marrow IgM repertoire in a similar manner as aging but less strongly. Immun Ageing. 2023;20(1):64. doi:10.1186/s12979-023-00393-1.

59.

Aviles

Belay

Vance

Sonnenfeld

Effects of space flight conditions on the function of the immune system and catecholamine production simulated in a rodent model of hindlimb unloading. Neuroimmunomodulation. 2005;12(3):173-81. doi:10.1159/000084850.

60.

Lieberthal

Sambamurthy

Scanzello

CR.

Inflammation in joint injury and post-traumatic osteoarthritis. Osteoarthritis Cartilage. 2015;23(11):1825-34. doi:10.1016/j.joca.2015.08.015.

61.

Khella

Horvath

Asgarian

Rolauffs

Hart

ML.

Anti-inflammatory therapeutic approaches to prevent or delay post-traumatic osteoarthritis (PTOA) of the knee joint with a focus on sustained delivery approaches. Int J Mol Sci. 2021;22(15):8005. doi:10.3390/ijms22158005.

62.

Punzi

Galozzi

Luisetto

Favero

Ramonda

Oliviero

, et al. Post-traumatic arthritis: overview on pathogenic mechanisms and role of inflammation. RMD Open. 2016;2(2):e000279. doi:10.1136/rmdopen-2016-000279.

63.

Visigalli

Strangio

Palmieri

Manduca

Hind limb unloading of mice modulates gene expression at the protein and mRNA level in mesenchymal bone cells. BMC Musculoskelet Disord. 2010;11:147. doi:10.1186/1471-2474-11-147.

64.

Egloff

Hügle

Valderrabano

Biomechanics and pathomechanisms of osteoarthritis. Swiss Med Wkly. 2012;142:w13583. doi:10.4414/smw.2012.13583.

65.

Hawliczek

Brix

Al Mutawa

Alsuwaidi

Du Plessis

Gao

, et al. Hind-limb unloading in rodents: current evidence and perspectives. Acta Astronautica. 2022;195:574-82. doi:10.1016/j.actaastro.2022.03.008.

66.

Qaisar

Karim

Elmoselhi

AB.

Muscle unloading: a comparison between spaceflight and ground-based models. Acta Physiol. 2020;228(3):e13431. doi:10.1111/apha.13431.

67.

Han

Zhu

Rat hindlimb unloading down-regulates insulin like growth factor-1 signaling and AMP-activated protein kinase, and leads to severe atrophy of the soleus muscle. Appl Physiol Nutr Metab. 2007;32(6):1115-23. doi:10.1139/H07-102.

68.

Baehr

West

DWD

Marshall

Marcotte

Baar

Bodine

SC.

Muscle-specific and age-related changes in protein synthesis and protein degradation in response to hindlimb unloading in rats. J Appl Physiol. 2017;122(5):1336-50. doi:10.1152/japplphysiol.00703.2016.

69.

Qaisar

Bhaskaran

Premkumar

Ranjit

Natarajan

Ahn

, et al. Oxidative stress-induced dysregulation of excitation-contraction coupling contributes to muscle weakness. J Cachexia Sarcopenia Muscle. 2018;9(5):1003-17. doi:10.1002/jcsm.12339.

70.

Morgan

Cowburn

Farrow

Carter

Cazzola

Walhin

, et al. Understanding the role of physical activity on the pathway from intra-articular knee injury to post-traumatic osteoarthritis disease in young people: a scoping review protocol. BMJ Open. 2023;13(3):e067147. doi:10.1136/bmjopen-2022-067147.

71.

Rajalekshmi

Agrawal

DK.

Advancing osteoarthritis research: insights from rodent models and emerging trends. J Orthop Sports Med. 2025;7(1):110-28. doi:10.26502/josm.511500187.

72.

Leite

CBG

Fricke

Tavares

Nshimiyimana

Mekhail

Kilgallen

, et al. Maresin 1-LGR6 axis mitigates inflammation and posttraumatic osteoarthritis after transection of the anterior cruciate ligament in mice. Osteoarthritis Cartilage. 2025;33(7):861-73. doi:10.1016/j.joca.2025.03.005.

73.

Racine

Aaron

RK.

Post-traumatic osteoarthritis after ACL injury. R I Med J. 2014;97(11):25-8.

74.

Cheung

DiLallo

Feeley

Lansdown

DA.

Osteoarthritis and ACL reconstruction-myths and risks. Curr Rev Musculoskelet Med. 2020;13(1):115-22. doi:10.1007/s12178-019-09596-w.

75.

Swärd

Frobell

Englund

Roos

Struglics

Cartilage and bone markers and inflammatory cytokines are increased in synovial fluid in the acute phase of knee injury (hemarthrosis)–a cross-sectional analysis. Osteoarthritis Cartilage. 2012;20(11):1302-8. doi:10.1016/j.joca.2012.07.021.

76.

Dwivedi

Flaman

Alaybeyoglu

Struglics

Frank

Chubinskya

, et al. Inflammatory cytokines and mechanical injury induce post-traumatic osteoarthritis-like changes in a human cartilage-bone-synovium microphysiological system. Arthritis Res Ther. 2022;24(1):198. doi:10.1186/s13075-022-02881-z.

77.

Cunha

Solomon

DJ.

ACL prehabilitation improves postoperative strength and motion and return to sport in athletes. Arthrosc Sports Med Rehabil. 2022;4(1):e65-e69. doi:10.1016/j.asmr.2021.11.001.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.21 MB