Abstract

   Surgical transection of the anterior cruciate ligament (ACL) in the
   porcine model leads to posttraumatic osteoarthritis if left untreated.
   However, a recently developed surgical treatment, bridge-enhanced ACL
   repair, prevents further cartilage damage. Since the synovial fluid
   bathes all the intrinsic structures of knee, we reasoned that a
   comparative analysis of synovial fluid protein contents could help to
   better understand the observed chondroprotective effects of the
   bridge-enhanced ACL repair. We hypothesized that post-surgical changes
   in the synovial fluid proteome would be different in the untreated and
   repaired knees, and those changes would correlate with the degree of
   cartilage damage. Thirty adolescent Yucatan mini-pigs underwent
   unilateral ACL transection and were randomly assigned to either no
   further treatment (ACLT, n = 14) or bridge-enhanced ACL repair (BEAR, n
   = 16). We used an isotopically labeled high resolution LC MS/MS-based
   proteomics approach to analyze the protein profile of synovial fluid at
   6 and 12 months after ACL transection in untreated and repaired porcine
   knees. A linear mixed effect model was used to compare the normalized
   protein abundance levels between the groups at each time point.
   Bivariate linear regression analyses were used to assess the
   correlations between the macroscopic cartilage damage (total lesion
   area) and normalized abundance levels of each of the identified
   secreted proteins. There were no significant differences in cartilage
   lesion area or quantitative abundance levels of the secreted proteins
   between the ACLT and BEAR groups at 6 months. However, by 12 months,
   greater cartilage damage was seen in the ACLT group compared to the
   BEAR group (p = 0.005). This damage was accompanied by differences in
   the abundance levels of secreted proteins, with higher levels of
   Vitamin K-dependent protein C (p = 0.001), and lower levels of
   Apolipoprotein A4 (p = 0.021) and Cartilage intermediate layer protein
   1 (p = 0.049) in the ACLT group compared to the BEAR group. There were
   also group differences in the secreted proteins that significantly
   changed in abundance between 6 and 12 months in ACLT and BEAR knees.
   Increased concentration of Ig lambda-1 chain C regions and decreased
   concentration of Hemopexin, Clusterin, Coagulation factor 12 and
   Cartilage intermediate layer protein 1 were associated with greater
   cartilage lesion area. In general, ACLT knees had higher concentrations
   of pro-inflammatory proteins and lower concentrations of
   anti-inflammatory proteins than BEAR group. In addition, the ACLT group
   had a lower and declining synovial concentrations of CILP, in contrast
   to a consistently high abundance of CILP in repaired knees. These
   differences suggest that the knees treated with bridge-enhanced ACL
   repair may be maintaining an environment that is more protective of the
   extracellular matrix, a function which is not seen in the ACLT knees.

Introduction

   Anterior cruciate ligament (ACL) injuries are has been linked to
   increased risk of posttraumatic osteoarthritis (OA) in humans and
   animal models [[34]1–[35]3]. Synovial fluid has been an attractive
   source to identify new biomarkers for monitoring joint health and a
   better understanding of the disease pathophysiology. This attraction is
   primarily due to the fact the synovial fluid bathes all the intrinsic
   structures of diarthrodial joints, including articular cartilage and
   synovium, both of which have shown to be actively involved in OA
   development [[36]4, [37]5]. Additionally, alterations in these
   structures due to OA may be directly reflected in the composition of
   synovial fluid, which could be correlated to disease severity and
   progression. Recent advances in high-throughput and sensitive mass
   spectrometry (MS)-based approaches have facilitated protein profiling
   of complex biological fluids including synovial fluid. As such, this
   technology has emerged as a powerful and reproducible technique to
   identify proteins involved in disease etiology and pathogenesis, as
   well as potential biomarkers for a range of diseases, including
   arthritis [[38]6–[39]11].

   Recently, a biologically augmented ACL repair procedure,
   bridge-enhanced ACL repair, has shown to be successful in reducing
   macroscopic evidence of posttraumatic OA following ACL injury in
   porcine knees [[40]12]. This new surgical technique uses a combination
   of a novel extracellular matrix-based scaffold to augment a suture
   repair of the torn ACL [[41]13]. Using the porcine ACL transection
   model, where the untreated knee progresses to posttraumatic OA and the
   repaired knee promotes cartilage preservation, allows us to compare
   these two different consequences of an identical surgical injury and
   potentially identify mechanisms and biomarkers for posttraumatic OA
   development.

   Compared to other animal models, the porcine knee has been shown to be
   closest to the human based on its size, anatomy and functional
   dependency on the ACL [[42]14]. Furthermore, porcine knee develops
   posttraumatic OA following ACL transection in a pattern similar to that
   reported in humans, but at a faster rate, with the joint changes at 1
   year reflective of those seen at 10–15 years after ACL reconstruction
   in humans [[43]12]. This faster onset of post-traumatic OA allows for
   more rapid assessment of factors that may influence the development of
   posttraumatic OA following ACL injury and treatment. The high degree of
   similarity between human and porcine synovial fluid [[44]15]. further
   justifies the porcine knee as a suitable model to study the biology of
   OA.

   In the current work, we used an isotopically labeled high resolution LC
   MS/MS-based proteomics approach to analyze the protein profile of
   synovial fluid at 6 and 12 months after ACL transection in untreated
   and repaired porcine knees. Our primary aim was to determine how the
   synovial fluid proteome differs between the two groups in an effort to
   identify candidate proteins that may be associated with the development
   of posttraumatic OA. We hypothesized that the development of
   macroscopic cartilage damage following surgical ACL transection would
   be accompanied by differential changes in synovial fluid proteome in
   untreated knees compared to repaired joints.

Materials and methods

Study design

   This study was approved by the Institutional Animal Care and Use
   Committee at Boston Children's Hospital. The study was designed as a
   randomized controlled large animal trial with cross-sectional outcome
   assessments at two post-injury time points. The study was designed as a
   randomized controlled large animal trial with cross-sectional outcome
   assessments at two post-injury time points. A total of 30 adolescent
   Yucatan mini-pigs (ages 14–16 months) underwent unilateral ACL
   transection and were randomly treated with bridge-enhanced ACL repair
   (BEAR, n = 16) or left untreated (ACLT, n = 14). Animals were obtained
   from LoneStar Laboratory Swine (Sioux Center, IA, USA). A computer
   based random permutation of the complete set of animal identifiers was
   used to randomize treatment allocation and side of unilateral surgery.
   Half of the animals within each treatment group were euthanized at 6
   months, while the other half was followed up to 12 months post-injury.
   The histological, biomechanical and OA-related outcomes for these
   animals have been previously reported by our group [[45]12, [46]16].
   For the current study, synovial fluid from an additional 6 healthy
   intact age-matched adolescent Yucatan minipigs was also used as a
   control.

Surgical procedure

   All animals were acclimated to the animal care facility environment for
   a minimum of 7 days prior to any surgical procedures. Animals allocated
   to ACLT or BEAR groups underwent ACL surgery under general anesthesia
   and postoperative housing as previously reported [[47]12, [48]16]. In
   brief, the ACL was transected surgically and for the animals assigned
   to BEAR group, the torn ACL was repaired using an extracellular
   matrix–based scaffold soaked with autologous blood as previously
   described [[49]17]. After surgery, all animals were housed in
   individualized pens and checked multiple times a day for 4 weeks. They
   were then shipped to a farm for long-term care (Coyote Consulting Corp
   Inc, Douglas, Massachusetts). Full weightbearing status was achieved
   within 48 to 72 hours and the animals were allowed ad libitum activity.
   Two animals (both in the ACLT group) developed subcutaneous abscesses
   near the jaw that were treated with short-term oral antibiotics. These
   animals were included in the study as there was no visual evidence of
   joint synovitis at the time of dissection. In the BEAR group, one
   animal (12 months) was shipped to the external holding facility at 2
   weeks rather than 4 weeks postoperatively, a deviation in the
   postoperative rehabilitation, thus it was excluded from the analysis.
   Also, one BEAR animal (6 months) had a missing synovial fluid sample,
   thus was excluded from this study. The total number of animals,
   analyzed in this study, was 7 in each group at each time point.

   After 6 and 12 months, synovial fluid was aspirated from the injured
   knee joints under anesthesia. For control animals, synovial fluid was
   obtained under anesthesia within 1 week of arrival to the animal care
   facility. Samples were centrifuged at 3,000g at room temperature for 10
   min and the supernatant stored at −80°C. Following aspiration, animals
   were euthanized and the knees were harvested and stored at -20°C.

Proteomics analysis

Sample preparation

   A multiplexed Isobaric Tag for Relative and Absolute Quantitation
   (iTRAQ) [[50]18] coupled with liquid chromatography with tandem mass
   spectrometry (LC-MS/MS) was used to identify the changes in synovial
   fluid protein levels. iTRAQ is a chemical labeling method, which uses
   multiplexed isobaric tagging reagent to label peptide mixtures which
   allows post label mixing of the samples (after enzymatic digestion)
   without adding complexity to the MS analysis [[51]19]. This will help
   minimizing the quantitation variability during sample preparation prior
   to LC MS/MS data collection. The iTRAQ-based quantitative proteomics
   approach has been widely used to identify biomarkers for several
   diseases including end stage OA and rheumatoid arthritis [[52]7, [53]8,
   [54]11].

   Comparison of the peak areas and resultant peak ratios for MS/MS
   reporter ions was used to measure the relative abundance of proteins.
   Synovial fluid from each individual sample was reduced with DTT,
   alkylated with iodoacetamide, and digested with trypsin at 37°C
   overnight. Digested samples were separated across five different sets
   for comparison with each set consisting of seven unique samples ([55]S1
   Table). A pooled sample consisting of equal amount of proteins from all
   35 total samples was made to serve as a common eighth sample across the
   five different sets of comparison. Per each comparison set, equal
   amounts of total peptides were aliquoted and labeled with the 8-plex
   iTRAQ reagents (iTRAQ Reagents Multiplex kit, Applied Biosystems, CA),
   then pooled, dried and stored until analyzed by mass spectrometry.
   Total digested peptides in each sample were quantified by Amino Acid
   Analysis (AAA; Hitachi Model L-8900) prior to iTRAQ labeling to ensure
   equal amounts of total peptides for each sample.

LC-MS/MS analysis

   LC-MS/MS analysis of the iTRAQ labeled samples peptides were carried
   out using AB SCIEX TripleTOF 5600 mass spectrometer (SCIEX, Framingham,
   MA) coupled to a NanoACQUITY UPLC system (Waters Inc., Milford, MA).
   The dried peptide samples were dissolved in 70% formic acid/water,
   diluted with 13 μl 0.1% trifluoroacetic acid (TFA) and quantitated at
   A280 absorption on a Nanodrop (ThermoFisher Scientific, Wilmington,
   DE). Peptides were loaded on a NanoACQUITY Symmetry C18 UPLC Trap
   column, washed in 9% Buffer A (0.1% formic acid in water) then run for
   160 minutes over a linear gradient 99%A to 65% A at 500 nL/min. The
   parameters utilized on the 5600 Triple-TOF were: 2300 V IonSpray
   Voltage Floating, Ion Source Gas 1 of 10, Curtain Gas of 20, Interface
   Heater Temperature 120, with a declustering potential of 60. MS cycle
   time was 250 milliseconds, with a maximum 20 MS/MS spectra taken each
   with a 100-millisecond accumulation time, and collision energy adjusted
   for iTRAQ reagents.

Protein abundance level calculation and analysis

   The ProteinPilot peptide summary was filtered to only include peptides
   with zero mis-cleavages, high confidence peptide identifications, and
   two or more iTRAQ reporter ion ratios measured. We then performed a
   cyclic Lowess normalization [[56]20] on the remaining peptides to
   compensate for any differences between iTRAQ labels. All peptides
   across the 5 sets were merged and normalized to the common pooled
   sample. This was done by subtracting the common pooled sample,
   replicated in each set, from the other 7 samples in each set. The
   purpose of this adjustment is to remove set-specific effects to
   facilitate comparisons between the experimental groups (i.e. Intact,
   ACLT and BEAR) and time points (i.e. 6 and 12 months) across all the
   sets. A linear mixed effect model was used to compare the normalized
   protein abundance levels between the groups at each time point (limma
   package in R v3.3.1, The R Foundation for Statistical Computing)
   [[57]21]. Benjamini and Hochberg method was used to adjust the p-values
   to control the false discovery rate [[58]22]. P≤0.05 considered
   statistically significant and used to select differentially abundant
   proteins between the groups (i.e. Intact, ACLT 6 months, ACLT 12
   months, BEAR 6 months and BEAR 12 months). Bivariate linear regression
   analyses were used to assess the correlations between the macroscopic
   cartilage damage (total lesion area assessed at 6 or 12 months after
   ACLT and BEAR) and normalized abundance levels of each identified
   secreted proteins.

Pathway enrichment analysis

   The differentially abundant proteins were used to analyze the
   enrichment of specific pathways using the Process Networks ontology in
   the MetaCore bioinformatics suite (Thomson Reuters) [[59]23]. Sus
   scrufa Ensembl identifiers and, if not recognized, gene symbols of the
   corresponding gene for each protein were used for as input for the
   analysis. Pathways were considered significantly enriched when they
   contain at least 2 unique proteins and adjusted P values (false
   discovery rate; FDR) were less than 0.05.

Results

   As previously reported, macroscopic cartilage damage was observed in
   the ACLT knees, primarily across the medial femoral condyle [[60]12].
   While at 6 months no difference in total cartilage lesion area was
   observed between ACLT and BEAR knees, ACLT knees had a significantly
   larger total lesion area at 12 months compared to the BEAR knees
   ([61]Fig 1) [[62]12].

Fig 1. Development of macroscopic cartilage damage following ACL transection.

   [63]Fig 1
   [64]Open in a new tab

   Total cartilage lesion area at 6 and 12 months after untreated ACL
   injury (ACLT) and bridge-enhanced ACL repair (BEAR). Data is presented
   as Mean ± SD. P value is derived from a one-way ANOVA with posthoc
   Bonferroni correction for multiple comparisons (adopted and modified
   with permission from Murray MM and Fleming BC, Am J Sports Med 2013
   [[65]12]) Macroscopic cartilage damage was assessed by measuring the
   cartilage lesions across the femoral condyles and tibial plateau in
   medial and lateral compartments using India ink staining and calipers
   [[66]12].

Most abundant secreted proteins found in the synovial fluid after ACL
transection

   A total of 1,340 unique peptides corresponding to 232 proteins were
   identified across all 34 samples ([67]S2 Table). Of the 232 detected
   proteins, 80 (34%) were secreted extracellular proteins. The
   subcellular localization patterns of the identified proteins were
   similar to those reported in knee synovial fluid proteins of humans
   with knee OA [[68]24]. The top 20 most abundant secreted proteins in
   the synovial fluid of each surgical group are listed in [69]Table 1. Of
   the 232 detected proteins, 65 (28%) were significantly differentially
   abundant in at least one of the comparisons between the experimental
   groups (i.e. ACLT, BEAR and Intact) and / or between the time points
   ([70]S3 Table), and 33 (51%) of these were secreted extracellular
   proteins.

Table 1. Top 20 most abundant secreted proteins detected in the synovial
fluid in each surgical group.

   The uniquely abundant proteins in each group are highlighted in bold.
   Abundant Proteins in the ACLT Group at 6 Months
   Rank Protein Gene Rank Protein Gene
   1 Peptidylprolyl isomerase A PPIA 11 Serotransferrin TF
   2 Vitamin K-dependent protein C PROC 12 Aggrecan core protein ACAN
   3 Lactadherin MFGE8 13 Corticosteroid-binding globulin SERPINA6
   4 Cartilage intermediate layer protein 1 CILP 14
   Alpha-2-HS-glycoprotein AHSG
   5 Coagulation Factor 12 F12 15 Protein AMBP AMBP
   6 Glucose-6-phosphate isomerase GPI 16 Hemopexin HPX
   7 Alpha-1-antitrypsin SERPINA1 17 Complement component C7 C7
   8 Pituitary adenylate cyclase-activating polypeptide ADCYAP1 18 Serum
   albumin ALB
   9 Haptoglobin HP 19 Apolipoprotein E APOE
   10 Inhibitor of carbonic anhydrase ICA 20 Clusterin CLU
   Abundant Proteins in the BEAR Group at 6 Months
   Rank Protein Gene Rank Protein Gene
   1 Peptidylprolyl isomerase A PPIA 11 Serotransferrin TF
   2 Vitamin K-dependent protein C PROC 12 Complement component C7 C7
   3 Plasminogen activator inhibitor 1 SERPINE1 13 Apolipoprotein C3 APOC3
   4 Cartilage intermediate layer protein 1 CILP 14 Apolipoprotein E APOE
   5 Lactadherin MFGE8 15 Inhibitor of carbonic anhydrase ICA
   6 Glucose-6-phosphate isomerase GPI 16 Serum albumin ALB
   7 Coagulation Factor 12 F12 17 Protein AMBP AMBP
   8 Corticosteroid-binding globulin SERPINA6 18 Hemopexin HPX
   9 Alpha-1-antitrypsin SERPINA1 19 Alpha-2-HS-glycoprotein AHSG
   10 Elafin PI3 20 Clusterin CLU
   Abundant Proteins in the ACLT Group at 12 Months
   Rank Protein Gene Rank Protein Gene
   1 Cystatin-B CSTB 11 Hyaluronan and proteoglycan link protein 1 HAPLN1
   2 Protein S100-A12 S100A12 12 Peptidylprolyl isomerase A PPIA
   3 Complement C5 C5 13 Aggrecan core protein ACAN
   4 Lactotransferrin LTF 14 Alpha-1-antitrypsin SERPINA1
   5 von Willebrand factor VWF 15 Cathepsin D CTSD
   6 Vitamin K-dependent protein C PROC 16 Prothrombin F2
   7 Prophenin-2 17 Complement factor D CFD
   8 Insulin-like growth factor-binding protein 2 IGFBP2 18 Prophenin-1
   9 Complement C1q subcomponent subunit A C1QA 19 Decorin DCN
   10 Chitinase-3-like protein 1 CHI3L1 20 Thyroxine-binding globulin
   SERPINA7
   Abundant Proteins in the BEAR Group at 12 Months
   Rank Protein Gene Rank Protein Gene
   1 Hyaluronan and proteoglycan link protein 1 HAPLN1 11 Protein S100-A12
   S100A12
   2 Cystatin-B CSTB 12 Complement C1q subcomponent subunit A C1QA
   3 Cartilage intermediate layer protein 1 CILP 13 Complement factor D
   CFD
   4 von Willebrand factor VWF 14 Apolipoprotein A4 APOA4
   5 Annexin A2 ANXA2 15 Insulin-like growth factor-binding protein 2
   IGFBP2
   6 Complement C5 C5 16 Clusterin CLU
   7 Cathepsin D CTSD 17 Alpha-2-HS-glycoprotein AHSG
   8 Apolipoprotein M APOM 18 Prophenin-1
   9 Annexin A1 ANXA1 19 Alpha-1-antitrypsin SERPINA1
   10 Aggrecan core protein ACAN 20 Lactotransferrin LTF
   [71]Open in a new tab

Treatment related changes in synovial fluid proteome

   At 6 months, a total of 27 secreted proteins were significantly
   differentially abundant between intact group and surgical groups (ACLT
   and/or BEAR; [72]Table 2). There were no significant differences in the
   normalized abundance levels of the detected secreted proteins between
   the ACLT and BEAR groups at 6 months.

Table 2. Secreted proteins that are significantly different in abundance in
ACLT and/or BEAR knees compared to intact group at 6 months.

   Fold change values represent the ratio of detection in the ACLT or BEAR
   group versus intact group; thus a fold change greater than 1 reflects
   higher protein abundance in the synovial fluid of the ACLT or BEAR
   group and a fold change less than 1 reflects higher protein abundance
   in the synovial fluid of the intact group.
   Protein Gene ACLT / Intact BEAR / Intact
   Fold Change P-Value Fold Change P-Value
   Differentially Abundant Proteins in both ACLT and BEAR Groups Compared
   to Intact
   Vitamin K-dependent protein C PROC 6 1.03E-06 7.35 6.91E-08
   Cartilage intermediate layer protein 1 CILP 3.82 5.51E-03 5.35 8.34E-04
   Coagulation Factor XII F12 2.71 2.74E-04 2.67 3.34E-04
   Alpha-1-antitrypsin SERPINA1 1.62 8.92E-04 1.56 1.80E-03
   Clusterin CLU 1.6 6.52E-05 1.65 2.66E-05
   Inhibitor of carbonic anhydrase ICA 1.6 1.02E-04 1.47 8.87E-04
   Hemopexin HPX 1.59 2.16E-04 1.52 6.56E-04
   Serotransferrin TF 1.42 6.18E-05 1.5 7.23E-06
   Apolipoprotein A4  APOA4 1.38 2.59E-02 1.63 1.22E-03
   Protein AMBP AMBP 1.37 1.23E-02 1.31 2.99E-02
   Serum albumin ALB 1.13 3.68E-02 1.18 4.55E-03
   Transthyretin TTR 0.83 2.77E-02 0.79 8.35E-03
   Inter-α trypsin inhibitor heavy chain 2 ITIH2 0.8 1.99E-02 0.76
   5.95E-03
   Complement factor B CFB 0.69 3.64E-02 0.72 4.43E-02
   Ig lambda-1 chain C regions IGLC1 0.59 2.36E-03 0.59 2.16E-03
   Complement factor D CFD 0.58 3.05E-04 0.61 1.17E-03
   Ficolin-2 FCN2 0.39 5.27E-03 0.54 3.98E-02
   Differentially Abundant Proteins in ACLT Compared to Intact
   Haptoglobin HP 3.21 8.33E-04
   Coagulation factor V F5 0.09 4.33E-02
   Differentially Abundant Proteins in BEAR Compared to Intact
   Peptidylprolyl isomerase A PPIA 11.53 1.74E-02
   Apolipoprotein C3 APOC3 1.75 4.57E-03
   Apolipoprotein E APOE 1.37 2.13E-02
   Apolipoprotein A1 APOA1 1.33 2.08E-02
   Prothrombin F2 0.73 1.13E-02
   Vitronectin VTN 0.71 5.80E-03
   [73]Open in a new tab

   At 12 months, a total of 16 secreted proteins were significantly
   differentially abundant between intact group and surgical groups (ACLT
   and/or BEAR; [74]Table 3). Compared to the BEAR group, the ACLT group
   had higher normalized abundance levels of vitamin K-dependent protein C
   (by 2.4 fold, P = 0.00126) along with lower normalized abundance levels
   of apolipoprotein A4 (by 0.7 fold, P = 0.0213) and cartilage
   intermediate layer protein (by 0.35 fold, P = 0.049).

Table 3. Secreted proteins that are significantly different in abundance in
ACLT and/or BEAR knees compared to intact group at 12 months.

   Fold change values represent the ratio of detection in the ACLT or BEAR
   group versus intact group; thus a fold change greater than 1 reflects
   higher protein abundance in the synovial fluid of the ACLT or BEAR
   group and a fold change less than 1 reflects higher protein abundance
   in the synovial fluid of the intact group.
   Protein Gene ACLT / Intact BEAR / Intact
   Fold Change P-Value Fold Change P-Value
   Differentially Abundant Proteins in both ACLT and BEAR Groups Compared
   to Intact
   Trypsin-1 PRSS1 2.23 1.06E-02 2.24 8.52E-03
   Haptoglobin HP 1.96 4.03E-02 1.91 4.32E-02
   Actin beta ACTB 1.52 1.58E-02 1.65 3.55E-03
   Clusterin CLU 1.43 1.28E-03 1.7 7.33E-06
   Apolipoprotein A4  APOA4 1.36 3.17E-02 1.86 5.79E-05
   Apolipoprotein A1 APOA1 1.29 3.71E-02 1.43 3.90E-03
   Vitronectin VTN 0.77 3.28E-02 0.64 4.63E-04
   Inter-α trypsin inhibitor heavy chain family member 4 ITIH4 0.63
   6.81E-03 0.55 6.32E-04
   Differentially Abundant Proteins in ACLT Compared to Intact
   Serotransferrin TF 1.2 2.14E-02
   Differentially Abundant Proteins in BEAR Compared to Intact
   Cartilage intermediate layer protein CILP 2.83 5.01E-02
   Annexin A1 ANXA1 2.48 4.36E-02
   Plasminogen PLG 0.84 4.88E-02
   Inter-α trypsin inhibitor heavy chain 1 ITIH1 0.8 1.69E-02
   Inter-α trypsin inhibitor heavy chain 2 ITIH2 0.8 1.67E-02
   Ig lambda-1 chain C regions IGLC1 0.68 1.73E-02
   Vitamin K-dependent protein C PROC 0.44 1.99E-03
   [75]Open in a new tab

   There were 20 secreted synovial fluid proteins with significant fold
   changes between the 6 to 12-month time points in ACLT and/or BEAR knees
   ([76]Table 4). Nine proteins had significant fold changes from 6 to 12
   months after surgery in both groups. Five proteins had a significant
   fold change from 6 to 12 months only after untreated ACL transection.
   Six proteins had a significant fold change from 6 to 12 months only
   after bridge-enhanced ACL repair.

Table 4. Secreted proteins that are significantly different in abundance
between 6 and 12 months after surgery.

   Differentially abundant in both ACLT and BEAR
   Protein Gene ACLT (12 / 6 months) BEAR (12 / 6 months)
   Fold Change P-Value Fold Change P-Value
   Increased from 6 months to 12 months
   Complement factor D CFD 1.80 8.97E-05 1.81 4.98E-05
   Transthyretin TTR 1.29 3.36E-03 1.37 2.69E-04
   Decreased from 6 months to 12 months
   Serotransferrin TF 0.85 2.93E-02 0.72 5.78E-05
   Serum albumin ALB 0.85 4.13E-03 0.76 1.05E-05
   Inhibitor of carbonic anhydrase ICA 0.74 5.26E-03 0.77 1.09E-02
   Apolipoprotein E APOE 0.73 1.61E-02 0.68 3.14E-03
   Protein AMBP AMBP 0.73 9.04E-03 0.69 2.07E-03
   Coagulation Factor 12 F12 0.60 3.46E-02 0.55 1.18E-02
   Vitamin K-dependent Protein C PROC 0.17 1.47E-06 0.06 8.04E-10
   Differentially abundant only in ACLT
   Protein Gene ACLT (12 / 6 months)
   Fold Change P-Value
   Increased from 6 months to 12 months
   Insulin-like growth factor-binding protein 2 IGFBP2 1.80 2.63E-02
   Ig lambda-1 chain C regions IGLC1 1.45 2.20E-02
   Thyroxine-binding globulin SERPINA7 1.45 1.24E-02
   Decreased from 6 months to 12 months
   Hemopexin HPX 0.72 4.98E-03
   Cartilage intermediate layer protein 1 CILP 0.26 5.46E-03
   Differentially abundant only in BEAR
   Protein Gene BEAR (12 / 6 months)
   Fold Change P-Value
   Increased from 6 months to 12 months
   Annexin A1 ANXA1 3.82 2.81E-03
   Decreased from 6 months to 12 months
   Alpha-1-antitrypsin SERPINA1 0.73 1.51E-02
   Complement component C7 C7 0.58 2.73E-02
   Apolipoprotein C3 APOC3 0.56 1.87E-03
   Leukocyte elastase inhibitor SERPINB1 0.42 3.84E-02
   Peptidylprolyl isomerase A PPIA 0.07 6.66E-03
   [77]Open in a new tab

   Differentially abundant proteins enriched 7 biological pathways
   (Process Network ontology) including terms related to blood
   coagulation, proteolysis, inflammation and cell adhesion ([78]Table 5).

Table 5. Biological pathways enriched by differentially abundant proteins at
6 months.

   Pathway FDR Proteins
   Blood Coagulation 1.18E-04 Vitamin K-dependent protein C, Prothrombin,
   Plasminogen, Coagulation Factor V, Coagulation Factor XII,
   Alpha-1-antitrypsin
   Proteolysis (ECM Remodeling) 3.50E-04 Plasminogen, Prothrombin,
   Vitronectin, Coagulation Factor XII, Trypsin I, Clusterin,
   Alpha-1-antitrypsin
   Inflammation (Kallikrein-Kinin System) 7.63E-04 Inter-α trypsin
   inhibitor heavy chain
   family member 4, Prothrombin, Plasminogen, Coagulation Factor V,
   Coagulation Factor XII, Alpha-1-antitrypsin
   Inflammation (Protein C Signaling) 7.63E-04 Vitamin K-dependent protein
   C, Actin Beta, Prothrombin, Plasminogen, Coagulation Factor V
   Inflammation (Complement System) 7.63E-04 Complement Component C7,
   Ficolin, Clusterin, Complement Factor B, Complement Factor D
   Cell Adhesion (Platelet-Endothelium-Leucocyte Interactions) 1.79E-03
   Vitamin K-dependent protein C, Prothrombin, Plasminogen, Coagulation
   Factor V, Vitronectin, Coagulation Factor XII
   Proteolysis (Connective Tissue Degradation) 4.51E-02 Plasminogen,
   Vitronectin, Trypsin 1, Alpha-1-antitrypsin
   [79]Open in a new tab

Associations between protein levels and total area of macroscopic articular
cartilage damage

   Among all the identified secreted proteins, notable associations (R^2 ≥
   0.1) were only found between the total area of the macroscopic
   cartilage damage across the femoral condyles and tibial plateau and the
   normalized abundance levels of Ig lambda-1 chain C regions (r = 0.52),
   Hemopexin (r = -0.40), Clusterin (r = -0.35), Coagulation factor 12 (r
   = -0.32) and Cartilage intermediate layer protein 1 (r = -0.33);
   [80]Fig 2.

Fig 2. Associations between macroscopic cartilage damage and secreted
proteins concentrations.

   [81]Fig 2
   [82]Open in a new tab

   Correlations between macroscopic cartilage damage area and the
   normalized abundance levels of (A) Ig lambda-1 chain C regions, (B)
   Hemopexin, (C) Clusterin, (D) Coagulation factor 12 and (E) Cartilage
   intermediate layer protein 1 for both ACLT and BEAR groups at 6 and 12
   months.

Discussion

   The results of this study support our hypothesis that development of
   macroscopic cartilage damage following an ACL injury and subsequent
   surgery are accompanied with changes in synovial fluid proteome with
   different responses in untreated knees compared to repaired joints.
   There was no significant difference in cartilage lesion area or
   quantitative protein abundance levels between the two treatment groups
   at 6 months. However, by 12 months after surgery, there was
   significantly greater cartilage damage in the knees with an untreated
   ACL transection than in those knees that underwent bridge-enhanced ACL
   repair ([83]Fig 1). This damage was accompanied by differences in the
   proteome between the groups at 12 months, with the ACLT group having a
   higher concentration of Vitamin K-dependent Protein C (2.4 fold), and a
   decreased content of Apolipoprotein A4 (0.7 fold) and Cartilage
   intermediate layer protein 1 (0.35 fold) when compared to the BEAR
   knees. In addition, there was also a difference in the proteins which
   significantly changed in abundance between 6 and 12 months ([84]Table
   4), with Insulin-like growth factor-binding protein 2 (IGFBP2), Ig
   lambda-1 chain C regions (IGLC1), Hemopexin and CILP changing in the
   ACLT group, and significant changes in Annexin A1, alpha-1 antitrypsin,
   Complement C7, Apolipoprotein C3, Leukocyte elastase inhibitor and
   Peptidylprolyl isomerase A (PPIA) found in the BEAR group. Increased
   levels of IGLC1 and decreased levels of Hemopexin, Clusterin,
   Coagulation factor 12 and CILP were also found to correlate with
   greater cartilage lesion area when the knees from both groups were
   pooled ([85]Fig 2).

   While there were no statistically significant differences in protein
   abundance between the surgical groups at 6 months after surgery, the
   makeup of the synovial fluid proteome, and specifically the top 20 most
   abundant proteins in the fluid, were similar but not identical in the
   two surgical groups. Seventeen of the 20 most abundant secreted
   proteins ([86]Table 1) were the same in ACLT and BEAR groups, 15 of
   which have been previously reported to be abundant in synovial fluid of
   human knees with OA [[87]8, [88]11, [89]24]. For the six differences
   (three in each group) in the top 20 secreted proteins, the ACLT knees
   had 2 additional pro-inflammatory proteins (Haptoglobin and Pituitary
   adenylate cyclase-activating polypeptide) while the BEAR group had two
   protease inhibitors at 6 months (Plasminogen activator inhibitor 1 and
   Elafin). The pro-inflammatory role of Haptoglobin, Pituitary adenylate
   cyclase-activating polypeptide (PACAP), Plasminogen activation and
   Elastase are well documented [[90]25–[91]28]. High synovial fluid
   concentrations of Haptoglobin, Plasminogen (or Plasmin) and Elastase
   have been reported in arthritic knees [[92]11, [93]24, [94]29–[95]31].
   PACAP-deficiency in mice has shown to result in lower inflammation in
   early arthritis followed by increased immune cell function and bone
   formation (e.g. osteophytes) in late arthritis [[96]32]. The presence
   of Haptoglobin and PACAP among the most abundant proteins in the ACLT
   group in addition to the presence of Plasminogen activator inhibitor 1
   and Elafin among the most abundant proteins in the BEAR group at 6
   months might be indicative of a more pro-inflammatory environment in
   ACLT knees at early stages of the disease. Further studies would be
   needed to validate this hypothesis.

   In addition to proteases and protease inhibitors, Apolipoprotein C3 and
   Aggrecan core protein (Aggrecan) were the other two secreted proteins
   which were uniquely present in the BEAR or ACLT top 20 abundance lists,
   respectively, at 6 months ([97]Table 1). Apolipoprotein C3 has
   previously been identified in the synovial fluid of human knees with OA
   [[98]24], and its plasma levels have been found to be elevated in
   patients with non-progressing radiographic knee OA compared to those
   who progress [[99]33]. This is reflected by our finding of
   Apolipoprotein C3 among the most abundant proteins in the
   non-progressive BEAR group and not in the progressive ACLT group at 6
   months ([100]Table 1). Aggrecan has also been previously detected in
   synovial fluid of knees with OA [[101]11, [102]24]; however, in this
   case, increased Aggrecan in the synovial fluid has been associated with
   greater cartilage damage in osteoarthritic joints [[103]34–[104]37].
   This is reflected in our study where Aggrecan was among the most
   abundant proteins in the ACLT group and not in BEAR or intact groups
   ([105]Table 1).

   At 12 months after surgery, there were significant differences in
   protein abundance levels between the BEAR and ACLT groups, with an
   increase in Vitamin K-dependent protein C and decreases in
   Apolipoprotein A4 and CILP seen in the ACLT group. Vitamin K-dependent
   protein C is an activator of several matrix metalloproteinases (MMPs),
   including MMP2, 9 and 13, and has shown to be significantly elevated in
   the synovium and synovial fluid of patients with rheumatoid arthritis
   (RA) and OA [[106]38]. While Apolipoprotein A4 has been detected in the
   synovial fluid of human knees with OA [[107]8, [108]24], their role in
   OA pathogenesis is not well understood and warrants further studies.
   Considering recent reports on the role of lipid metabolism in the OA
   development [[109]39, [110]40], it is possible that apolipoprotein
   proteins affects knee OA progression by regulating the lipid metabolism
   and efflux. CILP is a large secreted glycoprotein with major role in
   cartilage scaffolding and new cartilage formation [[111]41]. Increased
   CILP synthesis by chondrocytes has also been reported in early-stage OA
   [[112]42, [113]43]. which can be indicative of joint response to repair
   damaged cartilage [[114]44]. The consistently higher abundance of CILP
   in BEAR knees compared to intact (at 6 and 12 months) and ACLT (at 12
   months) knees may suggest a more chondroprotective environment in
   repaired knees (Tables [115]2 & [116]3).

   In addition, at 12 months, the top 20 most abundant proteins in the
   ACLT knees had 5 additional pro-inflammatory proteins (Vitamin
   K-dependent protein C, Prothrombin, PPIA and Chitinase-3-like protein
   1) and 3 fewer anti-inflammatory proteins (Clusterin, Alpha-2-HS
   glycoprotein and Annexin A1; [117]Table 1). The pro-inflammatory role
   of Vitamin K-dependent protein C, Prothrombin, PPIA and
   Chitinase-3-like protein 1, as well as anti-inflammatory role of
   Clusterin, Alpha-2-HS glycoprotein (Fetuin-A) and Annexin A1 in
   arthritic joints are well documented [[118]25–[119]28,
   [120]45–[121]61]. Elevated levels of these pro-inflammatory proteins
   have been reported in serum and synovial fluid of patients with RA and
   OA [[122]8, [123]24, [124]49, [125]62–[126]65]. Prophenin-2 is a
   porcine specific Cathelicidin antimicrobial peptide, with similar
   pro-inflammatory properties as LL-37 (the only identified Cathelicidin
   antimicrobial peptide in humans), which is regulated by macrophages,
   polymorphonuclear leukocytes, and keratinocytes [[127]66]. A recent
   study has shown strong overexpression of Cathelicidin in joints of
   patients with RA and rats with pristane-induced arthritis [[128]67]. A
   recent proteomic analysis has also reported LL-37 among identified
   synovial fluid proteins in the patients with knee OA [[129]24]. The
   presence of pro-inflammatory proteins among the most abundant proteins
   in the ACLT group and the anti-inflammatory proteins among the most
   abundant proteins in the BEAR group at 12 months suggests there may be
   a greater pro-inflammatory environment in ACLT knees at a later stage
   of the disease. This is further supported by the observed significantly
   lower abundance of the Vitamin K-dependent protein C in BEAR knees
   compared to ACLT and intact groups as well as higher abundance of
   Annexin A1 in the BEAR knees compared to intact group.

   In comparing the proteomes at 6 and 12 months after surgery ([130]Table
   4), there were significant increases in IGFBP2 and IGLC1 levels, as
   well as significant decreases in CILP and Hemopexin levels in the
   synovial fluid of ACLT knees from 6 to 12 months. IGFP2 is synthesized
   by synoviocytes and chondrocytes and regulates Insulin-like growth
   factor (IGF) [[131]68, [132]69], a key promoter of cartilage growth
   [[133]70, [134]71]. Increased levels of IGFBPs have been reported in
   the synovial fluid of arthritic joints [[135]72, [136]73], and serum
   concentration has been correlated with RA disease severity
   [[137]74–[138]76]. Hemopexin has a negative modulatory function on
   T-cell differentiation [[139]77, [140]78], and exogenous administration
   of Hemopexin has recently been shown to result in increased
   glycosaminoglycan deposition [[141]79]. In contrast to increased
   synovial concentrations of pro-inflammatory proteins (IGFBP2 and IGLC1)
   in the ACLT knees, there were significant decreases in the synovial
   concentrations of two pro-inflammatory proteins (PPIA and complement
   C7) in the BEAR knees from 6 to 12 months.

   Biological pathways enriched by the changes in synovial fluid protein
   content were related to blood coagulation, proteolysis, inflammation
   and cell adhesion ([142]Table 5). Among those, proteolysis (ECM
   remodeling) and inflammation (complement pathway) pathways have
   consistently shown to play an active role in OA pathophysiology
   [[143]8, [144]80–[145]83]. The role of remaining 5 enriched pathways in
   the development of OA is less studied. Recent comparative analysis of
   synovial fluid in patients with healthy knees with those with knee OA
   (both early and late stages) have shown significant enrichment of blood
   coagulation pathway by differentially abundant proteins between the
   groups [[146]8]. Our group has also shown enrichment of cell adhesion
   (platelet-endothelium-leucocyte interactions) and proteolysis
   (connective tissue degradation) pathways by differentially expressed
   genes in the synovium and cartilage within 2 weeks after ACL injury in
   a minipig model of posttraumatic OA [[147]80, [148]81]. These findings,
   in addition to abovementioned evidence supporting the involvement of
   the proteins / genes, which enriched these pathways, in OA
   pathophysiology underscore their importance as potential targets to
   mediate the biologic joint response to injury in an effort to lower the
   risk of posttraumatic OA.

Limitations

   Unbiased LC MS/MS proteomics techniques are primarily limited to
   detection of more highly abundant proteins within a complex biological
   matrix. Antibody-based depletion methods could have been used to
   improved our ability to quantify lower abundance level proteins by
   removing the top abundance level proteins. However, they can also
   result in removal of lower abundance level proteins and ultimately pose
   challenges in accurate quantification of protein contents [[149]84]. In
   our discovery workflow, we wanted to capture a “global” representation
   of proteins in the synovial fluid, as such chose to avoid
   antibody-based depletion. To better compensate and delve deeper into
   the synovial fluid proteome, additional in-depth and/or targeted
   approaches are required to further study less abundant proteins that
   are likely present but not detectable or quantifiable using this
   technique (i.e. Lubricin, matrix metalloproteinases and cytokines). We
   have previously used similar MS-based proteomics approach to study the
   synovial fluid proteome of healthy porcine knees [[150]15]. The
   substantial overlap between those findings and identified proteins here
   is reassuring and supports our MS-based protein discovery approach.
   However, further efforts are essential for orthogonal validation of the
   identified proteins in individual samples compared to the pooled
   samples in independent cohorts. Another limitation is the
   cross-sectional nature of the study. While the cross-sectional time
   points have enabled us to macroscopically assess the cartilage health,
   longitudinal analyses are required to better characterize the changes
   in synovial fluid proteome in response to joint degeneration over time.
   Finally, the current results only support associations between
   cartilage damage and certain synovial fluid proteins in early
   posttraumatic OA. Further functional studies are required to
   investigate the mechanistic nature of these associations and to
   determine the potential application of suggested targets as biomarkers
   for OA progression or as disease modifiers for development of novel
   therapeutics.

Conclusions

   Knees treated with a surgical procedure that results in posttraumatic
   OA (ACLT group) had higher concentrations of pro-inflammatory proteins
   and lower concentrations of anti-inflammatory proteins than knees
   treated with a surgical procedure that does not result in
   post-traumatic OA (bridge-enhanced ACL repair). In addition, the group
   developing posttraumatic OA had a lower and declining synovial
   concentrations of CILP, in contrast to a consistently high abundance of
   CILP in repaired knees. These differences suggest that the knees
   treated with bridge-enhanced ACL repair may be maintaining an
   environment that is more protective of the extracellular matrix, a
   function which is not seen in the ACLT knees.

Supporting information

   S1 Table. iTRAQ labeling of the synovial fluid samples.

   (XLSX)
   [151]Click here for additional data file.^ (10KB, xlsx)
   S2 Table. Detected synovial fluid proteins.

   (XLSX)
   [152]Click here for additional data file.^ (40.5KB, xlsx)
   S3 Table. Significant differentially abundant proteins between groups.

   (XLSX)
   [153]Click here for additional data file.^ (43.4KB, xlsx)
   S1 File. ARRIVE guidelines checklist.

   (PDF)
   [154]Click here for additional data file.^ (1.1MB, pdf)

Acknowledgments