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. Author manuscript; available in PMC: 2013 Dec 1.
Published in final edited form as: Arthritis Rheum. 2012 Dec;64(12):3993–4003. doi: 10.1002/art.37682

The biophysical mechanisms of altered hyaluronan concentration in synovial fluid after anterior cruciate ligament transection

William J McCarty 1, Justin C Cheng 1, Bradley C Hansen 1, Tomonori Yamaguchi 2, Gary S Firestein 3, Koichi Masuda 2, Robert L Sah 1,2,4
PMCID: PMC3510334  NIHMSID: NIHMS402884  PMID: 22933328

Abstract

Objectives

The residence time of hyaluronan (HA) in the synovial fluid (SF) of knee joints was investigated using the rabbit anterior cruciate ligament transection (ACLT) model. The aims were to assess at 7 and 28 days after surgery for non-operated (NonOp), ACLT, and SHAM groups: 1) HA molecular mass (Mr) distribution in SF, 2) endogenous replenishment of HA after saline washout, 3) HA residence times in SF, and 4) synovium and subsynovium cellularity.

Methods

Adult NZW rabbits underwent ACLT or SHAM surgeries on one hind limb, while each contralateral limb was a NonOp control. At 7 or 28d after surgery, joints were aspirated for SF, lavaged with saline, injected with saline or polydisperse HA, and sampled over 8hrs. Fluid samples were analyzed for HA concentration and Mr distribution to calculate HA residence times.

Results

HA Mr-distributions showed 1) loss of high-Mr HA at day 7, and a shift towards a lower-Mr HA distribution at day 28, 2) endogenous replenishment of high-Mr HA after washout, and 3) Mr-dependent HA loss, particularly at day 7 after ACLT. The residence time of HA decreased with Mr (~27hrs for 7000–2500kDa to ~7hrs for 250–50kDa) and at day 7 after ACLT (~70% decrease). The subsynovium of ACLT joints contained 4) increased cellularity and neovascularization at 7 and 28 days.

Conclusions

The residence time of HA in SF is transiently decreased after ACLT, suggesting a biophysical transport mechanism for the altered SF composition post-injury or during inflammation.

Keywords: hyaluronan, synovial fluid, synovitis, ACLT, transport, lubricant

Introduction

Synovial fluid (SF) is an ultrafiltrate of plasma with additional molecules secreted by local cell populations. Hyaluronan (HA) is a major component of SF that is secreted in high molecular mass (Mr) form (~4–6MDa, ~2–4mg/mL) (1–4) mostly by synoviocytes (5) in synovium, the inner lining of the joint. HA is also found on the intraarticular surface of synovium, providing substantial resistance to fluid and macromolecular outflow (6), and helping to maintain the concentration of HA in SF. The protein composition of SF reflects its origins as plasma, though the distribution of those proteins (7–10) differs from plasma due to the size-selective nature of synovium (11,12), which allows higher flux of smaller molecules.

The composition of SF is normally in dynamic equilibrium through the processes of convection from plasma to SF, secretion of molecules by local cells, diffusion out of SF to the lymphatics or cellular uptake, accumulation at surfaces, and degradation within the joint cavity. Plasma, including small proteins and metabolites, is filtered into SF from capillaries. Larger molecules are secreted locally, such as HA, and are mainly removed from SF by diffusion through synovium to the lymphatics (13,14), but also by accumulating at tissue surfaces (15,16) and by degradation in SF (13). A balance normally exists between secretion and loss processes that maintain a steady-state concentration of high-Mr HA in SF.

Injury and pathological conditions alter the protein and HA composition of SF due to shifts in the diffusion and transport rates of molecules out of SF. The permeability of synovium to proteins in joints with arthritis is increased (17,18), with proportionately larger increases in permeability for larger proteins (19). Protein flux between the microvasculature and SF, measured by a variety of techniques including the SF to serum ratio (19), injection of radioactive tracers and sampling (20,21), and tracer injection with clearance measurements (17,22,23), consistently show increased protein flux with synovitis or other indices of joint inflammation. For HA, the steady-state concentration is typically decreased and the Mr-distribution shifts towards lower Mr species after joint injury (24) and in rheumatoid arthritis (25,26). HA residence time in the joint might be expected to also be decreased, analogous to protein transport, though results from tracer studies involving injection of radiolabeled high-Mr HA and analysis of serum and SF are mixed. A type II collagen injection model in sheep showed SF half-life decreasing to 55% of normal (from ~21 to ~12hrs) after 2wks (27), while in a partial meniscectomy model of osteoarthritis (OA) in rabbits, high-Mr HA half-life tended to increase to 135% of normal at 4wks before returning to baseline by 12wks (28). HA residence times for the rabbit anterior cruciate ligament transection (ACLT) model of joint injury and OA (29–31) have not been reported.

The diffusion rate of molecules out of SF is determined by their molecular mass and shape, as well as the cellularity and extracellular matrix of synovium. Synovium is distinguished by densely populated synoviocytes 1–3 layers deep, though the cells are not laminar, do not share tight junctions, and lack a basement membrane (32,33). Molecular size-dependent diffusive transport occurs between cells in the highly fibrillar interstitial matrix (34,35) that has an effective pore size of ~20–90nm (12,36). Direct manipulation of the matrix content by digesting synovium matrix (37) and stretching the matrix through increased intra-articular pressure (38) delineated the importance of matrix molecules in determining synovium resistance to fluid flow and molecular diffusion. Pathological changes to synovial matrix, due to cell infiltration or synovitis, may be responsible for the increased protein, and possibly HA, permeability observed.

Thus the hypothesis of this study was that HA loss from SF is Mr-dependent and is increased after ACLT compared to sham operated (SHAM) and non-operated (NonOp) joints. The aims were to assess at 7 and 28 days after surgery for non-operated (NonOp), ACLT, and SHAM groups: 1) HA concentration and Mr distribution in synovial fluid (SF), 2) endogenous replenishment of HA after saline washout, 3) HA retention over 8hrs in SF via HA residence times, and 4) synovium and subsynovium cellularity.

Methods

Study Design

The residence time of HA in the SF of knee joints was investigated using the rabbit ACLT model. All animal procedures were approved by the local IACUC committee. Young adult (11–13mo) New Zealand white rabbits (N=26 rabbits, n=52 knees) underwent ACLT (N=20) or SHAM (N=6) surgeries on their right hind limb, while each left limb was a NonOp control. At 7 (N=12 ACLT, N=6 SHAM) or 28days (N=8 ACLT) after surgery, joints were aspirated for SF, lavaged with saline, injected with saline or polydisperse HA, and flexed. Directly after flexing (t=0+) or at 1, 3, or 8hrs after injection, 50μL of joint fluid was withdrawn (Table S1). Rabbits were euthanized after the 8hr time point and fluid samples and intact joints were stored at −80°C. Samples at 0+, 1, 3, and 8hrs were analyzed for HA concentration (cHA) and the HA Mr distribution to calculate HA residence time for various bins of Mr. SF and serum samples were also analyzed HA concentration. Synovium samples were analyzed for cell density.

Surgeries and Transport Study

ACLT or SHAM surgeries were performed. Rabbits were anesthetized, intubated, and the right hind limb was shaved and cleaned. The patella was displaced laterally and a ~3cm long incision medial to the patellar ligament was made through the skin and joint capsule. The infrapatellar fat pad was displaced and the ACL exposed. For ACLT surgeries, the ACL was then cut using curved, fine-tip scissors and the transection verified by Lachman test. ACLT and SHAM groups were then rinsed with saline, the patella was realigned, the capsule was closed with 2–0 suture, and then the skin was closed with 4–0 suture.

Bolus injection and longitudinal sampling of knee fluid were performed on operated and NonOp hind limbs at 7 or 28d after surgery. Rabbits were anesthetized and neat SF was withdrawn using a 22g needle. Joints were lavaged 3x with 0.5mL saline, injected with 0.5mL of saline or 2.5mg/mL polydisperse HA (2500–50kDa, Lifecore Biomedical LLC, Chaska, MN), and flexed 10x. Additionally, 3 NonOp joints were injected with a FITC-labeled HA preparation (39) that included HA in the 4000kDa range (Healon®) to track high-Mr HA as well. Samples of the joint fluid (50μL) were taken with a 25g needle at t=0+ or at 1, 3, and 8hrs after injection. Blood was drawn for serum after the 8hr time point and the animals were euthanized. The hind limbs were removed and the SF, joint fluid, serum, and limbs were stored at −80°C until processing.

HA concentration, Mr distribution, and Transport Analysis

Joint fluids were analyzed for cHA and Mr. Portions of SF and joint fluid were digested overnight at 37°C with Proteinase K, loaded with 1μL per sample (0.3–3μg HA) in a 1% agarose gel, and electrophoresed at 150V in tris-acetate EDTA buffer. Gels were fixed in 25% isopropanol, stained overnight in Stainsall (Sigma-Aldrich, Saint Louis, MO), destained in water, and imaged on a lightbox. Hyalose monodisperse HA standards (Lifecore Biomedical) at 4000, 2400, 1156, 450, 262, 160, and 31kDa (200ng each) were loaded as a calibration ladder. Gel distribution analysis was validated on test gels by loading 0.1, 0.3, 1, and 3μg of 51, 1680, and 4000kDa HA separately and combined in equal parts. Distributions varied by <5% (average 2%) per bin for different loads, independent of size. Combined distribution data were consistent with the sum of the individual distributions (deviations averaged 6%, and were <15% per bin). The efficiency of the proteinase K digestion was tested by staining gels of SF after digestion for protein with SYPRO Ruby (Life Technologies, Grand Island, NY), indicating no visible protein bands (<0.1ng/μL protein). Gels with samples of FITC-HA were scanned on a fluorescent scanner (Storm, GE Healthcare, Piscataway, NJ). A custom MATLAB (Mathworks Inc, Natick, MA) program was used to quantify the % HA intensity (40) in Mr bins of 7000–2500, 2500–1000, 1000–500, 500–250, and 250–50kDa. The cHA in each joint fluid and serum sample was determined by digesting portions with Proteinase K, inhibition of enzyme activity by boiling for 10min, and then using an ELISA-like assay (Corgenix, Broomfield, CO or R&D Systems, Minneapolis, MN). Total cHA was multiplied by % intensity to give the cHA in each Mr bin.

The residence time constants for HA by Mr were calculated. The endogenous secretion of HA was approximated by the average cHA measured after saline injection, the majority of which was localized in the 7000–2500 bin. This endogenous secretion was subtracted from the total cHA after polydisperse HA injection at each time point. A 2-parameter exponential decay was fit through the remaining cHA at 0+, 1, 3, and 8hrs for all bins except the 7000–2500 bin (due to the high secretion rate compared to injected cHA) to calculate the best-fit residence time constant (τ) for each group.

Synovium Histology

The synovium and subsynovium matrix structure and cellularity were determined. Limbs were thawed at 4°C and samples of synovium were harvested from the medial and lateral distal patellar regions. Samples were fixed overnight in 4% paraformaldehyde, and either dehydrated and embedded in paraffin or embedded in Optimal Cutting Temperature medium, and sectioned at 10 or 100μm thickness, respectively. Thin sections were stained with H&E or alcian blue, or were probed for HA (HA binding protein-HRP, Corgenix), CD4+ lymphocytes (ab25804, Abcam, Cambridge, MA), or CD11b+ macrophages (ab8878, Abcam), and imaged at 20 or 40× on an inverted microscope (Nikon Instruments, Melville, NY). Thick sections were stained with 2μg/mL propidium iodide in saline and imaged with 20× objective, 0.75μm square voxel size, and 1024×1024 pixels on a confocal microscope (Leica Microsystems, Wetzlar, Germany).

Statistical Analysis

The data are presented as mean±SEM. The fixed effects of SURGERY (NonOp, ACLT, or SHAM) and DAY (7 or 28d post-op) and repeated effects of TIME (1, 3, or 8hrs) and Mr (by bin) on cHA after injection were assessed by 4-way ANOVA. The fixed effect of GROUP (NonOp, ACLT d7, SHAM d7, or ACLT d28) and repeated effect of Mr on steady-state neat SF cHA were assessed by 2-way ANOVA with Dunnett post-hoc tests to determine differences from the NonOp group within each Mr bin. The fixed effect of GROUP on time constant and serum cHA was assessed by 1-way ANOVA with Dunnett post-hoc tests to determine differences from the NonOp group. Significance was set as P<0.05 and statistical analyses were performed using Systat 10.2 (Systat Software Inc., Chicago, IL).

Results

HA concentration and Mr-distribution in synovial fluid after surgery

The Mr-distribution of HA in SF shifted towards lower Mr HA after surgery. The cHA in SF varied with Mr (P<0.0001) and GROUP (P<0.0001), with an interaction (P<0.0001, Fig. 1). The total cHA was decreased from ~2.5mg/mL by 47% for ACLT and 49% for SHAM at day 7, and by 26% for ACLT day 28. Both ACLT and SHAM day 7 groups contained decreased high-Mr HA (7000–2500kDa, P<0.001, Fig. 1A, B). By day 28, the ACLT group still contained less high-Mr HA (P<0.01), but also contained more lower-Mr (1000–50kDa) HA (P<0.01–0.05, Fig. 1D–F) than the NonOp group.

Figure 1.

Figure 1

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(A) HA Mr distribution in SF is altered after surgery. Quantification (B–F) showed decreased high-Mr HA is present at day 7 after ACLT or SHAM surgery. By day 28, in addition to decreased high-Mr, there is increased lower Mr HA present. *: P<0.05, **: P<0.01, ***: P<0.001.

Residence time of hyaluronan in synovial fluid

After washout and injection of lower-Mr HA, the HA Mr-distribution showed endogenous replenishment of high-Mr HA, while lower-Mr HA was lost from SF, in particular at day 7 after ACLT. Rabbit SF normally contains mainly high Mr HA (>4000kDa) that, after washout, was replenished over time for both NonOp and ACLT joints (Fig. 2 a–g). After washout, HA with a Mr-distribution lower than the typical endogenous profile, was injected and resampled at t=0+, showing a moderate dilution effect, but similar size distribution to the injection profile for both NonOp and ACLT joints (Fig. 2 h–j). Distributions determined at 1, 3, and 8hrs after injection showed Mr-dependent loss over time, with exacerbated loss occurring in ACLT joints 7d after surgery (Fig. 2 k–v).

Figure 2.

Figure 2

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Mr distribution of HA in SF (a) or in joint fluids sampled over time after saline lavage (b–g) or HA injection (h–j) for NonOp and ACLT rabbit knees at day 7 (k–p) or 28 (q–v). Replenishment of high-Mr HA occurred in all joints, likely due to secretion. Compared to NonOp controls, ACLT samples had less HA content at day 7, though this effect is lessened by day 28. HA loss from SF is Mr-dependent, with faster loss at lower Mr.

The cHA breakdown by Mr of each sample was determined to allow quantitative comparisons between surgical groups and time after surgery. After washout and injection (Fig. 3A), the HA distributions at t=0+ were similar for NonOp and ACLT joints (P=0.82, Fig. 3B), diluted on average to ~60% injection concentration. The total cHA by Mr bin was divided into the endogenous HA (top, open bars) and injected (bottom, filled bars, Fig. 2C and D), with the endogenous concentration mainly found in the 7000–2500kDa bin (Fig. 3.C.i and D.i). The injected HA varied with SURGERY (P<0.0001), DAY post-surgery (P<0.05), Mr (P<0.0001), and TIME post-injection (P<0.0001), with interactions of Mr*SURGERY (P<0.01), TIME*DAY (P<0.0001), and Mr*TIME (P<0.01). By 8hrs post-injection on day 7, cHA in the 2500–1000kDa bin had dropped to 75% of injection for NonOp compared to 37% for ACLT and 57% for SHAM groups (Fig. 3.C.ii) and in the 250–50kDa bin to 48% for NonOp compared to 24% for ACLT and 45% for SHAM groups (Fig. 3.C.v). However, by 8hrs post-injection on day 28 the differences between NonOp and ACLT were smaller, as HA concentration in the 2500–1000kDa bin had dropped to 85% of injection for NonOp compared to 68% for ACLT (Fig. 3.D.ii) and in the 250–50kDa bin to 55% for NonOp and 43% for ACLT (Fig. 3.D.v).

Figure 3.

Figure 3

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HA concentrations by Mr bin (i–v) in joint fluid samples over a time course after HA injection (A, B) in NonOp, ALCT, or SHAM knees at day 7 (C) or 28 (D). Open bars indicate endogenous replenishment, and closed bars injected HA concentration. Dotted lines indicate normal SF values. Significant effects: surgery (P<0.0001), day post-surgery (P<0.05), Mr (P<0.0001), and time post-injection (P<0.0001).

The residence time of HA decreased with Mr and after surgery. Plotted as % of injected over sampling times, the increased loss for ACLT compared to SHAM and NonOp at day 7 were apparent (Fig. 4A), though by day 28 ACLT and NonOp are similar (Fig. 4B). The residence time constants, which describe the slope of the HA loss, by Mr-bin ranged from ~27hrs for the NonOp 2500–1000kDa bin to ~7hrs for the 250–50kDa bin (Fig. 4.C.i–C.iv), and were decreased at day 7 for all Mr after ACLT (by 64–75%). In the 2500–1000kDa bin, SHAM was also decreased by (by 41%) compared to NonOp (Fig. 4.C), indicating a major effect of the ACLT and minor effect of the surgical procedure itself. By day 28, the time constants for ACLT were similar to NonOp controls (P=0.39–0.50).

Figure 4.

Figure 4

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HA loss over time by Mr bin (i–iv) at day 7 (A) and 28 (B), with calculated residence time constants (C). Residence time for HA increased with Mr, and decreased at day 7 post-ACLT, though that decrease is recovered by day 28. SHAM controls were similar to NonOp at low Mr, and between ACLT and NonOp at higher Mr. *: P<0.05, **: P<0.01.

The Mr-dependence of HA loss for high-Mr HA was confirmed using FITC-HA. High-Mr FITC-labeled HA was cleared from the joint fluid slower than mid-range HA, while low-Mr HA was quickly lost (Fig. S1).

Synovium and subsynovium matrix structure and cell density

Microscopy images showed altered synovium structure and increased cell density of lymphocytes and macrophages in the subsynovium after surgery. Compared to NonOp tissue, synovium thickening and loss of matrix staining occurred at ACLT day 7; by ACLT day 28, the matrix intensity had returned, though the synovium lining was still thickened (Fig. 5). The subsynovium of ACLT joints contained substantial cellular density and also evidence of neovascularization at days 7 and 28 (Fig. 5), as well as positive staining for lymphocytes and macrophages. Although the increased transport rates of HA out of the joint after ACLT appeared to be resolved by day 28, the increased cell density of subsynovium was still apparent, though no longer expressing CD4 and CD11b markers (Fig. 5).

Figure 5.

Figure 5

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Microscopy images of synovium and subsynovium from NonOp (A) and ACLT joints at day 7 (B) and 28 (C) show increased synovium thickness and decreased matrix content and cellular infiltration by lymphocytes and macrophages, as well as increased cell density and neovascularization after surgery.

Serum HA concentration

The cHA in blood serum was also transiently increased after surgery. Treatment (normal, ACLT d7, SHAM d7, or ACLT d28) significantly affected serum HA content (P<0.05, Fig. S2). Serum HA concentration almost doubled from ~21ng/mL to ~41ng/mL in both ACLT and SHAM groups at day 7, before returning to ~22ng/mL at day 28.

Discussion

These results demonstrate that the residence time of HA in SF is transiently decreased after ACLT, suggesting a biophysical mechanism for the alterations in SF HA Mr distribution, and SF composition in general after injury or during inflammation. The HA Mr-distribution shifted to lower Mr HA after surgery (Fig. 1). Direct sampling of SF over time after washout and injection indicated accumulation of high Mr species, while lower Mr HA diffused out of SF, especially at day 7 after ACLT (Fig. 2). Quantification of these gels confirmed the effects of HA Mr, surgery, day after surgery, and time after injection on HA loss from SF (Fig. 3). The time constants for HA residence in SF by Mr are consistent with previously reported times, and demonstrate an effect both of the surgical insult, as well as a more intense effect of ACLT itself in decreasing HA residence times (Fig. 3, 4). Decreased density and loss of matrix associated with synovial thickening and subsynovial cell infiltration after surgery (Fig. 5) likely decreases the resistance to HA loss through synovium to the lymphatics, while the systemic HA turnover via plasma concentration (Fig. S2), an additional measure of inflammation, was also increased at day 7. Together, these data suggest increased HA transport due to an inflammatory state as a biophysical mechanism underlying the loss of high-Mr HA from SF after injury and during inflammatory pathologies, though other inflammatory changes, such as increased lymphatic drainage, local degradation, and biosynthetic changes may also contribute.

The in vivo data provide a self-consistent description of the transport of HA from SF, despite inherent limitations in this type of animal study. Animal numbers were limited, and neat SF was sampled repeatedly from individual knees. Although 26 rabbits and 52 knees were utilized, they were divided among 13 groups in order to investigate the effects of surgery and time after surgery on HA transport. Despite limited sample numbers, sufficient power was achieved to show significant effects and confirm the hypothesis. In addition, endogenous noise in the high-Mr bins confounded the transport measurements, though FITC-labeling of HA confirmed similar transport trends as measured quantitatively for lower Mr bins (Fig. S1). The rate of FITC-HA loss from the joint is difficult to quantify and compare to the concentration-based HA data due to the confounding effects of loss of label, quenching of the fluorescence in vivo, and unknown conversion between scanned fluorescence intensity and concentration. Nevertheless, the qualitative trends confirming Mr-dependent efflux of HA from SF were observed. Repeated sampling of the SF required multiple needle sticks through the joint capsule, which could have increased the HA loss from SF. However, the needle sampling did not appear to markedly affect HA loss, since large holes in synovium would have negated the Mr-dependence, which was not the case. Finally, homeostasis reflects loss through degradation, transport, and cellular phagocytosis. Although degradation and cellular uptake were not measured directly, the uniformity and concentration of high-Mr HA that accumulated after saline washout suggests the time-scale for degradation or uptake was longer than for diffusion, implying the data reported here were dominated by the diffusion effects.

By ACLT day 28, the loss rates of HA were similar to NonOp values, though the steady-state cHA from aspirated SF show increased cHA in the low-Mr ranges. Together, these findings suggest an additional mechanism increasing the low-Mr cHA. Two possibilities are degradation of high-Mr HA due to increased hyaluronidase or reactive oxygen species activity after surgery or increased synthesis of smaller HA by synoviocytes. After aspiration and saline injection, replenishment of HA does not show higher concentrations of low-Mr HA (Fig. 3, open bars), suggesting that degradation is primarily responsible for the shift.

Although endogenous replenishment appeared similar between saline or HA injected joints within the same experimental group, analysis at later time points after injection would be needed to determine the effect of saline vs HA injection on replenishment. As indicated by the dotted lines in Fig. 3.C.i, after 8hrs, only ~26–40% of the steady-state concentration had returned, as the time constant for replenishment is ~14hrs. Sampling at later points in time, after 2 to 3 time constants, would be necessary to determine whether the rates were truly different.

The results are consistent with a biophysical mechanism explaining the shift to lower-Mr HA in SF after injury and during inflammatory pathologies (Fig. 6). Normally, the Mr-dependent residence time of HA in SF accompanied by the secretion of high-Mr HA leads to a steady-state, with SF containing mostly high-Mr HA (Fig. 1); low-Mr HA, created by HA turnover diffuses relatively quickly out of SF. After injury or during inflammation, the synovium becomes more permeable to HA (Figs. 2–4), likely due to cellular infiltration displacing and digesting the extracellular matrix barrier to molecular efflux through synovium, decreasing the residence time of high-Mr HA. Faster diffusive efflux of high-Mr HA, coupled with decreased secretion rates and possibly increased degradation rates, would shift the steady-state Mr distribution to lower Mr, as observed experimentally. Although the cell infiltration was still present at day 28, the synovial matrix production between days 7 and 28 appears to have replenished the extracellular matrix barrier (Fig. 5), with a co-incident restoration of residence times. In addition to major inflammatory events such as injury or during rheumatoid arthritis, this mechanism may also be responsible for altered SF composition in OA, where an inflammatory component is increasingly recognized (41).

Figure 6.

Figure 6

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Schematic representation of the biophysical processes, including secretion, degradation, and loss, contributing to altered HA Mr distribution in pathological SF.

The cHA variable includes the effects of volume, which is typically altered after injury and during inflammation. The aspirated volume of neat SF is one approximation of the total SF volume, although it is difficult to accurately measure. Larger volumes of neat fluid were aspirated from the knee after surgery, increasing from ~20±13μL (mean±SD) for NonOp to 290±120 for ACLT day 7 and 540±335 for ACLT day 28. However, the transport study was conducted after fluid aspiration and injection of 500μL for each group, normalizing the volume at time zero. Dilution effects are unlikely to be confounding the interpretation of the transport data as the volumes were highest at day 28, when the transport rates and concentrations were similar to NonOp values. Although the cHA and Mr-distribution shifted to lower-Mr species by ACLT day 28, the total mass of HA of all sizes, due to the increase in fluid volume, was increased.

Understanding the mechanisms leading to altered SF HA composition, such as altered transport rates due to inflammation, is important since SF function is determined by composition. Changes to the HA concentration and Mr in SF alter the biophysical properties of the fluid, including viscosity and viscoelastic properties (42,43), as well as the lubrication function (44). SF effusion is characteristic of inflammatory pathology, and increased plasma filtration rates into SF, which depend on SF hydrostatic pressure (45) and joint capsule strains (46) that vary with flexion, likely contribute to decreased HA concentration. In addition to HA, lubricant molecules secreted locally into SF, such as lubricin, likely also have decreased residence time in SF. A decreased barrier to diffusion out of SF also indicates decreased resistance to convective and diffusive molecular flux from plasma into SF. This may explain the increased concentration of higher Mr plasma proteins, including from the inter-α-trypsin inhibitor family, that are found in pathological SF (47,48).

HA in SF is modified with various HA binding proteins (HABP), especially during inflammation, which likely affects the residence time of HA in vivo. To characterize the HA transport independent of HABP effects, the samples here were incubated with Proteinase K to digest protein. The calculated residence time rates may be an underestimate of the residence time in vivo due to the decrease in Mr after protein removal. The HA distributions were similar overall with and without proteinase K digestion, though additional light, high-Mr signal in the area between the well and main hyper-intense band at ~4000kDa was apparent before digestion (data not shown). These high-Mr species are probably SHAP-HA aggregates (47,48), HA modified by inter-alpha-trypsin inhibitor heavy chains, which likely develop over time on exogenously injected HA as well.

The transport mechanisms suggested by these results indicate potential avenues, and pitfalls, for therapeutic interventions. The Mr-dependent HA residence times in SF in NonOp and after ACLT provide an estimate of the residence time for other proteoglycans and proteins in SF, and highlight the difficulty for localized retention of injected pharmaceuticals, especially during the inflammatory response phases following injury. The inflammatory state of the synovial lining may be a potential target for intervention to restore the normal homeostasis of SF. Early clinical intervention that decreases the synovial inflammatory response and restores lubrication following injury (49,50) may be able to limit or retard the progression from injury to post-traumatic osteoarthritis.

This work investigated the state of transport of HA in the joint at various times after ACLT, demonstrating the Mr-dependent rate of HA loss likely due to inflammatory cell infiltration into the synovial and subsynovial joint capsule lining. Such altered transport rates help explain the altered HA content and general compositional changes in SF after injury and during inflammatory pathologies. In addition, these data describe the molecular conditions under which any pharmaceutical intervention must function, as well as suggest potential targets for clinical intervention.

Supplementary Material

Supp Fig S1
Supp Fig S2
Supp Table S1

Acknowledgements

This work was supported by grants from the National Institute of Arthritis, Musculoskeletal and Skin Diseases and a Ruth L. Kirschstein National Research Service Award predoctoral fellowship from the National Institute on Aging (for WJM).

The authors have no commercial financial support, benefits, or interests to report.

Footnotes

The authors have no professional or financial conflicts of interest to disclose.

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Supp Table S1
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