Kimberly A. Waller

Prevention of Cartilage Degeneration and Restoration of Chondroprotection by Lubricin Tribosupplementation in the Rat Following Anterior Cruciate Ligament Transection

OBJECTIVE To investigate whether cartilage degeneration is prevented or minimized following intraarticular injections of lubricin derived from human synoviocytes in culture, recombinant human PRG4 (rhPRG4), or human synovial fluid (SF) in a rat model of anterior cruciate ligament (ACL) injury. METHODS Unilateral ACL transection (ACLT) was performed in Lewis rats (n = 45). Nine animals were left untreated. The remaining rats were given intraarticular injections (50 microl/injection) of either phosphate buffered saline (PBS) (n = 9), human synoviocyte lubricin (200 microg/ml; n = 9), rhPRG4 (200 microg/ml; n = 9), or human SF lubricin (200 microg/ml; n = 9) twice weekly beginning on day 7 after injury. Joints were harvested on day 32 after injury. Histologic analysis was performed using Safranin O-fast green staining, and articular cartilage degeneration was graded using the Osteoarthritis Research Society International (OARSI)-modified Mankin criteria. Histologic specimens were immunoprobed for lubricin and sulfated glycosaminoglycans. A 24-hour urine collection was performed on days 17 and 29 postinjury, and urinary C-terminal telopeptide of type II collagen (CTX-II) levels were measured. RESULTS Treatment with human synoviocyte lubricin resulted in significantly lower OARSI scores for cartilage degeneration compared with no treatment or PBS treatment (P < 0.05). Increased immunostaining for lubricin in the superficial zone chondrocytes and on the surface of cartilage was observed in lubricin-treated, but not untreated or PBS-treated, joints. On day 17, urinary CTX-II levels in human synoviocyte lubricin- and human SF lubricin-treated animals were significantly lower than those in untreated animals (P = 0.005 and P = 0.002, respectively) and in PBS-treated animals (P = 0.002 and P < 0.001, respectively). CONCLUSION After treatment with any of the 3 types of lubricin evaluated in this study, a reduction in cartilage damage following ACLT was evident, combined with a reduction in type II collagen degradation. Our findings indicate that intraarticular lubricin injection following an ACL injury may be beneficial in retarding the degeneration of cartilage and the development of posttraumatic OA.

Role of lubricin and boundary lubrication in the prevention of chondrocyte apoptosis

Osteoarthritis is a complex disease involving the mechanical breakdown of articular cartilage in the presence of altered joint mechanics and chondrocyte death, but the connection between these factors is not well established. Lubricin, a mucinous glycoprotein encoded by the PRG4 gene, provides boundary lubrication in articular joints. Joint friction is elevated and accompanied by accelerated cartilage damage in humans and mice that have genetic deficiency of lubricin. Here, we investigated the relationship between coefficient of friction and chondrocyte death using ex vivo and in vitro measurements of friction and apoptosis. We observed increases in whole-joint friction and cellular apoptosis in lubricin knockout mice compared with wild-type mice. When we used an in vitro bovine explant cartilage-on-cartilage bearing system, we observed a direct correlation between coefficient of friction and chondrocyte apoptosis in the superficial layers of cartilage. In the bovine explant system, the addition of lubricin as a test lubricant significantly lowered the static coefficient of friction and number of apoptotic chondrocytes. These results demonstrate a direct connection between lubricin, boundary lubrication, and cell survival and suggest that supplementation of synovial fluid with lubricin may be an effective treatment to prevent cartilage deterioration in patients with genetic or acquired deficiency of lubricin.

The impact of anterior cruciate ligament injury on lubricin metabolism and the effect of inhibiting tumor necrosis factor α on chondroprotection in an animal model

OBJECTIVE To examine the effects of anterior cruciate ligament transection (ACLT) in a rat model on lubricin metabolism and its relationship to markers of inflammation and cartilage damage, and to determine whether blocking the metabolic effects of tumor necrosis factor alpha (TNFalpha) by etanercept increases the chondroprotection provided by lubricin. METHODS Unilateral ACLT was performed in Lewis rats. Levels of lubricin, TNFalpha, interleukin-1beta (IL-1beta), and sulfated glycosaminoglycans (sGAG) in synovial fluid (SF) lavage specimens and synovial tissue lubricin gene expression were evaluated at 1 week and 4 weeks following ACLT. Histologic evaluation of articular cartilage included staining with lubricin-specific monoclonal antibody 9G3 and Safranin O. The percentage of lubricin staining on the surface of articular cartilage in weight-bearing areas was estimated by digital imaging. Blocking of TNFalpha was performed using etanercept, which was administered subcutaneously at a dose of 0.5 mg/kg around the ACL-transected joints, using different dosing strategies. The ACL-transected and contralateral joints of these rats were harvested 4 weeks following surgery. RESULTS Four weeks following ACLT, SF lubricin concentrations and the percentage of cartilage surface lubricin staining were significantly lower in the injured joints compared with the contralateral joints. A significant decrease in synovial tissue lubricin gene expression was associated with elevated TNFalpha and IL-1beta concentrations in SF lavage samples. With all of the etanercept treatment strategies, blocking of TNFalpha significantly increased the amount of lubricin bound to cartilage, coupled with a significant decrease in sGAG release. However, changes in the concentrations of lubricin in SF were variable. CONCLUSION Blocking TNFalpha resulted in a chondroprotective effect, exemplified by increased lubricin deposition on articular cartilage and a decrease in sGAG release from articular cartilage in an animal model of posttraumatic arthritis.

The biology of Lubricin: Near frictionless joint motion

Lubricin is a surface-active mucinous glycoprotein secreted in the synovial joint that plays an important role in cartilage integrity. In healthy joints, lubricin molecules coat the cartilage surface, providing boundary lubrication and preventing cell and protein adhesion. Arthropathy occurring in patients with joint trauma, inflammatory arthritis or genetically mediated lubricin deficiencies have insufficient lubricin to prevent damage to articular cartilage. Recent studies in lubricin null joints indicate that lubricin (Prg4) plays a role in preventing damage to the superficial zone and preservation of chondrocytes. Progress in the production of recombinant forms of lubricin and the successes of lubricin supplementation in small animal models identify rhPRG4 as a potential therapeutic for patients with transient lubricin deficiency in the setting of trauma or autoimmune arthritis.

Prevention of cartilage degeneration and gait asymmetry by lubricin tribosupplementation in the rat following anterior cruciate ligament transection.

OBJECTIVE To investigate whether cartilage degeneration is prevented or minimized in a rat model of anterior cruciate ligament (ACL) injury following a single dose-escalated intraarticular injection of lubricin derived from human synoviocytes in culture. METHODS Unilateral ACL transection (ACLT) of the right hind limb was performed in Lewis rats (n = 56). Control animals underwent a capsulotomy alone, leaving the ACL intact (n = 11). Intraarticular injections (50 μl/injection) of phosphate buffered saline (PBS; n = 14 rats) and human synoviocyte lubricin (1,600 μg/ml; n = 14 rats) were performed on day 7 postsurgery. Animals were killed on day 70 postsurgery. Histologic specimens were immunoprobed for lubricin and sulfated glycosaminoglycans. Urinary C-telopeptide of type II collagen (CTX-II) levels were measured on days 35 and 70 postsurgery. Hind limb maximum applied force was determined using a variable resistor walkway to monitor quadruped gait asymmetries. RESULTS Increased immunostaining for lubricin in the superficial zone and on the surface of cartilage was observed in lubricin-treated and control animals but not in PBS-treated or untreated animals with ACLT. On days 35 and 70 after surgery, urinary CTX-II levels in human synoviocyte lubricin-treated animals were lower than in untreated and PBS-treated animals (P < 0.005 and P < 0.001, respectively). Animals with ACLT treated with human synoviocyte lubricin and control animals distributed their weight equally between hind limbs compared to PBS-treated or untreated animals (P < 0.01). CONCLUSION Our findings indicate that a single intraarticular injection of concentrated lubricin following ACLT reduces type II collagen degradation and improves weight bearing in the affected rat joint. These findings support the practice of tribosupplementation with lubricin for retarding cartilage degeneration and possibly the development of posttraumatic osteoarthritis.

Preventing Friction-induced Chondrocyte Apoptosis: Comparison of Human Synovial Fluid and Hylan G-F 20

Objective. Symptomatic osteoarthritis (OA) is a common painful disease with limited treatment options. A rising number of patients with OA have been treated with intraarticular injections of hyaluronic acid, including the high-molecular-weight hylan G-F 20, which is injected following arthrocentesis. We investigated the effectiveness of hylan G-F 20 to lower coefficient of friction (COF) and prevent chondrocyte apoptosis in vitro. Methods. A disc-on-disc bovine cartilage bearing was used to measure the static and kinetic COF when lubricated with hylan G-F 20, human synovial fluid (HSF), and phosphate buffered saline (PBS). Following friction testing, we stained paraffin-embedded sections of these cartilage bearings for activated caspase-3, a marker of apoptosis. Results. Bearings lubricated with hylan G-F 20 had kinetic COF values that were similar to bearings lubricated with PBS, but significantly higher than those lubricated with HSF. There were no significant differences in static COF values in bearings lubricated with hylan G-F 20 as compared to PBS or HSF. However, bearings lubricated with HSF had significantly lower static COF values compared to bearings lubricated with PBS. The mean percentage of caspase-3-positive chondrocytes in the superficial and upper intermediate zones of bearings lubricated with hylan G-F 20 was significantly higher compared to that of bearings lubricated with HSF or unloaded controls, but significantly lower than in those lubricated with PBS. Conclusion. These findings indicate that joint lubrication may prevent chondrocyte apoptosis by lowering the COF. Further, removal of synovial fluid prior to hylan G-F 20 injection may be detrimental to cartilage health.

Lubricin Restoration in a Mouse Model of Congenital Deficiency

Congenital deficiency of the principal boundary lubricant in cartilage (i.e., lubricin, encoded by the gene PRG4) increases joint friction and causes progressive joint failure. This study was undertaken to determine whether restoring lubricin expression in a mouse model would prevent, delay, or reverse the disease process caused by congenital deficiency.

Intra-articular Recombinant Human Proteoglycan 4 Mitigates Cartilage Damage After Destabilization of the Medial Meniscus in the Yucatan Minipig.

Background: Lubricin, or proteoglycan 4 (PRG4), is a glycoprotein responsible for joint boundary lubrication. PRG4 has been shown previously to be down-regulated after traumatic joint injury such as a meniscal tear. Preliminary evidence suggests that intra-articular injection of PRG4 after injury will reduce cartilage damage in rat models of surgically induced posttraumatic osteoarthritis. Objective: To determine the efficacy of intra-articular injection of full-length recombinant human lubricin (rhPRG4) for reducing cartilage damage after medial meniscal destabilization (DMM) in a preclinical large animal model. Study Design: Controlled laboratory study. Methods: Unilateral DMM was performed in 29 Yucatan minipigs. One week after DMM, animals received 3 weekly intra-articular injections (3 mL per injection): (1) rhPRG4 (1.3 mg/mL; n = 10); (2) rhPRG4+hyaluronan (1.3 mg/mL rhPRG4 and 3 mg/mL hyaluronan [~950 kDA]; n = 10); and (3) phosphate-buffered saline (PBS; n = 9). Hindlimbs were harvested 26 weeks after surgery. Cartilage integrity was evaluated by use of macroscopic (India ink) and microscopic (safranin O–fast green and hematoxylin and eosin) scoring systems. Secondary outcomes evaluated via enzyme-linked immunosorbent assay (ELISA) included PRG4 levels in synovial fluid, carboxy-terminal telepeptide of type II collagen (CTX-II) concentrations in urine and serum, and interleukin 1β (IL-1β) levels in synovial fluid and serum. Results: The rhPRG4 group had significantly less macroscopic cartilage damage in the medial tibial plateau compared with the PBS group (P = .002). No difference was found between the rhPRG4+hyaluronan and PBS groups (P = .23). However, no differences in microscopic damage scores were observed between the 3 groups (P = .70). PRG4 production was elevated in the rhPRG4 group synovial fluid compared with the PBS group (P = .033). The rhPRG4 group presented significantly lower urinary CTX-II levels, but not serum levels, when compared with the PBS (P = .013) and rhPRG4+hyaluronan (P = .011) groups. In serum and synovial fluid, both rhPRG4 (P = .006; P = .017) and rhPRG4+hyaluronan groups (P = .009; P = .03) presented decreased IL-1β levels. Conclusion: All groups exhibited significant cartilage degeneration after DMM surgery. However, animals treated with rhPRG4 had the least amount of cartilage damage and less inflammation, providing evidence that intra-articular injections of rhPRG4 may slow the progression of posttraumatic osteoarthritis. Clinical Relevance: Patients with meniscal trauma are at high risk for posttraumatic osteoarthritis. This study demonstrates that an intra-articular injection regimen of rhPRG4 may attenuate cartilage damage after meniscal injury.

Friction-Induced Mitochondrial Dysregulation Contributes to Joint Deterioration in Prg4 Knockout Mice

Deficiency of PRG4 (lubricin), the boundary lubricant in mammalian joints, contributes to increased joint friction accompanied by superficial and upper intermediate zone chondrocyte caspase-3 activation, as shown in lubricin-null (Prg4−/−) mice. Caspase-3 activity appears to be reversible upon the restitution of Prg4 either endogenously in vivo, in a gene trap mouse, or as an applied lubricant in vitro. In this study we show that intra-articular injection of human PRG4 in vivo in Prg4−/− mice prevented caspase-3 activation in superficial zone chondrocytes and was associated with a modest decrease in whole joint friction measured ex vivo using a joint pendulum method. Non-lubricated Prg4−/− mouse cartilage shows caspase cascade activation caused by mitochondrial dysregulation, and significantly higher levels of peroxynitrite (ONOO− and −OH) and superoxide (O−2) compared to Prg4+/+ and Prg4+/− cartilage. Enzymatic activity levels of caspase 8 across Prg4 mutant mice were not significantly different, indicating no extrinsic apoptosis pathway activation. Western blots showed caspase-3 and 9 activation in Prg4−/− tissue extracts, and the appearance of nitrosylated Cys163 in the active cleft of caspase-3 which inhibits its enzymatic activity. These findings are relevant to patients at risk for arthrosis, from camptodactyl-arthropathy-coxa vara-pericarditis (CACP) syndrome and transient lubricin insufficiency due to trauma and inflammation.

Transcriptional Profiling of Articular Cartilage in a Porcine Model of Early Post-traumatic Osteoarthritis†

To identify the molecular pathophysiology present in early post‐traumatic osteoarthritis (PTOA), the transcriptional profile of articular cartilage and its response to surgical PTOA induction were determined. Thirty six Yucatan minipigs underwent anterior cruciate ligament (ACL) transection and were randomly assigned in equal numbers to no further treatment, reconstruction or ligament repair. Cartilage was harvested at 1 and 4 weeks post‐operatively and histology and RNA‐sequencing were performed and compared to controls. Microscopic cartilage scores significantly worsened at 1 (p = 0.028) and 4 weeks (p = 0.001) post‐surgery relative to controls, but did not differ between untreated, reconstruction or repair groups. Gene expression after ACL reconstruction and ACL transection were similar, with only 0.03% (including SERPINB7 and CR2) and 0.2% of transcripts (including INHBA) differentially expressed at 1 and 4 weeks respectively. COL2A1, COMP, SPARC, CHAD, and EF1ALPHA were the most highly expressed non ribosomal, non mitochondrial genes in the controls and remained abundant after surgery. A total of 1,275 genes were differentially expressed between 1 and 4 weeks post‐surgery. With the treatment groups pooled, 682 genes were differentially expressed at both time‐points, with the most significant changes observed in MMP1, COCH, POSTN, CYTL1, and PTGFR. This study confirmed the development of a microscopic PTOA stage after ACL surgery in the porcine model. Upregulation of multiple proteases (including MMP1 and ADAMTS4) were found; however, the level of expression remained orders of magnitude below that of extracellular matrix protein‐coding genes (including COL2A1 and ACAN). In summary, genes with established roles in PTOA as well as novel targets for specific intervention were identified.