Introduction: Articular cartilage (AC) is an avascular tissue with precise polarity and organization. The three distinct zones are: surface, middle and deep. The production and accumulation of the superficial zone protein (SZP), also known as lubricin, by the surface zone is a characteristic feature of AC. To date, there is a wealth of evidence showing differentiation of AC from mesenchymal stem cells. Most studies that described chondrogenic differentiation did not focus on AC with characteristic surface marker SZP/lubricin. The present investigation was initiated to determine the induction of SZP/lubricin in skeletal muscle-derived mesenchymal stem/progenitor cells (MDMSCs) by transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7). Methods: MDMSCs were cultured as a monolayer at a density of 1 × 10⁵ cells/well in 12-well tissue culture plates. Cell cultures were treated for 3, 7 and 10 days with TGF-β1 and BMP-7. The medium was analyzed for SZP. The cells were used to isolate RNA for RT-PCR assays for SZP expression. Results: The SZP/lubricin increased in a time-dependent manner on Days 3, 7 and 10 in the medium. As early as Day 3, there was a three-fold increase in response to 3 ng/ml of TGF-β1 and 300 ng/ml of BMP-7. This was confirmed by immunochemical localization of SZP as early as Day 3 after treatment with TGF-β1. The expression of SZP mRNA was enhanced by TGF-β1. Conclusions: The present investigation demonstrated the efficient and reproducible induction of SZP/lubricin accumulation by TGF-β1 and BMP-7 in skeletal MDMSCs. Optimization of the experimental conditions may permit the utility of MDMSCs in generating surface zone-like cells with phenotypic markers of AC and, therefore, constitute a promising cell source for tissue engineering approaches of superficial zone cartilage.

Introduction: Pre-clinical testing of hemiarthroplasty devices requires that the tribological conditions present in vivo with live cartilage be closely duplicated. A current limitation in the tribological testing of live cartilage involves the use of cell-culture media as lubricant. Study Aim: to develop and test a new hyaluronan-phospholipid based medium (HA–phospholipid medium) that combines the rheological and frictional properties of synovial fluid with the nourishing properties of culture media to keep cells alive. Materials and Methods: The HA–phospholipid medium consisted of culture medium with added phospholipid dipalmitoylphosphatidylcholine (0.3 mg/mL), and hyaluronic acid (2.42 mg/mL). A standard cell culture medium was used as the control. The rheology of each medium was determined using a flat plate configuration. Bovine calf cartilage was used to assess cell viability and friction in each medium. For friction measurements, a cobalt-chrome alloy ball was articulated against cartilage disks immersed in medium. Results: Lipid vesicles 0.1 to 50 μm in diameter were identified in the HA–phospholipid medium. Cartilage cell viability was significantly higher in the HA–phospholipid medium (62% ± 8%, 95% CI) than in control medium (49.5% ± 5%) (p = 0.009). The HA–phospholipid medium exhibited strong shear-thinning behavior, similar to synovial fluid, with viscosities ~100-fold higher at 10 s⁻¹ and 5-fold higher at 20,000 s⁻¹ than the approximately Newtonian control medium. The HA–phospholipid medium also yielded 20% lower friction values than the control medium after one hour of testing. Conclusions: The rheological and friction results indicate that the HA–phospholipid medium is superior to the control cell culture medium in emulating the shear thinning and lubricative properties of natural synovial fluid, making it more clinically relevant for in vitro wear and friction testing with live cartilage.

It is well established that the total protein concentration and albumin-to-globulin ratio influence the wear of ultra-high molecular polyethylene (UHMWPE, “polyethylene”) in joint prostheses. A factor on wear not yet studied, but of possible clinical relevance, is protein cleavage. Such cleavage is expected in the presence of an inflammatory response and as a result of wear processes at the articular interface. The aim of this study was to compare the tribological behavior of polyethylene articulated against an orthopedic wrought CoCrMo alloy for three lubricants: cleaved albumin, uncleaved albumin, and newborn calf serum (control). We hypothesized that the cleavage of albumin will increase the friction and wear rate of polyethylene, with a concomitant roughening of the polymer surface and the generation of larger wear debris particles. Cleavage of the bovine albumin into five fragments was performed by digestion with cyanogen bromide. In pin-on-flat (POF) wear tests of polyethylene pins made of Ticona GUR^® 1020/1050 against CoCrMo alloy discs, the cleaved albumin led to the lowest polyethylene wear and highest friction coefficients, whereas albumin led to the highest wear rates. In knee simulator tests, the albumin lubricant also led to a 2.7-fold increase in the tibial insert wear rate compared to the regular bovine serum lubricant (a wear rate for the cleaved albumin could not be obtained). The generated polyethylene wear particles were of increasing size and fibrillar shape in going from serum to albumin to cleaved albumin, although only the shape achieved statistical significance. Unlike bovine serum, cleaved albumin led to wear scars for both the POF and simulator wear tests that closely emulated the morphological features observed on explanted polyethylene tibial inserts from total knee replacements. We posit that the smaller protein fragments can more efficiently adsorb on the surfaces of both the polyethylene and the metal, thus offering protection against wear, while at the same time leading to an increase in friction, particle size, and particle elongation, as the protein fragment films interact adhesively during sliding. The results of this study have implications for pre-clinical wear testing methodology as they suggest that albumin concentration may be more pertinent than total protein concentration for wear testing polyethylene.

Total Joint Replacement (TJR) devices undergo standardized wear testing in mechanical simulators while submerged in a proteinaceous testing solution to mimic the environmental conditions of artificial joints in the human body. Typically, bovine calf serum is used to provide the required protein content. However, due to lot-to-lot variability, an undesirable variance in testing outcome is observed. Based on an earlier finding that yellowish-orange serum color saturation is associated with wear rate, we examined potential sources of this variability, by running a comparative wear test with bilirubin; hemin; and a fatty acid, oleic acid, in the lubricant. All these compounds readily bind to albumin, the most abundant protein in bovine serum. Ultrahigh molecular weight polyethylene (UHMWPE) pins were articulated against CoCrMo discs in a pin-on-disc tribometer, and the UHMWPE wear rates were compared between lubricants. We found that the addition of bilirubin increased wear by 121%, while hemin had a much weaker, insignificant effect. When added at the same molar ratio as bilirubin, the fatty acid tended to reduce wear. Additionally, there was a significant interaction with respect to bilirubin and hemin in that UHMWPE wear rate decreased with increasing fatty acid concentration. We believe the conformational change in albumin by binding bilirubin makes it more likely to form molecular bridges between UHMWPE and the metal counterface, thus increasing adhesive wear. However, fatty acids compete for binding sites on albumin, and can prevent this conformational change. Hence, the protein is stabilized, and the chance for albumin to form bridges is lowered. Ultimately, UHMWPE wear rate is driven by the competitive binding of bilirubin and fatty acid to albumin.

Lubricin is a secreted, mucin-like glycoprotein and proteoglycan abundant in synovial fluid that provides boundary lubrication and prevents cell adhesion in synovial joints. The antilubricin S6.79 monoclonal antibody recognizes an O-linked glycopeptide epitope in lubricin’s mucin domain. The central, long mucin domain of lubricin is extensively O-glycosylated with Gal(β1-3)GalNAc-O-Ser/Thr, and about two thirds of the O-glycosylated sites are capped with sialic acid. Our aim was to determine whether removal of sialic acid by sialidase could improve the detection of lubricin in a number of human tissues using the S6.79 monoclonal antibody. Sialidase treatment caused a dramatic increase in antibody reactivity in human pericardium, splenic capsule and trabeculae, plasma, serum, eye sleep extract, and liver sinusoids. Sialidase had minimal effect on S6.79 antibody reactivity with lubricin in synovial fluid and synovial tissue. These observations suggest that the origin of lubricin in blood may be different from that in synovial fluid and that desialylation of lubricin is essential for unmasking epitopes within the mucin domain.