Cartilage immunoprivilege depends on donor source and lesion location.

The ability to repair damaged cartilage is a major goal of musculoskeletal tissue engineering. Allogeneic (same species, different individual) or xenogeneic (different species) sources can provide an attractive source of chondrocytes for cartilage tissue engineering, since autologous (same individual) cells are scarce. Immune rejection of non-autologous hyaline articular cartilage has seldom been considered due to the popular notion of "cartilage immunoprivilege". The objective of this study was to determine the suitability of allogeneic and xenogeneic engineered neocartilage tissue for cartilage repair. To address this, scaffold-free tissue engineered articular cartilage of syngeneic (same genetic background), allogeneic, and xenogeneic origin were implanted into two different locations of the rabbit knee (n=3 per group/location). Xenogeneic engineered cartilage and control xenogeneic chondral explants provoked profound innate inflammatory and adaptive cellular responses, regardless of transplant location. Cytological quantification of immune cells showed that, while allogeneic neocartilage elicited an immune response in the patella, negligible responses were observed when implanted into the trochlea; instead the responses were comparable to microfracture-treated empty defect controls. Allogeneic neocartilage survived within the trochlea implant site and demonstrated graft integration into the underlying bone. In conclusion, the knee joint cartilage does not represent an immune privileged site, strongly rejecting xenogeneic but not allogeneic chondrocytes in a location-dependent fashion. This difference in location-dependent survival of allogeneic tissue may be associated with proximity to the synovium. STATEMENT OF SIGNIFICANCE: Through a series of in vivo studies this research demonstrates that articular cartilage is not fully immunoprivileged. In addition, we now show that anatomical location of the defect, even within the same joint compartment, strongly influences the degree of the resultant immune response. This is one of the first investigations to show that (1) immune tolerance to allogeneic tissue engineered cartilage and (2) subsequent implant survival are dependent on the implant location and proximity to the synovium.

ERK activation is required for hydrostatic pressure-induced tensile changes in engineered articular cartilage.

The objective of this study was to identify ERK 1/2 involvement in the changes in compressive and tensile mechanical properties associated with hydrostatic pressure treatment of self-assembled cartilage constructs. In study 1, ERK 1/2 phosphorylation was detected by immunoblot, following application of hydrostatic pressure (1 h of static 10 MPa) applied at days 10-14 of self-assembly culture. In study 2, ERK 1/2 activation was blocked during hydrostatic pressure application on days 10-14. With pharmacological inhibition of the ERK pathway by the MEK1/ERK inhibitor U0126 during hydrostatic pressure application on days 10-14, the increase in Young's modulus induced by hydrostatic pressure was blocked. Furthermore, this reduction in Young's modulus with U0126 treatment during hydrostatic pressure application corresponded to a decrease in total collagen expression. However, U0126 did not inhibit the increase in aggregate modulus or GAG induced by hydrostatic pressure. These findings demonstrate a link between hydrostatic pressure application, ERK signalling and changes in the biomechanical properties of a tissue-engineered construct.

Biomechanical evaluation of suture-holding properties of native and tissue-engineered articular cartilage.

The purpose of this study was to determine suture-holding properties of tissue-engineered neocartilage relative to native articular cartilage. To this end, suture pull-out strength was quantified for native articular cartilage and for neocartilages possessing various mechanical properties. Suture-holding properties were examined in vitro and in vivo. Neocartilage from bovine chondrocytes was engineered using two sets of exogenous stimuli, resulting in neotissue of different biochemical compositions. Compressive and tensile properties and glycosaminoglycan, collagen, and pyridinoline cross-link contents were assayed (study 1). Suture pull-out strength was compared between neocartilage constructs, and bovine and leporine native cartilage. Uniaxial pull-out test until failure was performed after passing 6-0 Vicryl through each tissue (study 2). Subsequently, neocartilage was implanted into a rabbit model to examine short-term suture-holding ability in vivo (study 3). Neocartilage glycosaminoglycan and collagen content per wet weight reached 4.55 ± 1.62% and 4.21 ± 0.77%, respectively. Tensile properties for neocartilage constructs reached 2.6 ± 0.77% MPa for Young's modulus and 1.39 ± 0.63 MPa for ultimate tensile strength. Neocartilage reached ~ 33% of suture pull-out strength of native articular cartilage. Neocartilage cross-link content reached 50% of native values, and suture pull-out strength correlated positively with cross-link content (R² = 0.74). Neocartilage sutured into rabbit osteochondral defects was successfully maintained for 3 weeks. This study shows that pyridinoline cross-links in neocartilage may be vital in controlling suture pull-out strength. Neocartilage produced in vitro with one-third of native tissue pull-out strength appears sufficient for construct suturing and retention in vivo.

Tribological altruism: A sacrificial layer mechanism of synovial joint lubrication in articular cartilage.

Boundary lubrication is characterized by sliding surfaces separated by a molecularly thin film that reduces friction and wear of the underlying substrate when fluid lubrication cannot be established. In this study, the wear and replenishment rates of articular cartilage were examined in the context of friction coefficient changes, protein loss, and direct imaging of the surface ultrastructure, to determine the efficiency of the boundary lubricant (BL) layer. Depletion of cartilage lubricity occurred with the concomitant loss of surface proteoglycans. Restoration of lubrication by incubation with synovial fluid was much faster than incubation with culture media and isolated superficial zone protein. The replenishment action of the BL layer in articular cartilage was rapid, with the rate of formation exceeding the rate of depletion of the BL layer to effectively protect the tissue from mechanical wear. The obtained results indicate that boundary lubrication in articular cartilage depends in part on a sacrificial layer mechanism. The present study provides insight into the natural mechanisms that minimize wear and resist tissue degeneration over the lifetime of an organism.

Effects of TGF-ß1 on alternative splicing of Superficial Zone Protein in articular cartilage cultures.

OBJECTIVE: Superficial Zone Protein (SZP) is expressed by the superficial zone chondrocytes and is involved in boundary lubrication of the articular cartilage surface. SZP protein expression is dependent on anatomical location and is regulated by the transforming growth factor-ß (TGF-ß) pathway. The hypothesis of this study was that between load-bearing, and non-load-bearing locations, of the femoral medial condyle alternative splice isoforms of SZP are different, and regulated by TGF-ß1. METHODS: Using reverse transcription-polymerase chain reaction (RT-PCR) we identified differentially expressed SZP alternative splicing. Using recombinant proteins of the N-terminal region produced from these isoforms, we identified differences in binding to heparin and the extracellular matrix. RESULTS: We identified a novel splice form of SZP (isoform E), lacking exons 2-5. Differences in alternative splicing were observed between anterior load-bearing locations of the femoral medial condyle (M1) compared to the posterior non-load-bearing location (M4). TGF-ß1 increased splicing out of exons 4 and 5 encoding a heparin binding domain. The minimal induction time for changes in splicing by TGF-ß1 at the M1 location was 1h, although this did change total SZP mRNA levels. Inhibition of Smad3 phosphorylation inhibited TGF-ß1 induced splicing, and SZP protein expression. Recombinant proteins corresponding to isoforms upregulated by TGF-ß1 had reduced binding. The SZP dimerization domain is located within exon 3. CONCLUSIONS: In conclusion, alternative splicing of SZP is regulated by TGF-ß1 signaling and may regulate SZP interaction with heparin/heparan sulfate or other components in the extracellular matrix of articular cartilage by splicing out of the heparin binding domain.

Atomic force microscope investigation of the boundary-lubricant layer in articular cartilage.

OBJECTIVE: To determine the roles of superficial zone protein (SZP), hyaluronan (HA), and surface-active phospholipids (SAPL) in boundary lubrication of articular cartilage through systematic enzyme digestion using trypsin, hyaluronidase, and phospolipase-C (PLC) surface treatments. METHODS: The friction coefficient of articular cartilage surfaces was measured with an atomic force microscope (AFM) before and after enzyme digestion. Surface roughness, adhesion, and stiffness of the articular surface were also measured to determine the mechanism of friction in the boundary lubrication regime. Histology and transmission electron microscopy were used to visualize the surface changes of treatment groups that showed significant friction changes after enzyme digestion. RESULTS: A significant increase in the friction coefficient of both load-bearing and non load-bearing regions of the joint was observed after proteolysis by trypsin. Treatment with trypsin, hyaluronidase, or PLC did not affect the surface roughness. However, trypsin treatment decreased the adhesion significantly. Results indicate that the protein component at the articular cartilage surface is the main boundary lubricant, with SZP being a primary candidate. The prevailing nanoscale deformation processes are likely plastic and/or viscoelastic in nature, suggesting that plowing is the dominant friction mechanism. CONCLUSIONS: The findings of this study indicate that SZP plays an intrinsic and critical role in boundary lubrication at the articular surface of cartilage, whereas the effects of HA and SAPL on the tribological behavior are marginal.