This protocol describes the preparation, biotribological testing, and analysis of osteochondral cylinders sliding against metal implant material. Outcome measures included in this protocol are metabolic activity, gene expression and histology.
Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are now available for the shoulder, hip, ankle and the first metatarsalphalangeal joint. While providing pain reduction and clinical improvement, progressive degenerative changes of the opposing cartilage are observed in many patients. The mechanisms leading to this damage are not fully understood. This protocol describes a tribological experiment to simulate a metal-on-cartilage pairing and comprehensive analysis of the articular cartilage. Metal implant material is tested against bovine osteochondral cylinders as a model for human articular cartilage. By applying different loads and sliding speeds, physiological loading conditions can be imitated. To provide a comprehensive analysis of the effects on the articular cartilage, histology, metabolic activity and gene expression analysis are described in this protocol. The main advantage of tribological testing is that loading parameters can be adjusted freely to simulate in vivo conditions. Furthermore, different testing solutions might be used to investigate the influence of lubrication or pro-inflammatory agents. By using gene expression analysis for cartilage-specific genes and catabolic genes, early changes in the metabolism of articular chondrocytes in response to mechanical loading might be detected.
The treatment of osteochondral defects is demanding and requires surgery in many cases. For focal osteochondral lesions in middle-aged patients, focal metallic implants are a viable option, especially after the failure of primary treatment, like bone marrow stimulation (BMS) or autologous chondrocyte implantation (ACI)1. Partial surface replacements can be considered salvage procedures that can reduce pain and improve the range of motion2. These implants are typically composed of a CoCrMo alloy and are available in different sizes and offset configurations to match the normal anatomy3. While initially developed for defects on the medial femoral condyle in the knee, such implants are now available and in use for the hip, ankle, shoulder, and elbow4,5,6. For a satisfactory outcome, it is crucial to assess the mechanical joint alignment and condition of the opposing cartilage. Furthermore, correct implantation without protrusion of the implant has been shown to be fundamental7.
Clinical studies demonstrated excellent short-term results in terms of pain reduction and improvement of function in middle-aged patients for various locations5,6,8. Compared with allograft implantation, focal metal implants allow early weight bearing. However, the opposing articular cartilage showed accelerated wear in a considerable number of patients9,10. Hence, even with proper placement, in many cases degeneration of the native cartilage seems inevitable, while the underlying mechanisms remain unclear. Similar degenerative changes have been observed after bipolar hemiarthroplasty of the hip11 and are increased with activity and loading12.
Tribological experiments provide the possibility to study such pairings in vitro and simulate different loading situations occurring under physiological conditions13. The use of osteochondral pins offers a simple geometry model to investigate the tribology of articular cartilage sliding against native cartilage or any implant material14 and might further be used in whole joint simulation models15. Metal-on-cartilage pairings show accelerated cartilage wear, extracellular matrix disruption, and decreased cell viability in the superficial zone compared with a cartilage-on-cartilage pairing16. Damage to the cartilage occurred mainly in the form of delamination between the superficial and middle zones17. However, the mechanisms leading to cartilage degeneration are not fully understood. This protocol provides a comprehensive analysis of the biosynthetic activity of articular cartilage. By the determination of metabolic activity and gene expression levels of catabolic genes, early indications for cartilage breakdown might be identified. The advantage of in vitro tribological experiments is that loading parameters can be adjusted to imitate various loading conditions.
Hence, the following protocol is suitable to simulate a metal-on-cartilage pairing, representing an experimental hemiarthroplasty model.
1. Preparation of metal cylinders
2. Harvesting of osteochondral cylinders
3. Tribological testing
4. Analysis
NOTE: Osteochondral cylinder are analyzed for metabolic activity and gene expression to investigate biological activity; histology is performed to study cartilage surface integrity and the underlying matrix.
The contact area and contact pressure must be confirmed using a pressure measurement film (Figure 1). Physiological loading condition can be confirmed by comparing with reference imprints for defined contact pressures. During testing, the coefficient of friction is monitored constantly. With a migrating contact area, a low friction coefficient can be maintained for at least 1 h (Figure 2). Using Safranin O staining the extracellular matrix composition and structure can be determined (Figure 3). The intensity of Safranin O staining is proportional to the proteoglycan content. Fast Green counterstains the non-collagen sites and provides a clear contrast to the Safranin O staining. The proteoglycan content varies over the articular surface but should be uniform throughout the tissue section in baseline samples (Figure 3A). Control samples submerged in the testing solution show extraction of GAGs, which can be counteracted by mechanical loading (Figure 3B, 3C). Metabolic activity of the bovine articular chondrocytes is independent of the harvesting site, but shows an increase with mechanical loading compared with unloaded controls (Figure 4). The gene expression levels of cartilage-specific genes (COL2A1, ACAN) increase with physiological loading conditions, whereas catabolic genes (COL1A1 and MMP13) are upregulated with stationary contact area (Figure 5).
Volume (µl) | |
Transcriptor RT Reactions Buffer 5x conc. | 6 |
Protector RNase Inhibitor 40U/µl | 0.75 |
Deoxynucleotide Mix 10 mM each | 3 |
Random Hexamer Primer 600 µM | 3 |
Transcriptor Reverse Transcriptase 20 U/µl | 0.75 |
MS2 RNA (0,8 µg/µl) | 0.375 |
Nuclease free distilled water | 0.125 |
Total volume | 14 |
Table 1: Reagents for a single reaction for cDNA synthesis.
Volume (µl) | |
FastStart Probe Master 2X | 5 |
Hydrolysis Probe 2,5 µM | 1 |
Left PrimerGAPDH 5 µM | |
Right Primer GAPDH 5 µM | |
Nuclease free distilled water | 3 |
Total Master Mix | 9 |
Table 2: Reagents for the Master Mix for a single PCR.
Figure 1: Pressure measurement of the initial contact area at the metal-cartilage interface before testing. Due to the convexity of the metal cylinder and the articular surface and its elastic properties, the initial contact area is elliptical. During sliding, this initial contact area moves with a stroke of 2 mm, resulting in a larger area that is exposed to mechanical loading; scale bar = 2 mm. Please click here to view a larger version of this figure.
Figure 2: Time-depended coefficient of friction (duration 1 hour) for seven samples tested at 8 mm/s sliding speed and 1 N load (2 MPa contact pressure). Each colored line represents the COF of one osteochondral cylinder. The observed variability is within the limits for biological samples. Please click here to view a larger version of this figure.
Figure 3: Histological cross-sections of bovine osteochondral samples stained with Safranin-O and Fast Green. (A) Baseline samples show high GAG content throughout the articular cartilage. (B) Control sample submerged in testing solution without mechanical loading show less Safranin-O staining in the middle zone, indication an extraction of proteoglycans. (C) Tested samples show higher GAG content compared with controls, indicating mechanical stimulation; scale bar = 250 µm Please click here to view a larger version of this figure.
Figure 4: Metabolic activity of bovine articular chondrocytes after tribological testing with different loading variations and controls. The horizontal dotted line represents baseline levels. The nonparametric Kruskal–Wallis test was performed for comparison between testing groups followed by Dunn’s post hoc test in case of significance. *p < 0.05. This figure has been modified from Stotter et al.18. Please click here to view a larger version of this figure.
Figure 5: Gene expression of cartilage-specific genes after tribological testing with different loading conditions and controls. COL2A1=collagen type 2; ACAN=aggrecan; COL1A1= collagen type 1; MMP13= matrix metalloproteinase 13. The expression levels were normalized to the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase). The horizontal dotted lines represent baseline expression levels. The nonparametric Kruskal–Wallis test was performed for comparison between testing groups followed by Dunn’s post hoc test in case of significance. *p < 0.05. This figure has been modified from Stotter et al.18. Please click here to view a larger version of this figure.
Focal metallic implants represent a salvage procedure for osteochondral defects, especially in middle-aged patients and after failed primary treatment. Although clinical studies demonstrated promising short-term results, one observed complication is damage to the opposing, native cartilage10. Cadaver and biomechanical studies show clear evidence that proper implantation with flat or slightly recessed positioning maintains natural contact pressures19. Tribological experiments provide a possibility to test various cartilage pairings in vitro. In such, loading conditions, lubrication, material pairings and duration might be adjusted as desired.
Bovine cartilage is available in high quantity at the local abattoir. The cellularity and the zonal structure are very similar to the human femoral condyles20. However, proteoglycan content is site-specific, whereas gene expression levels have been shown to be uniform over the articular surface. In this protocol, osteochondral plugs were harvested from the weight bearing area. Cartilage thickness, collagen architecture and resulting tribological properties show regional differences over the articular surface16. The limitation of using osteochondral plugs in an unconfined loading setup with a disrupted collagen network and changed fluid pressurization compared with whole joint models need to be considered.
In the majority of tribological studies, PBS alone is used as testing solution to generate more robust data. PBS is a buffer solution with isotonic osmolarity and helps to maintain a constant pH during biological experiments. Using PBS with hyaluronic acid provides boundary lubrication and reduced friction21. Accordingly, synovial fluid reduces the friction coefficient and improves fluid pressurization compared with saline22. The friction coefficient depends on various system properties, demonstrated by the classic Stribeck Curve. The Stribeck Curve relates the friction coefficient and viscosity, speed and load and presents the basic lubrication regimes: boundary, mixed, and hydrodynamic lubrication. Boundary lubrication can be obtained with PBS alone as lubricating fluid, but loading parameters would need to be adjusted accordingly. The COF delivered from the tests are average values over the stroke. Thus, it can be assumed that different lubrication conditions occur during the cycle. During standstill at reversal position, boundary conditions might be prevailing, while mixed lubrication might be predominant during sliding. Based on absolute duration during the sliding cycle, the latter would have had more influence on the mean COF value.
To investigate physiological conditions occurring in joints during daily activities, loading conditions can be adjusted accordingly in the tribometer software. Pressure sensitive measurements should be used to confirm the desired contact pressures. Reported femorotibial contact pressures range between 1 MPa during standing and up to 10 MPa during downhill running23. With a focal resurfacing, implant pressures are just slightly elevated compared with healthy joints24. Reported relative sliding velocities during the gait cycle are reported up to 100 mm/s with high variations during the different phases. This means that relative joint movements exceed the velocities that can be applied in this tribological setup. To mimic natural kinematic conditions and contact pressures in healthy knee joints, loading conditions range from 1 to 10 MPa contact pressure and 5 to 100 mm/s sliding speed. However, while high loads can be applied in this experimental setup, the range of sliding velocities is limited. Pathological loading conditions, both overload and inadequate loads, might also be simulated. Low sliding velocities or static loading equate immobilization, while higher loads represent nonphysiological mechanical stimulation.
As enzymatic digestion can affect the expression of cartilage-specific genes, a nonenzymatic tissue homogenization is described in this protocol. During cDNA synthesis, in addition to the instructions, RNA from bacteriophage MS2 is added for stabilization purposes. Gene expression levels, but not proteins, were analyzed to detect early changes in the biosynthetic activity of articular chondrocytes. In addition to histological sections and metabolic activity, these assays provide comprehensive information on the effects of mechanical loading on articular cartilage.
The authors have nothing to disclose.
This research was funded by NÖ Forschungs- und Bildungsges.m.b.H. and the provincial government of Lower Austria through the Life Science Calls (Project ID: LSC15-019) and by the Austrian COMET Program (Project K2 XTribology, Grant No. 849109).
Amphotericin B | Sigma‐Aldrich Chemie GmbH | A-2942-100ML | |
buffered formaldehyde solution 4% | VWR | 97131000 | |
Cell Proliferation Kit II (XTT) | Roche Diagnostics | 11465015001 | XTT-based ex vivo toxicology assay |
CoCrMo raw material | Acnis International | CoCrMo rods 6mm in diameter | |
CryoStar NX70 Cryostat | Thermo Fischer Scientific | cryosectioning device | |
dimethyl sulfoxide (DMSO) | Sidma-Aldrich Chemie | D 2438-10ML | |
Dulbecco’s modified Eagle’s medium | Sigma‐Aldrich Chemie GmbH | medium | |
fetal bovine serum | Gibco | ||
Hyaluronic acid | Anika Therapeutics Inc. | component of lubricating solution | |
iCycler | BioRad | thermal cycler | |
Leica microscope DM‐1000 | Leica | microscope for histology | |
LightCycler 480 Sealing Foil | Roche Diagnostics | ||
LightCycler 96 | Roche Diagnostics | thermal cycler for PCR | |
MagNA Lyser Green Beads | Roche Diagnostics | 3358941001 | |
Osteochondral Autograft Transfer System (OATS) | Arthrex Inc. | cutting tube for harvesting osteochondral cylinders | |
osteosoft | Merck | 1017279010 | decalcifier-solution |
Penicillin /Streptomycin | Sigma‐Aldrich Chemie GmbH | P4333-100ML | |
phosphate‐buffered saline | Sigma‐Aldrich Chemie GmbH | PBS | |
Prescale Low Pressure | Fujifilm | pressure indicating film | |
RNeasy Fibrous Tissue Kit | QIAGEN | 74404 | |
Synergy 2 | BioTek Instruments | plate reader | |
Tetra‐Falex MUST | Falex Tribology | Tribometer | |
Tissue‐ Tek O.C.T. | SAKURA | 4583 | embedding formulation |
Transcriptor First Strand cDNA Synthesis Kit | Roche Diagnostics | 40897030001 | |
β-mercaptoethanol | Sidma-Aldrich Chemie | M3148 |