Bovine Stifle Joints Research Articles

Quantitative spectral micro-CT of a CA4+ loaded osteochondral sample with a tabletop system

Micro-computed tomography (μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}CT) is the gold standard for nondestructive 3D imaging of biomedical samples in the centimeter scale, but it has limited effectiveness in revealing intricate soft tissue details due to the limited attenuation contrast. Radiopaque contrast agents that accumulate in the structures of interest are employed to enhance their visibility. However, the increased attenuation provided by the contrast agents does not guarantee discrimination among tissues. This issue can be solved by spectral μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}CT (Sμ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}CT) systems employing small-pixel chromatic photon-counting detectors. These detectors, combined with material decomposition algorithms, allow the generation of high-resolution material-specific 3D maps. This work aims to demonstrate the potential of photon-counting X-ray Sμ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}CT on osteochondral samples loaded with a cationic iodinated contrast agent (CA4+) at a spatial resolution below 50 μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}m, and to compare the results against a conventional μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}CT system. An osteochondral sample extracted from a bovine stifle joint was loaded with CA4+ and imaged with a novel multimodal X-ray imaging system, featuring a 62 μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}m pixel CdTe spectral detector (Pixirad1-PixieIII). After material decomposition, quantitative 3D density maps of iodine and hydroxyapatite were reconstructed. The same sample was also scanned with a commercial μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}CT scanner with matched spectrum and exposure time. SCTμ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document} images at a (measured) spatial resolution comparable with the commercial scanner (∼\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\sim$$\\end{document}45 μ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upmu$$\\end{document}m) were obtained. Spectral images allowed for a fully automatic segmentation of cartilage and subchondral bone. The unambiguous discrimination between iodine and hydroxyapatite revealed a more realistic representation of proteoglycan distribution compared to conventional imaging.

Response of Articular Cartilage to Hyperosmolar Stress: Report of an Ex Vivo Injury Model

Background: Physiological 0.9% saline is commonly used as an irrigation fluid in modern arthroscopy. There is a growing body of evidence that a hyperosmolar saline solution has chondroprotective effects, especially if iatrogenic injury occurs. Purpose: To (1) corroborate the superiority of a hyperosmolar saline solution regarding chondrocyte survival after mechanical injury and (2) observe the modulatory response of articular cartilage to osmotic stress and injury. Study Design: Controlled laboratory study. Methods: Osteochondral explants were isolated from bovine stifle joints and exposed to either 0.9% saline (308 mOsm) or hyperosmolar saline (600 mOsm) and then damaged with a sharp dermatome blade to attain a confined full-thickness cartilage injury site, incubated in the same fluids for another 3 hours, and transferred to chondropermissive medium for further culture for 1 week. Chondrocyte survival was assessed by confocal imaging, while the cellular response was evaluated over 1 week by relative gene expression for apoptotic and inflammatory markers and mediator release into the medium. Results: The full-thickness cartilage cut resulted in a confined zone of cell death that mainly affected superficial zone chondrocytes. Injured samples that were exposed to hyperosmolar saline showed less expansion of cell death in both the axial (P < .007) and the coronal (P < .004) plane. There was no progression of cell death during the following week of culture. Histological assessment revealed an intact cartilage matrix and normal chondrocyte morphology. Inflammatory and proapoptotic genes were upregulated on the first days postexposure with a notable downregulation toward day 7. Mediator release into the medium was concentrated on day 3. Conclusion: This in vitro cartilage injury model provides further evidence for the chondroprotective effect of a hyperosmolar saline irrigation fluid, as well as novel data on the capability of articular cartilage to quickly regain joint homeostasis after osmotic stress and injury. Clinical Relevance: Raising the osmolarity of an irrigating solution may be a simple and safe strategy to protect articular cartilage during arthroscopic surgery.

Anisotropy of T2 and T1ρ relaxation time in articular cartilage at 3 T.

The anisotropy of R2 and R1ρ relaxation rates in articular cartilage contains information about the collagenous structure of the tissue. Here we determine and study the anisotropic and isotropic components of T2 and T1ρ relaxation parameters in articular cartilage with a clinical 3T MRI device. Furthermore, a visual representation of the topographical variation in anisotropy is given via anisotropy mapping. Eight bovine stifle joints were imaged at 22 orientations with respect to the main magnetic field using T2, continuous-wave (CW) T1ρ, and adiabatic T1ρ mapping sequences. Relaxation rates were separated into isotropic and anisotropic relaxation components using a previously established relaxation anisotropy model. Pixel-wise anisotropy values were determined from the relaxation-time maps using Michelson contrast. The relaxation rates obtained from the samples displayed notable variation depending on the sample orientation, magnetization preparation, and cartilage layer. R2 demonstrated significant anisotropy, whereas CW-R1ρ (300 Hz) and CW-R1ρ (500 Hz) displayed a low degree of anisotropy. Adiabatic R1ρ was largely isotropic. In the deep cartilage regions, relaxation rates were generally faster and more anisotropic than in the cartilage closer to the tissue surface. The isotropic relaxation rate components were found to have similar values regardless of measurement sequence. The fitted relaxation model for T2 and T1ρ demonstrated varying amounts anisotropy, depending on magnetization preparation, and studied the articular cartilage layer. Anisotropy mapping of full joints showed varying amounts of anisotropy depending on the quantitative MRI parameter and topographical location, and in the case of T2, showed systematic changes in anisotropy across cartilage depth.

N-TELOPEPTIDE DERIVED FROM TYPE II COLLAGEN UPREGULATES EXPRESSION OF BETA1 INTEGRIN AND MMPS IN CARTILAGE OF PATIENTS RECEIVING HIP OR KNEE ARTHROPLASTY

Previous studies showed that telo-peptides degraded from type II collagen, a type of collagen fragments, could induce cartilage damage in bovine stifle joints. We aim to investigate the role of integrins (ITGs) and matrix metalloproteinases (MMPs) in collagen fragment-induced human cartilage damage that is usually observed in osteoarthritis (OA). We hypothesized that N-telopeptide (NT) derived from type II collagen could up-regulate the expression of β1 integrin (ITGB1) and then MMPs that may lead to osteoarthritic cartilage damage.Human chondrocytes were isolated from femoral head or tibial plateau of patients receiving arthroplasty (N = 24). Primary chondrocyte cultures were either treated with 30 µM NT, or 30 µM scrambled NT (SN), or PBS, or left untreated for 24 hrs. Total proteins and RNAs were extracted for examination of expression of ITGB1 and MMPs-3&amp;13 with Western blotting and quantitative real-time PCR.Compared to untreated or PBS treated chondrocytes, NT-treated chondrocytes expressed significantly higher levels of ITGB1 and MMPs-3&amp;-13. However, SN also up-regulated expression of ITGB1 and MMP-13.ITGB1 and MMPs-3&amp;-13 might mediate the catalytic effect of NT, a type of collagen fragments, on human cartilage damage that is a hallmark of OA.

The Longer-Term Effects of a Single Bupivacaine Exposure on the Mechanical Properties of Native Cartilage Explants.

The purpose of this study was to determine the in vitro effects of a single exposure of bupivacaine on the mechanical properties of bovine cartilage explants at 3 weeks. Femoral condyle articular cartilage explants were aseptically harvested from juvenile bovine stifle joints before being exposed to chondrogenic medium containing 0.50% (wt/vol) bupivacaine, 0.25% (wt/vol) bupivacaine, or no medication (control) for 1 hour. Explants were then washed and maintained in culture in vitro for 3 weeks before testing. Cell viability, tensile and compressive mechanical properties, histological properties, and biochemical properties were then assessed. Explants exhibited a dose-dependent decrease in mean tensile Young's modulus with increasing bupivacaine concentration (9.86 MPa in the controls, 6.48 MPa in the 0.25% bupivacaine group [P = 0.048], and 4.72 MPa in the 0.50% bupivacaine group [P = 0.005]). Consistent with these results, collagen content and collagen crosslinking decreased with bupivacaine exposure as measured by mass spectrometry. Compressive properties of the explants were unaffected by bupivacaine exposure. Explants also exhibited a trend toward dose-dependent decreases in viability (51.2% for the controls, 47.3% for the 0.25% bupivacaine-exposed group, and 37.0% for the 0.50% bupivacaine-exposed group [P = 0.072]). Three weeks after 1-hour bupivacaine exposure, the tensile properties of bovine cartilage explants were significantly decreased, while the compressive properties remained unaffected. These decreases in tensile properties corresponded with reductions in collagen content and crosslinking of collagen fibers. Physicians should be judicious regarding the intra-articular administration of bupivacaine in native joints.

HMGB1 can activate cartilage progenitor cells in response to cartilage injury through the CXCL12/CXCR4 pathway.

Recent studies have suggested that cartilage progenitor cells (CPCs) could be activated and differentiated into chondrocytes to produce matrix and to restore the integrity of damaged cartilage after injury. However, the mechanism involved in CPC activation upon damage is still unclear. This study aims to investigate the role of high mobility group box chromosomal protein 1 (HMGB1) in both activation and migration of CPCs during cartilage injury. Explants harvested from mature bovine stifle joints were used for impact injury. The proliferation and migration of CPCs were examined via confocal imaging. Gene and protein expression of Hmbg1, Cxcl12, and Cxcr4 was also examined by quantitative polymerase chain reaction (qPCR), ELISA, and western blot. Each experiment was repeated 3 times. ANOVA and Student's t-test were performed for statistical analysis. HMGB1 released from dead and damaged chondrocytes after an impact injury could activate CPCs in the superficial zone of cartilage and promote their migration and proliferation to injury sites. However, the block of HMGB1 activation with its specific binding inhibitor glycyrrhizin inhibits the proliferation and migration of CPCs. Further investigations demonstrate that HMGB1 promotes CPCs migration through the pathway of C-X-C motif chemokine 12 (CXCL12) and its receptor CXCR4. Quantitative analysis of HMGB1 in cell culture medium also indicates that CPCs may have a self-activation property after the HMGB1 released from dead cells has been exhausted. HMGB1 is a pivotal factor that could enhance the migration and proliferation of CPCs through the CXCL12/CXCR4 pathway after cartilage injury, which could provide useful information for cartilage repair and osteoarthritis treatment.

Osteochondral Explant Isolation and Culture Under a Compression and Shear Bioreactor.

Osteochondral explants harvested from different species are valuable preclinical ex vivo models for tissue engineering research. In this chapter, we describe the isolation of osteochondral plugs from bovine stifle joints, followed by defect creation, and plug preparation in a straightforward manner before mechanical loading using a compression and shear bioreactor. The method can be adapted to isolate osteochondral plugs from any animal species and to load explants in any type of bioreactor.

A Cationic Contrast Agent in X-ray Imaging of Articular Cartilage: Pre-Clinical Evaluation of Diffusion and Attenuation Properties.

The aim of this study was the preliminary assessment of a new cationic contrast agent, the CA4+, via the analysis of spatial distribution in cartilage of ex vivo bovine samples, at micrometer and millimeter scale. Osteochondral plugs (n = 18) extracted from bovine stifle joints (n = 2) were immersed in CA4+ solution up to 26 h. Planar images were acquired at different time points, using a microCT apparatus. The CA4+ distribution in cartilage and saturation time were evaluated. Tibial plates from bovine stifle joints (n = 3) were imaged with CT, before and after 24 h-CA4+ bath immersion, at different concentrations. Afterward, potential CA4+ washout from cartilage was investigated. From microCT acquisitions, the CA4+ distribution differentiated into three distinct layers inside the cartilage, reflecting the spatial distribution of proteoglycans. After 24 h of diffusion, the iodine concentration reached in cartilage was approximately seven times that of the CA4+ bath. The resulting saturation time was 1.9 ± 0.9 h and 2.6 ± 2.9 h for femoral and tibial samples, respectively. Analysis of clinical CT acquisitions confirmed overall contrast enhancement of cartilage after 24 h immersion, observed for each CA4+ concentration. Distinct contrast enhancement was reached in different cartilage regions, depending on tissue’s local features. Incomplete but remarkable washout of cartilage was observed. CA4+ significantly improved cartilage visualization and its qualitative analysis.

Mechanical characterization of articular cartilage degraded combinedly with MMP-1 and MMP-9

Matrix metalloproteinases (MMPs) degrade the extracellular matrix (ECM) subsequently damaging cartilage and altering biomechanical properties. Collectively, MMPs cleave every ECM macromolecule. However, MMPs present complex substrate interactions and digest differing ECM components making it difficult to understand the individual role each MMP plays in cartilage degradation. To understand the combined impact MMPs have on cartilage biomechanical properties, MMPs from two subfamilies: collagenase and gelatinase were investigated. Three ratios of MMP-1 (c) and MMP-9 (g), c1:g1, c1:g0 and c0:g1 were considered. Cartilage plugs (n = 30) were collected from the femoral condyles of 3 bovine stifle joints. In groups of 10, samples were treated with MMP-1, MMP-9, or a combination. Samples were subjected to indentation loading up to 20% bulk strain and were assessed mechanically and histologically to determine the degradative impact. Young's modulus and peak load were compared between the control and degraded explants. In comparison to samples degraded by MMP-1 or MMP-9 individually, cartilage degraded with both enzymes resulted in a 9–15% greater reduction in stiffness and peak load. Individually, MMP-1 and MMP-9 have a minimum effect on cartilage micromechanical properties, but synergistically the two enzymes digest ECM components and a much greater degradative effect is observed.

Near infrared spectroscopic evaluation of biochemical and crimp properties of knee joint ligaments and patellar tendon.

Knee ligaments and tendons play an important role in stabilizing and controlling the motions of the knee. Injuries to the ligaments can lead to abnormal mechanical loading of the other supporting tissues (e.g., cartilage and meniscus) and even osteoarthritis. While the condition of knee ligaments can be examined during arthroscopic repair procedures, the arthroscopic evaluation suffers from subjectivity and poor repeatability. Near infrared spectroscopy (NIRS) is capable of non-destructively quantifying the composition and structure of collagen-rich connective tissues, such as articular cartilage and meniscus. Despite the similarities, NIRS-based evaluation of ligament composition has not been previously attempted. In this study, ligaments and patellar tendon of ten bovine stifle joints were measured with NIRS, followed by chemical and histological reference analysis. The relationship between the reference properties of the tissue and NIR spectra was investigated using partial least squares regression. NIRS was found to be sensitive towards the water (R2CV = .65) and collagen (R2CV = .57) contents, while elastin, proteoglycans, and the internal crimp structure remained undetectable. As collagen largely determines the mechanical response of ligaments, we conclude that NIRS demonstrates potential for quantitative evaluation of knee ligaments.

Validation of watershed-based segmentation of the cartilage surface from sequential CT arthrography scans.

This study investigated the utility of a 2-dimensional watershed algorithm for identifying the cartilage surface in computed tomography (CT) arthrograms of the knee up to 33 minutes after an intra-articular iohexol injection as boundary blurring increased. A 2D watershed algorithm was applied to CT arthrograms of 3 bovine stifle joints taken 3, 8, 18, and 33 minutes after iohexol injection and used to segment tibial cartilage. Thickness measurements were compared to a reference standard thickness measurement and the 3-minute time point scan. 77.2% of cartilage thickness measurements were within 0.2 mm (1 voxel) of the thickness calculated in the reference scan at the 3-minute time point. 42% fewer voxels could be segmented from the 33-minute scan than the 3-minute scan due to diffusion of the contrast agent out of the joint space and into the cartilage, leading to blurring of the cartilage boundary. The traced watershed lines were closer to the location of the cartilage surface in areas where tissues were in direct contact with each other (cartilage-cartilage or cartilage-meniscus contact). The use of watershed dam lines to guide cartilage segmentation shows promise for identifying cartilage boundaries from CT arthrograms in areas where soft tissues are in direct contact with each other.

Hyperosmolar Ionic Solutions Modulate Inflammatory Phenotype and sGAG Loss in a Cartilage Explant Model.

The objective of this study was to compare the effects of hyperosmolar sodium (Na+), lithium (Li+) and potassium (K+) on catabolic and inflammatory osteoarthritis (OA) markers and sulfated glycosaminoglycan (sGAG) loss in TNF-α-stimulated cartilage explants. Explants from bovine stifle joints were stimulated with TNF-α for 1 day to induce cartilage degradation followed by supplementation with 50 mM potassium chloride (KCl), 50 mM lithium chloride (LiCl), 50 mM sodium chloride (NaCl), or 100 nM dexamethasone for an additional 6 days. We assessed the effect of TNF-α stimulation and hyperosmolar ionic treatment on sGAG loss and expression of OA-associated proteins: ADAMTS-5, COX-2, MMP-1, MMP-13, and VEGF. TNF-α treatment increased sGAG loss (P < 0.001) and expression of COX-2 (P = 0.018), MMP-13 (P < 0.001), and VEGF (P = 0.017) relative to unstimulated controls. Relative to activated controls, LiCl and dexamethasone treatment attenuated sGAG loss (P = 0.008 and P = 0.042, respectively) and expression of MMP-13 (P = 0.005 and P = 0.036, respectively). In contrast, KCl treatment exacerbated sGAG loss (P = 0.032) and MMP-1 protein expression (P = 0.010). NaCl treatment, however, did not alter sGAG loss or expression of OA-related proteins. Comparing LiCl and KCl treatment shows a potent reduction (P < 0.05) in catabolic and inflammatory mediators following LiCl treatment. These results suggest that these ionic species elicit varying responses in TNF-α-stimulated explants. Cumulatively, these findings support additional studies of hyperosmolar ionic solutions for potential development of novel intraarticular injections targeting OA.

Time- and depth-dependent changes to articular cartilage mitochondrial content occur during the 48 hours after injury

Purpose: Posttraumatic osteoarthritis (PTOA) is a degenerative joint disease after injury with no viable long-term treatments beyond joint arthroplasty and analgesia. It has recently been shown that impact injury and subsequent electron transport initiate a decrease in mitochondrial content in the superficial zone of articular cartilage, suggesting mitochondria play a role early in the development of PTOA. Electron transport inhibition after trauma has shown some promise; however, in published studies this approach is only effective if applied less than 24 hours after injury. Given that dysfunctional mitochondria have been observed as late as 4 weeks after injury and it has been reported several times that end-stage OA chondrocytes have mitochondrial dysfunction, we wanted to better understand the development and extent of mitochondrial dysfunction throughout cartilage after injury. We hypothesized injured areas deeper than the superficial zone would show decreases in mitochondrial content between 24 and 48 h after injury. Methods: Healthy bovine stifle (knee) joints with no signs of osteoarthritis were obtained from a local abattoir (Bud’s Custom Meats Riverside, IA). Matched 10 mm diameter osteochondral plugs were taken from the loaded portion of the femoral condyle. Once removed, the plugs were washed twice in HBSS with antibiotics then cultured in normal media (45% DMEM, 45% F12, 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2.5 μm/mL amphotericin B) and maintained at 37°C, 5% CO2, and 5% O2. After 24 h equilibration to culture conditions, the plugs were subject to either a sham (0 J/cm2) or a 2 J/cm2 impact delivered via a custom-built drop tower utilizing a 6 mm flat, beveled, stainless steel platen. After impact, the samples were placed in fresh porcine media and returned to the incubator. Prior studies had viewed plugs from above using confocal microscopy; in this case, to view the deeper zones of cartilage plugs were bisected slightly off center of the impact into a 5.9 mm section with an Isomet 1000 (Buehler, Lake Bluff, IL), then faced by trimming 0.5 mm of the cartilage with a single straight scalpel cut. These measured cuts (the combination of the isomet blade with and the cartilage removed during facing) provided a sagittal cross-section of the plug at the center of the impact site. The plugs were washed in phenol red free DMEM/F12 media and co-stained for 30 min in the incubator with 1 mg/mL Calcein Green AM and 200 nM MitoTracker Deep Red (both Life Technologies, Waltham, MA). The samples were then washed and imaged on an Olympus FV1000 (Shinjuku, Japan) confocal microscope. Images were taken at the middle of the impact site with a 10X objective lens and images were manually segmented according to either specific zones of the cartilage (superficial, transitional, radial, or deep zone) or by 50 μm increments in depth starting from the superficial zone (Figure 1). Within each zone or depth, a custom watershed-based 3D image analysis algorithm was used to segment live cells according to Calcein Green AM. Per-cell MitoTracker staining intensity for live cells was extracted and averaged by zone depth. Intensities of cells in injured plugs were normalized to their respective sham control. Two-way ANOVA with Tukey multiple comparisons was performed to determine significance between groups. Results: Live cell mitochondrial staining intensity in the superficial and transitional zones of the cartilage significantly increased in the first 24 h after injury compared to sham (Figure 2, n = 4, p < 0.05). However, 48 h after impact, live cell mitochondrial staining decreased significantly both in the superficial zone and in the transitional zone compared to 24 h after injury (p < 0.05). Mitochondrial content at 48 h decreased below control levels in the superficial and transitional zones of three out of four specimens. Analysis by either measured depth or cartilage zone each revealed this significant change from 24 to 48 h after injury. Conclusions: We observed a significant increase in live cell mitochondrial staining 24 h after impact compared to sham; however, this time point was marked by significant inter- and intra-specimen variation as shown in the variance in Figure 2. This acute increase in staining may indicate stimulation of the chondrocytes by the mechanical injury. However, we observed a substantial and significant decrease in live chondrocyte mitochondrial staining in the superficial and transitional zones by 48 h after injury. These changes must be verified via immunohistochemistry, but when compared to prior results they suggest dynamic changes in mitochondrial dysfunction through the depth of articular cartilage after injury.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Finite element modeling of osteochondral explant impacts predicts locations of cell death

Purpose: Post-traumatic osteoarthritis (PTOA) is a subset of osteoarthritis initiated by an injury such as a blunt impact. However, the local tissue mechanics associated with the earliest stages of PTOA remain undefined. Computational modeling techniques such as finite element analysis provide a means to evaluate mechanical environments that are challenging to measure experimentally. The objective of this work was to compare specimen-specific stresses and strains computed using finite element analysis to confocal microscopy images of chondrocyte viability. Methods: Four viable osteochondral explants (25 mm x 25 mm x 15 mm) were harvested from the tibial plateau of healthy bovine stifle joints (knee). Once removed, the explants were washed twice in HBSS, cultured in normal media (45% DMEM, 45% F12, 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2.5 μm/mL amphotericin B) and incubated at 37°C, 5% CO2, and 5% O2. The following day a surface scan of the articular surface of each explant (EinScan Pro 2X Pro; Shining 3D) and ultrasound measurements (Olympus 35DL) of cartilage thickness were acquired. Next, the explants were subject to a 2 J/cm2 impact using a 6 mm flat, beveled stainless-steel platen. After impact, the samples were placed in fresh porcine media and returned to the incubator for 24 h. Cartilage explants were cut with an Isomet 1000 (Buehler, Lake Bluff, IL) slightly off center of the impact and faced by trimming 0.5 mm of the cartilage with a scalpel. These provided a coronal plane cross-section of the explant through the center of the impact site. The explants were washed in phenol red free DMEM/F12 media and stained for 30 min in the incubator with 1 mg/mL Calcein Green AM and 200 nM MitoTracker Deep Red (Life Technologies, Waltham, MA). The samples were then washed and imaged on an Olympus FV1000 (Shinjuku, Japan) confocal microscope. Images were acquired on either edge of the impact, 1 mm inside each edge of the impact site, and in the center of the impact (Figure 1) using a 10X objective lens. An additional top-down view of the articular surface was acquired. Specimen-specific finite element models were generated using the surface scan, cartilage thickness measurements, and geometric measurements of each explant. Cartilage was modeled as hyperelastic with a first-order Ogden strain energy potential. Acceleration of the impact platen was measured (PCB Piezotronics accelerometer), and double integration was used to calculate platen displacement at max tissue compression and impact force. von Mises stress and normal z strain values were compared from corresponding nodes within confocal images (Figure 2). Cell death in the superficial zone and deeper regions of cartilage was assessed. Results: Cell death was present in the superficial zone in 4/4 peripheral edge locations, 3/4 locations interior to the impactor edge and 3/4 of the middle sites. There was no superficial cell death on the central edge of the impact zone. Locations experiencing > 40% strain were associated with high levels of cell death in the superficial zone. When strains exceeded 60%, cell death extended into the deeper zones of the cartilage. It was noteworthy that the highest strains were found at the 1-mm interior to the impact platen edge on all specimens. Von Mises stresses were consistently &gt 30 MPa on the edge of the impact site; however, no obvious relationships were identified between von Mises values and cell death in the superficial, middle, or deep zones. Conclusions: We found strong associations between cartilage strain and chondrocyte death. The highest normal z strain occurs just interior to the beveled edge of the impact platen, and not on the peripheral-most edge of the impact platen. While we did find associations between locations of elevated stress, strain, and cell death, the magnitudes of those values remain to be validated. While important, cell death is a relatively broad measure of injury and associations between cellular health and function and these mechanical measures should be explored.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Hemarthrosis alters mitochondrial dynamics in situ after traumatic injury

Purpose: Hemarthrosis occurs after a variety of joint injuries and contributes to rapid progression of posttraumatic osteoarthritis (PTOA). Similarly, mitochondrial responses to traumatic injury appear to modulate rate of PTOA progression. In vitro studies have shown that chondrocyte mitochondria are impaired by a variety of signaling molecules present in whole blood and that pro-inflammatory activity is responsive to manipulations of mitochondrial function. The combination of oxidizing mitochondria and oxidizing, labile iron derived from blood already associated with trauma may cause more severe mitochondrial damage in situ than previously characterized in agarose models. This study tested whether the addition of whole blood after injury would exacerbate losses in mitochondrial content observed after impact in a chelation-inhibitable fashion. Methods: Fresh, skeletally mature, bovine stifle (knee) joints were obtained from a local abattoir (Bud’s Custom Meats, Riverside, IA) and 1 cm diameter cylindrical osteochondral explants from the loaded articular surface of the femoral condyle were harvested. Explants were equilibrated for 1 day in 45% F-12/45% Dulbecco’s modified Eagle medium/10% fetal bovine serum at 37ºC with 5% O2 and 5% CO2. Explants were impacted with a 2 J impact via drop tower to the articular surface using a flat impermeable platen (or sham impact for controls) and placed into one of three solutions: normal media, normal media + 10% whole blood from the same animal, or normal media + 10% blood + the iron chelator desferrioxamine mesylate (DFO) (100 μM, Sigma-Aldrich). Samples were returned to the incubator in these solutions and 2 h later stained in serum free media with 1 μM calcein AM, a viability dye, and 200 nM MitoTracker Deep Red, a stain for mitochondria (both ThermoFisher Scientific). Cells were visualized with an Olympus FV1000 confocal microscope with a 20X objective immersed in phenol red-free media, as well as with a 100X immersion objective placed in contact with the impacted site. Images were analyzed using a custom-written two-part analysis routine developed in Matlab. Viable cells were automatically identified from the calcein AM-stained images using intensity thresholding and area-based shape measures, and resultant cell counts were expressed as viable cell density (cells/mm2). A mask image of only viable cells was applied to the MitoTracker images to extract mitochondrial staining intensity from viable cells. Data reported are the average results from explants obtained from 4 separate animals represented in all groups. Results: Impact resulted in a ∼50% reduction in viable cell density in the zone of impact across all groups and conditions. Total viable cell mitochondrial staining decreased after impact (p < 0.05) and this was not altered with whole blood addition after impact, Figure 1. A trend towards recovery of mitochondrial content was observed with whole blood and DFO after impact; however this has not reached statistical significance at n = 4 (p = 0.2 for reversing the effect of impact). A clear difference in mitochondrial network morphology was observed comparing samples with and without whole blood, Figure 2, with samples exposed to whole blood demonstrating a much more punctate, fragmented morphology. No significant differences in cell viability were observed after including blood in our studies, suggesting that impact itself was largely determinate of cell death at sites of impact. The mitochondrial phenotype after impact appears very different in the presence of hemarthrosis, with brighter and more punctate mitochondria than impact alone. Conclusions: Iron chelation may be effective in preventing loss of overall mitochondrial intensity, i.e. mitochondrial mass, but greater numbers of replicates will be needed to reach statistical significance. While DFO provided some recovery of intensity, it did not appear to rescue the morphology changes associated with exposure to blood. This study contributes to ongoing efforts in the field to improve the physiological relevance of in vitro models of cartilage injury by demonstrating a clear effect of whole blood on intracellular responses to mechanical injury.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Mechanically stimulated osteochondral organ culture for evaluation of biomaterials in cartilage repair studies

Surgical procedures such as microfracture or autologous chondrocyte implantation have been used to treat articular cartilage lesions; however, repair often fails in terms of matrix organization and mechanical behaviour. Advanced biomaterials and tissue engineered constructs have been developed to improve cartilage repair; nevertheless, their clinical translation has been hampered by the lack of reliable in vitro models suitable for pre-clinical screening of new implants and compounds.In this study, an osteochondral defect model in a bioreactor that mimics the multi-axial motion of an articulating joint, was developed. Osteochondral explants were obtained from bovine stifle joints, and cartilage defects of 4 mm diameter were created. The explants were used as an interface against a ceramic ball applying dynamic compressive and shear loading. Osteochondral defects were filled with chondrocytes-seeded fibrin-polyurethane constructs and subjected to mechanical stimulation. Cartilage viability, proteoglycan accumulation and gene expression of seeded chondrocytes were compared to free swelling controls. Cells within both cartilage and bone remained viable throughout the 10-day culture period. Loading did not wear the cartilage, as indicated by histological evaluation and glycosaminoglycan release. The gene expression of seeded chondrocytes indicated a chondrogenic response to the mechanical stimulation. Proteoglycan 4 and cartilage oligomeric matrix protein were markedly increased, while mRNA ratios of collagen type II to type I and aggrecan to versican were also enhanced. This mechanically stimulated osteochondral defect culture model provides a viable microenvironment and will be a useful pre-clinical tool to screen new biomaterials and biological regenerative therapies under relevant complex mechanical stimuli. Statement of SignificanceArticular cartilage lesions have a poor healing capacity and reflect one of the most challenging problems in orthopedic clinical practice. The aim of current research is to develop a testing system to assess biomaterials for implants, that can permanently replace damaged cartilage with the original hyaline structure and can withstand the mechanical forces long term.Here, we present an osteochondral ex vivo culture model within a cartilage bioreactor, which mimics the complex motion of an articulating joint in vivo. The implementation of mechanical forces is essential for pre-clinical testing of novel technologies in the field of cartilage repair, biomaterial engineering and regenerative medicine. Our model provides a unique opportunity to investigate healing of articular cartilage defects in a physiological joint-like environment.

Near Infrared Spectroscopic Evaluation of Ligament and Tendon Biomechanical Properties.

Knee ligaments and tendons are collagen-rich viscoelastic connective tissues that provide vital mechanical stabilization and support to the knee joint. Deterioration of ligaments has an adverse effect on the health of the knee and can eventually lead to ligament rupture and osteoarthritis. In this study, the feasibility of near infrared spectroscopy (NIRS) was, for the first time, tested for evaluation of ligament and tendon mechanical properties by performing measurements on bovine stifle joint ligament (N = 40) and patellar tendon (N = 10) samples. The mechanical properties of the samples were determined using a uniaxial tensile testing protocol. Partial least squares regression models were then developed to determine if morphological, viscoelastic, and quasi-static properties of the samples could be predicted from the NIR spectra. Best performance of NIRS in predicting mechanical properties was observed for toughness at yield point (median [Formula: see text], median normalized [Formula: see text]), toughness at failure point (median [Formula: see text], median normalized [Formula: see text]), and the ultimate strength of the ligament/tendon (median [Formula: see text], median normalized [Formula: see text]). Thus, we show that NIRS is capable of estimating ligament and tendon biomechanical properties, especially in parameters related to tissue failure. We believe this method could substantially enhance the currently limited arthroscopic evaluation of ligaments and tendons.

Non-Destructive Spectroscopic Assessment of High and Low Weight Bearing Articular Cartilage Correlates with Mechanical Properties.

Autologous articular cartilage (AC) harvested for repair procedures of high weight bearing (HWB) regions of the femoral condyles is typically obtained from low weight bearing (LWB) regions, in part due to the lack of non-destructive techniques for cartilage composition assessment. Here, we demonstrate that infrared fiber optic spectroscopy can be used to non-destructively evaluate variations in compositional and mechanical properties of AC across LWB and HWB regions. AC plugs (N = 72) were harvested from the patellofemoral groove of juvenile bovine stifle joints, a LWB region, and femoral condyles, a HWB region. Near-infrared (NIR) and mid-infrared (MIR) fiber optic spectra were collected from plugs, and indentation tests were performed to determine the short-term and equilibrium moduli, followed by gravimetric water and biochemical analysis. LWB tissues had a significantly greater amount of water determined by NIR and gravimetric assay. The moduli generally increased in tissues from the patellofemoral groove to the condyles, with HWB condyle cartilage having significantly higher moduli. A greater amount of proteoglycan content was also found in HWB tissues, but no differences in collagen content. In addition, NIR-determined water correlated with short-term modulus and proteoglycan content (R = -0.40 and -0.31, respectively), and a multivariate model with NIR data was able to predict short-term modulus within 15% error. The properties of tissues from LWB regions differ from HWB tissues and can be determined non-destructively by infrared fiber optic spectroscopy. Clinicians may be able to use this modality to assess AC prior to harvesting osteochondral grafts for focal defect repair.

Arthroscopic irrigation of the bovine stifle joint increases cartilage surface friction and decreases superficial zone lubricin

The purpose of this study was to determine the effects of arthroscopic irrigation on cartilage superficial zone lubricin and surface friction. Arthroscopic partial meniscectomy is one of the most commonly performed orthopedic surgeries in the United States, but rates of osteoarthritis progression following this procedure are high. The effect of arthroscopic irrigation on articular surface lubrication has not been previously considered as a contributing factor in outcomes after arthroscopy. Fourteen bovine stifle joints were randomized to receive arthroscopic irrigation (n=7) or no treatment (n=7). Full-thickness osteochondral explants from these joints underwent friction testing to measure static and dynamic coefficients of friction. Following mechanical testing, samples were fixed and stained for lubricin. Percent integrated density, a measure of the amount of lubricin in the superficial zone (0–100µm depth), was determined. Static and dynamic coefficients of friction were found to be significantly greater in arthroscopy specimens compared to controls (p=0.02 and p<0.001, respectively). Percent integrated density of lubricin in the superficial zone was significantly lower in arthroscopy specimens compared to controls (p<0.001).

Systematic arthroscopic investigation of the bovine stifle joint

The objective of the present study was to establish a protocol for arthroscopic exploration of the bovine stifle joint using craniomedial, caudolateral and caudomedial approaches. An anatomic and arthroscopic study using 26 cadaveric limbs from 13 non-lame adult dairy cows was performed. The craniomedial approach was created between the middle and medial patellar ligaments to investigate the cranial pouches of the stifle joint. The inter-condylar eminence, the proximal aspect of the medial femoral trochlear ridge and the lateral aspect of the lateral femoral condyle were used as starting points for systematic examination of the medial femorotibial, the femoropatellar and the lateral femorotibial joints, respectively.The observed structures were: the suprapatellar pouch, articular surfaces of the patella, femoral trochlear ridges, cruciate ligaments, menisci, and the meniscotibial ligaments. The arthroscopic portal for the caudomedial femorotibial pouch was about 6–8 cm caudal to the medial collateral ligament. The proximal and distal caudolateral femorotibial pouches were explored 3 cm and 1.5 cm caudal to the ipsilateral collateral ligament, respectively. The observed structures were the caudal aspect of femoral condyles, menisci, caudal cruciate ligament, popliteal tendon and the meniscofemoral ligament. Restricted joint size and risk of common peroneal nerve damage were the major limitations for exploration of the caudal femorotibial compartments. The study described the arthroscopic portals and normal intra-articular anatomy of the bovine stifle joint but further investigations are warranted to validate these techniques in clinical cases.