Equilibrium Compressive Modulus Research Articles

Compressive fatigue and endurance of juvenile bovine articular cartilage explants

Articular cartilage is an enduring tissue. For most individuals, articular cartilage facilitates a lifetime of pain-free ambulation, supporting millions of loading cycles from activities of daily living. Although early studies into osteoarthritis focused on the role of mechanical fatigue in articular cartilage degeneration, much is still unknown regarding its strength and endurance characteristics. The compressive strength of juvenile, bovine articular cartilage explants was determined to be loading rate-dependent, reaching a maximum strength of 29.5 ± 4.8 MPa at a strain rate of 0.10 %/sec. The fatigue and endurance properties of articular cartilage were characterized utilizing a material testing system, as well as a custom, validated instrument termed the two degrees-of-freedom endurance meter (endurometer). These instruments characterized fatigue in articular cartilage explants at loading levels ranging from 10 to 80 % strength (%S), up to 100,000 cycles. Cartilage explants displayed characteristics of fatigue – fatigue life increased as the loading magnitude decreased. All explants failed within 14,000 cycles at loading levels between 50 and 80 %S. At 10 and 20 %S, all explants endured 100,000 loading cycles. There was no significant difference in equilibrium compressive modulus between run-out explants and unloaded controls, although the pooled modulus increased in response to testing. Histological staining and biochemical assays revealed no material changes in collagen, sulfated glycosaminoglycan, or hydration content between unloaded controls and explants cyclically loaded to run-out. These results suggest articular cartilage may have a putative endurance limit of 20 %S (5.86 MPa), with implications for articular cartilage biomechanics and mechanobiology.

Contrast‐Enhanced Computed Tomography Scoring System for Distinguishing Early Osteoarthritis Disease States: A Feasibility Study

Early detection of osteoarthritis (OA) remains a diagnostic challenge owing to insensitive diagnostic techniques currently available. Herein a new semiquantitative scoring system, based upon contrast-enhanced computed tomographic (CECT) imaging, is described for further refinement of early OA disease staging. Trochlear ridge cartilage defects were surgically created in the femoropatellar joint of an adult horse (ACUC approved protocols). Seven weeks post-surgery, CECT imaging was performed on a clinical scanner after intra-articular injection of a cationic iodinated contrast agent, CA4+, into both injured and control femoropatellar joint compartments. The femoral cartilage surface was densely biopsied, and specimens were assessed for visual (Outerbridge score), functional (equilibrium compressive modulus), and biochemical (glycosaminoglycan content) measures of cartilage quality. Cartilage CECT attenuation was compared with cartilage quality measures using receiver operating characteristic curve analysis to establish attenuation thresholds for distinguishing among cartilage quality levels. CECT imaging identifies macroscopically damaged cartilage regions and in morphologically identical tissue provides moderately sensitive and specific semiquantitative segregation of cartilage quality based upon CECT attenuation, reflecting both glycosaminoglycan content and compressive stiffness of cartilage area under the curve (AUC = 0.83 [95% confidence interval [CI]: 0.72-0.93] for distinguishing poor quality and AUC = 0.76 [95% CI: 0.65-0.90] for distinguishing healthy quality cartilage). A semiquantitative 6-point scoring system-the Osteoarthritis Attenuation and Morphological Assessment (OAMA) score-is proposed as a tool for assessing cartilage quality from CECT images. The OAMA scoring system expands the current disease staging capability of early OA by inclusion of morphological, biochemical, and biomechanical assessments. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2138-2148, 2019.

2061 Acellular hyaluronic acid scaffold with growth factor delivery for cartilage repair in a large animal model

OBJECTIVES/SPECIFIC AIMS: Focal cartilage injuries of the knee joint are common and present a treatment challenge due to minimal intrinsic repair. Cartilage tissue engineering techniques currently used in clinical practice are expensive, cumbersome, and often ineffective in patients with mechanical or medical comorbidities. To address these issues, we developed an acellular nanofibrous scaffold with encapsulated growth factors designed to enhanced articular cartilage repair. Our goal is to evaluate this technology in vitro and pilot a large animal model for eventual translation into human subjects. METHODS/STUDY POPULATION: Hyaluronic acid (HA, 65 kDa) will be methacrylated (~40% modification, MeHA) and conjugated with cell-adhesive (RGD) groups. A solution of 4% wt/vol MeHA, 2% wt/vol polyethylene oxide (900 kDa), 0.05% wt/vol Irgacure 2959, and 0.005% wt/vol stromal cell-derived factor-1α (SDF-1α) and/or transforming growth factor-β3 (TGF-β3) will be prepared in ddH2O. The solution will be electrospun onto a rotating mandrel to achieve a dry scaffold thickness of 0.5 mm. The scaffold matt will be UV cross-linked and 5 mm-diameter samples will be cut out. Four groups of scaffolds will be prepared: MeHA, MeHA+SDF, MeHA+TGF, MeHA+SDF+TGF. All groups will be evaluated for fiber diameter, swell thickness, equilibrium compressive modulus, degradation rate, and growth factor release rate over 4 weeks (n=10). Scaffolds will also be seeded with juvenile porcine MSCs (5×104) in 200 μL of medium incubated for 24 hours. Seeded scaffolds will be evaluated for equilibrium compressive modulus, cell infiltration, and chondrogenesis at 4 and 8 weeks (n=10). Scaffolds will then be evaluated in a juvenile Yucatan minipig cartilage defect model. In total, 6 animals will undergo bilateral knee surgery to create four 4 mm-diameter full-thickness cartilage defects in each trochlear grove. All defects will receive microfracture to release marrow elements. Each knee will receive 2 scaffolds of the same group (replicates) with paired microfracture controls, resulting in a sample size of 3. Animals will be sacrificed at 12 weeks and defects will be evaluated via non-destructive indentation testing for mechanical properties, microCT for defect fill and subchondral bone morphology, and histology for ICRS II Visual Histological Assessment Scoring. RESULTS/ANTICIPATED RESULTS: Our preliminary studies have shown reliable replication of electrospun MeHA scaffolds. We anticipate cross-linking density to correlate positively with compressive modulus, and negatively with swell thickness, degradation rate, and growth factor release rate. We anticipate the addition of SDF-1α and TGF-β3 to increase cell infiltration and chondrogenesis, respectively, within seeded scaffolds. Similarly, we expect minipig defects treated with growth factor-releasing scaffolds to show greater mechanical properties, defect fill, and ICRS II score compared with MeHA scaffolds without growth factor. DISCUSSION/SIGNIFICANCE OF IMPACT: This study has the potential to show how an HA-based cell-free scaffold can be augmented with 2 growth factors that act synergistically to improve cartilage repair in a large animal model. This technology would improve upon the cell-free scaffolds already used clinically for autologous matrix-induced chondrogenesis and is directly translatable.

Mechanical viability of a thermoplastic elastomer hydrogel as a soft tissue replacement material.

Hydrogels are a class of synthetic biomaterials composed of a polymer network that swells with water and as such they have both an elastic and viscous component making them ideal for soft tissue applications. This study characterizes the compressive, tensile, and shear properties of a thermoplastic elastomer (TPE) hydrogel and compares the results to published literature values for soft tissues such as articular cartilage, the knee meniscus, and intervertebral disc components. The results show the TPE hydrogel material is viscoelastic, strain rate dependent, has similar surface and bulk properties, displays minimal damping under dynamic load, and has tension-compression asymmetry. When compared to other soft tissues it has a comparable equilibrium compressive modulus of approximately 0.5MPa and shear modulus of 0.2MPa. With a tensile modulus of only 0.2MPa though, the TPE hydrogel is inferior in tension to most collagen based soft tissues. Additional steps may be necessary to reinforce the hydrogel system and increase tensile modulus depending on the desired soft tissue application. It can be concluded that this material could be a viable option for soft tissue replacements.

Biomimetic 3D printed scaffolds for meniscus tissue engineering

The menisci distribute loads to protect the articular cartilage of the knee joint from excessive stress. Injuries to their avascular inner regions do not heal, disrupt function, and increase the risk for knee osteoarthritis. Meniscus tissue engineering aims to restore normal meniscus function by use of regenerated tissue on bioengineered scaffolds. The primary purpose of this study was to design and 3D print polycaprolactone scaffolds that recapitulate the shape and structural components of the meniscus extracellular matrix to provide a template and structural support for complete cell-based meniscus regeneration. A secondary aim was to characterize 3D printed polycaprolactone scaffold fibre architectures with varied fibre spacings, offsets between layers, and circumferential orientations in terms of their equilibrium compressive modulus. Meniscus scaffolds were produced layer-by-layer with a biomimetic fibre architecture inspired by the circumferentially- and radially- oriented collagen fibre bundles found in the native tissue. Suture tabs were incorporated to facilitate fixation at the meniscal horns and the coronary attachments. These scaffolds were designed to have 100% pore interconnectivity and 61% porosity. The equilibrium compressive modulus at 10% strain of the meniscus scaffold architecture was 18.8 ± 3.1MPa (mean ± standard deviation, n = 3) and ultimate load in a suture pull-out test on the anterior horn suture tab was 32N. The scaffolds produced in this study demonstrate a promising strategy for meniscus tissue engineering as they provide a complete biomimetic template for regeneration of functional components of the meniscus extracellular matrix.

* Constrained Cage Culture Improves Engineered Cartilage Functional Properties by Enhancing Collagen Network Stability.

When cultured with sufficient nutrient supply, engineered cartilage synthesizes proteoglycans rapidly, producing an osmotic swelling pressure that destabilizes immature collagen and prevents the development of a robust collagen framework, a hallmark of native cartilage. We hypothesized that mechanically constraining the proteoglycan-induced tissue swelling would enhance construct functional properties through the development of a more stable collagen framework. To test this hypothesis, we developed a novel "cage" growth system to mechanically prevent tissue constructs from swelling while ensuring adequate nutrient supply to the growing construct. The effectiveness of constrained culture was examined by testing constructs embedded within two different scaffolds: agarose and cartilage-derived matrix hydrogel (CDMH). Constructs were seeded with immature bovine chondrocytes and cultured under free swelling (FS) conditions for 14 days with transforming growth factor-β before being placed into a constraining cage for the remainder of culture. Controls were cultured under FS conditions throughout. Agarose constructs cultured in cages did not expand after the day 14 caging while FS constructs expanded to 8 × their day 0 weight after 112 days of culture. In addition to the physical differences in growth, by day 56, caged constructs had higher equilibrium (agarose: 639 ± 179 kPa and CDMH: 608 ± 257 kPa) and dynamic compressive moduli (agarose: 3.4 ± 1.0 MPa and CDMH 2.8 ± 1.0 MPa) than FS constructs (agarose: 193 ± 74 kPa and 1.1 ± 0.5 MPa and CDMH: 317 ± 93 kPa and 1.8 ± 1.0 MPa for equilibrium and dynamic properties, respectively). Interestingly, when normalized to final day wet weight, cage and FS constructs did not exhibit differences in proteoglycan or collagen content. However, caged culture enhanced collagen maturation through the increased formation of pyridinoline crosslinks and improved collagen matrix stability as measured by α-chymotrypsin solubility. These findings demonstrate that physically constrained culture of engineered cartilage constructs improves functional properties through improved collagen network maturity and stability. We anticipate that constrained culture may benefit other reported engineered cartilage systems that exhibit a mismatch in proteoglycan and collagen synthesis.

Contrast-Enhanced Computed Tomography Enables Quantitative Evaluation of Tissue Properties at Intrajoint Regions in Cadaveric Knee Cartilage.

ObjectiveThe aim of this study was to investigate whether the concentration of the anionic contrast agent ioxaglate, as quantitated by contrast-enhanced computed tomography (CECT) using a clinical cone-beam CT (CBCT) instrument, reflects biochemical, histological, and biomechanical characteristics of articular cartilage imaged in an ex vivo, intact human knee joint.DesignAn osteoarthritic human cadaveric knee joint (91 years old) was injected with ioxaglate (36 mg I/mL) and imaged using CBCT over 61 hours of ioxaglate diffusion into cartilage. Following imaging, the joint surfaces were excised, rinsed to remove contrast agent, and compressive stiffness (equilibrium and instantaneous compressive moduli) was measured via indentation testing (n = 17 sites). Each site was sectioned for histology and assessed for glycosaminoglycan content using digital densitometry of Safranin-O stained sections, Fourier transform infrared spectroscopy for collagen content, and morphology using both the Mankin and OARSI semiquantitative scoring systems. Water content was determined using mass change after lyophilization.ResultsCECT attenuation at all imaging time points, including those <1 hour of ioxaglate exposure, correlated significantly (P < 0.05) with cartilage water and glycosaminoglycan contents, Mankin score, and both equilibrium and instantaneous compressive moduli. Early time points (<30 minutes) also correlated (P < 0.05) with collagen content and OARSI score. Differences in cartilage quality between intrajoint regions were distinguishable at diffusion equilibrium and after brief ioxaglate exposure.ConclusionsCECT with ioxaglate affords biochemical and biomechanical measurements of cartilage health and performance even after short, clinically relevant exposure times, and may be useful in the clinic as a means for detecting early signs of cartilage pathology.

Nutrient Channels Aid the Growth of Articular Surface-Sized Engineered Cartilage Constructs.

Symptomatic osteoarthritic lesions span large regions of joint surfaces and the ability to engineer cartilage constructs at clinically relevant sizes would be highly desirable. We previously demonstrated that nutrient transport limitations can be mitigated by the introduction of channels in 10 mm diameter cartilage constructs. In this study, we scaled up our previous system to cast and cultivate 40 mm diameter constructs (2.3 mm overall thickness); 4 mm diameter and channeled 10 mm diameter constructs were studied for comparison. Furthermore, to assess whether prior results using primary bovine cells are applicable for passaged cells-a more clinically realistic scenario-we cast constructs of each size with primary or twice-passaged cells. Constructs were assessed mechanically for equilibrium compressive Young's modulus (EY), dynamic modulus at 0.01 Hz (G*), and friction coefficient (μ); they were also assessed biochemically, histologically, and immunohistochemically for glycosaminoglycan (GAG) and collagen contents. By maintaining open channels, we successfully cultured robust constructs the size of entire human articular cartilage layers (growing to ∼52 mm in diameter, 4 mm thick, mass of 8 g by day 56), representing a 100-fold increase in scale over our 4 mm diameter constructs, without compromising their functional properties. Large constructs reached EY of up to 623 kPa and GAG contents up to 8.9%/ww (% of wet weight), both within native cartilage ranges, had G* >2 MPa, and up to 3.5%/ww collagen. Constructs also exhibited some of the lowest μ reported for engineered cartilage (0.06-0.11). Passaged cells produced tissue of lower quality, but still exhibited native EY and GAG content, similar to their smaller controls. The constructs produced in this study are, to our knowledge, the largest engineered cartilage constructs to date which possess native EY and GAG, and are a testament to the effectiveness of nutrient channels in overcoming transport limitations in cartilage tissue engineering.

A mechanically robust injectable hydrogel scaffold for adipose-derived stem cell delivery for the treatment of peripheral arterial disease

Event Abstract Back to Event A mechanically robust injectable hydrogel scaffold for adipose-derived stem cell delivery for the treatment of peripheral arterial disease Stuart Young1, Lauren E. Flynn2 and Brian G. Amsden1 1 Queen's University, Department of Chemical Engineering, Canada 2 University of Western Ontario, Department of Chemical and Biochemical Engineering, Canada Introduction: Peripheral arterial disease can lead to critical limb ischemia with high risks of amputation and mortality. A promising treatment involves delivering adipose-derived stem cells (ASCs) to stimulate revascularization and functional recovery. However, when delivered via intramuscular injection in saline, substantial ASC loss occurs. To improve ASC retention and survival, a modular delivery gel was developed composed of: i) methacrylated glycol chitosan (MGC) functionalized with GGGGRGDS, and ii) a diacrylate triblock copolymer of poly(ethylene glycol) (PEG) and poly(trimethylene carbonate) (PTMC) to improve mechanical robustness. A thermal initiator system of ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED) was optimized for in situ gelation following injectable delivery. Methods: MGC was prepared as previously reported[1]. The N terminus of GGGGRGDS peptide was acrylated and conjugated to MGC via Michael addition. Acrylated PEG-PTMC of varying block lengths was prepared by ring-opening polymerization of TMC initiated by PEG (4, 10, or 20 kg·mol-1) (TMC:PEG MW = 1:10 – 1:2)) using HCl as a monomer activator, followed by reaction with acryloyl chloride. Polymer structure and functionalization were confirmed by 1H NMR and GPC. To form gels, APS and TEMED (2.5 to 10 mM) were added to solutions of MGC and PEG-PTMC. Initiation sensitivity and crosslinking time were assessed by rheometry at 20 °C and 37°C. Gels were equilibrated in PBS, after which the swelling ratio and mechanical properties were measured. Isolated bovine ASCs[2], expanded to passage 2, were suspended in the pre-polymer solutions. APS/TEMED was added, and the suspension injected via 25G needle into molds and crosslinked by heating (37°C). ASC viability (LIVE/DEAD staining) and metabolic activity (MTT assay) were monitored over 7 days. Results: The MGC had a 5% degree of methacrylation and 5% degree of peptide functionalization, while the PEG-PTMC had > 99% acrylation. Pre-polymer solutions showed no gelation over 10 min at 20 °C (Fig. 1a). Upon heating, gelation time was directly related to initiator concentration (Fig. 1b). The swelling, equilibrium compressive modulus, and maximum strain of PEG-PTMC hydrogels was dependent on the overall molecular weight and PEG/PTMC ratio (Fig. 2). By blending MGC with PEG-PTMC, gel stiffness was significantly reduced to the range of native tissue (~10-15 kPa), while improving elastic resiliency under dynamic loading. Further, the metabolic activity of encapsulated ASCs was maintained over 7 d of culture. Discussion: Blending of MGC with PEG-PTMC facilitated adjustment of gel mechanical properties while improving resiliency under dynamic loading. The APS/TEMED crosslinking approach provided an injectable delivery method with high stimuli sensitivity. Sustained ASC metabolic activity in culture suggests the gel could promote ASC survival and retention, thereby promoting their regenerative effects in ischemic regions.

Supplementation of Exogenous Adenosine 5′-Triphosphate Enhances Mechanical Properties of 3D Cell–Agarose Constructs for Cartilage Tissue Engineering

Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5'-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.

Regional variation in T1ρ and T2 times in osteoarthritic human menisci: correlation with mechanical properties and matrix composition

Changes in T1ρ and T2 magnetic resonance relaxation times have been associated with articular cartilage degeneration, but similar relationships for meniscal tissue have not been extensively investigated. This work examined relationships between T1ρ and T2 measurements and biochemical and mechanical properties across regions of degenerate human menisci. Average T1ρ and T2 relaxation times were determined for nine regions each of seven medial and 13 lateral menisci from 14 total knee replacement patients. Sulfated glycosaminoglycan (sGAG), collagen and water contents were measured for each region. Biomechanical measurements of equilibrium compressive, dynamic compressive and dynamic shear moduli were made for anterior, central and posterior regions. T1ρ and T2 times showed similar regional patterns, with longer relaxation times in the (radially) middle region compared to the inner and outer regions. Pooled over all regions, T1ρ and T2 times showed strong correlations both with one another and with water content. Correlations with biochemical content varied depending on normalization to wet or dry mass, and both imaging parameters showed stronger correlations with collagen compared to sGAG content. Mechanical properties displayed moderate inverse correlations with increasing T1ρ and T2 times and water content. Both T1ρ and T2 relaxation times correlated strongly with water content and moderately with mechanical properties in osteoarthritic menisci, but not as strongly with sGAG or collagen contents alone. While the ability of magnetic resonance imaging (MRI) to detect early osteoarthritic changes remains the subject of investigation, these results suggest that T1ρ and T2 relaxation times have limited ability to detect compositional variations in degenerate menisci.

Cationic agent contrast-enhanced computed tomography imaging of cartilage correlates with the compressive modulus and coefficient of friction

The aim of this study is to evaluate whether contrast-enhanced computed tomography (CECT) attenuation, using a cationic contrast agent (CA4+), correlates with the equilibrium compressive modulus (E) and coefficient of friction (μ) of ex vivo bovine articular cartilage. Correlations between CECT attenuation and E (Group 1, n = 12) and μ (Group 2, n = 10) were determined using 7 mm diameter bovine osteochondral plugs from the stifle joints of six freshly slaughtered, skeletally mature cows. The equilibrium compressive modulus was measured using a four-step, unconfined, compressive stress-relaxation test, and the coefficients of friction were determined from a torsional friction test. Following mechanical testing, samples were immersed in CA4+, imaged using μCT, rinsed, and analyzed for glycosaminoglycan (GAG) content using the 1,9-dimethylmethylene blue (DMMB) assay. The CECT attenuation was positively correlated with the GAG content of bovine cartilage (R(2) = 0.87, P < 0.0001 for Group 1 and R(2) = 0.74, P = 0.001 for Group 2). Strong and significant positive correlations were observed between E and GAG content (R(2) = 0.90, P < 0.0001) as well as CECT attenuation and E (R(2) = 0.90, P < 0.0001). The CECT attenuation was negatively correlated with the three coefficients of friction: CECT vs μ(static) (R(2) = 0.71, P = 0.002), CECT vs μ(static_equilibrium) (R(2) = 0.79, P < 0.001), and CECT vs μ(kinetic) (R(2) = 0.69, P = 0.003). CECT with CA4+ is a useful tool for determining the mechanical properties of ex vivo cartilage tissue as the attenuation significantly correlates with the compressive modulus and coefficient of friction.

Depth-Dependent Transverse Shear Properties of the Human Corneal Stroma

To measure the transverse shear modulus of the human corneal stroma and its profile through the depth by mechanical testing, and to assess the validity of the hypothesis that the shear modulus will be greater in the anterior third due to increased interweaving of lamellae. Torsional rheometry was used to measure the transverse shear properties of 6 mm diameter buttons of matched human cadaver cornea pairs. One cornea from each pair was cut into thirds through the thickness with a femtosecond laser and each stromal third was tested individually. The remaining intact corneas were tested to measure full stroma shear modulus. The shear modulus from a 1% shear strain oscillatory test was measured at various levels of axial compression for all samples. After controlling for axial compression, the transverse shear moduli of isolated anterior layers were significantly higher than central and posterior layers. Mean modulus values at 0% axial strain were 7.71 ± 6.34 kPa in the anterior, 1.99 ± 0.45 kPa in the center, 1.31 ± 1.01 kPa in the posterior, and 9.48 ± 2.92 kPa for full thickness samples. A mean equilibrium compressive modulus of 38.7 ± 8.6 kPa at 0% axial strain was calculated from axial compression measured during the shear tests. Transverse shear moduli are two to three orders of magnitude lower than tensile moduli reported in the literature. The profile of shear moduli through the depth displayed a significant increase from posterior to anterior. This gradient supports the hypothesis and corresponds to the gradient of interwoven lamellae seen in imaging of stromal cross-sections.

Effect of a mechanical stimulation bioreactor on tissue engineered, scaffold‐free cartilage

Achieving sufficient functional properties prior to implantation remains a significant challenge for the development of tissue engineered cartilage. Many studies have shown chondrocytes respond well to various mechanical stimuli, resulting in the development of bioreactors capable of transmitting forces to articular cartilage in vitro. In this study, we describe the production of sizeable, tissue engineered cartilage using a novel scaffold-free approach, and determine the effect of perfusion and mechanical stimulation from a C9-x Cartigen bioreactor on the properties of the tissue engineered cartilage. We created sizable tissue engineered cartilage from porcine chondrocytes using a scaffold-free approach by centrifuging a high-density chondrocyte cell-suspension onto an agarose layer in a 50 mL tube. The gross and histological appearances, biochemical content, and mechanical properties of constructs cultured in the bioreactor for 4 weeks were compared to constructs cultured statically. Mechanical properties were determined from unconfined uniaxial compression tests. Constructs cultured in the bioreactor exhibited an increase in total GAG content, equilibrium compressive modulus, and dynamic modulus versus static constructs. Our study demonstrates the C9-x CartiGen bioreactor is able to enhance the biomechanical and biochemical properties of scaffold-free tissue engineered cartilage; however, no additional enhancement was seen between loaded and perfused groups.

Evaluation of Negative Fixed-Charge Density in Tissue-Engineered Cartilage by Quantitative MRI and Relationship With Biomechanical Properties

Applying tissue-engineered cartilage in a clinical setting requires noninvasive evaluation to detect the maturity of the cartilage. Magnetic resonance imaging (MRI) of articular cartilage has been widely accepted and applied clinically in recent years. In this study, we evaluated the negative fixed-charge density (nFCD) of tissue-engineered cartilage using gadolinium-enhanced MRI and determined the relationship between nFCD and biomechanical properties. To reconstruct cartilage tissue, articular chondrocytes from bovine humeral heads were embedded in agarose gel and cultured in vitro for up to 4 weeks. The nFCD of the cartilage was determined using the MRI gadolinium exclusion method. The equilibrium modulus was determined using a compressive stress relaxation test, and the dynamic modulus was determined by a dynamic compression test. The equilibrium compressive modulus and dynamic modulus of the tissue-engineered cartilage increased with an increase in culture time. The nFCD value--as determined with the [Gd-DTPA(2-)] measurement using the MRI technique--increased with culture time. In the regression analysis, nFCD showed significant correlations with equilibrium compressive modulus and dynamic modulus. From these results, gadolinium-enhanced MRI measurements can serve as a useful predictor of the biomechanical properties of tissue-engineered cartilage.

Electrostatic and non-electrostatic contributions of proteoglycans to the compressive equilibrium modulus of bovine articular cartilage

This study presents direct experimental evidence for assessing the electrostatic and non-electrostatic contributions of proteoglycans to the compressive equilibrium modulus of bovine articular cartilage. Immature and mature bovine cartilage samples were tested in unconfined compression and their depth-dependent equilibrium compressive modulus was determined using strain measurements with digital image correlation analysis. The electrostatic contribution was assessed by testing samples in isotonic and hypertonic saline; the combined contribution was assessed by testing untreated and proteoglycan-depleted samples. Though it is well recognized that proteoglycans contribute significantly to the compressive stiffness of cartilage, results demonstrate that the combined electrostatic and non-electrostatic contributions may add up to more than 98% of the modulus, a magnitude not previously appreciated. Of this contribution, about two thirds arises from electrostatic effects. The compressive modulus of the proteoglycan-depleted cartilage matrix may be as low as 3 kPa, representing less than 2% of the normal tissue modulus; experimental evidence also confirms that the collagen matrix in digested cartilage may buckle under compressive strains, resulting in crimping patterns. Thus, it is reasonable to model the collagen as a fibrillar matrix that can sustain only tension. This study also demonstrates that residual stresses in cartilage do not arise exclusively from proteoglycans, since cartilage remains curled relative to its in situ geometry even after proteoglycan depletion. These increased insights on the structure–function relationships of cartilage can lead to improved constitutive models and a better understanding of the response of cartilage to physiological loading conditions.

Transient Exposure to Transforming Growth Factor Beta 3 Improves the Mechanical Properties of Mesenchymal Stem Cell–Laden Cartilage Constructs in a Density-Dependent Manner

Mesenchymal stem cells (MSCs) are an attractive cell source for cartilage tissue engineering and regenerative medicine. However, the use of these cells has been limited by their reduced ability to form functional tissue compared to chondrocytes when placed in three-dimensional culture systems. To optimize MSC functional chondrogenesis, we examined the effects of increasing seeding density and transient application of transforming growth factor beta 3 (TGF-beta3), two factors previously shown to improve growth of chondrocyte-based constructs. Chondrocytes seeded in agarose at 20 million cells/mL and MSCs seeded at 20 or 60 million cells/mL agarose were cultured for 7 weeks under continuous or transient application of TGF-beta3. In the transient group, cell-laden constructs were exposed to TGF-beta3 for the initial 3 weeks, followed by 4 weeks of culture in medium without TGF-beta3. Compressive properties, biochemical content, and gene expression were assessed at 3, 5, and 7 weeks. Matrix distribution and collagen type was determined using histology and immunohistochemistry, and chondrogenic and osteogenic markers were assessed using real-time polymerase chain reaction. When maintained continuously with TGF-beta3, chondrocyte-seeded constructs achieved a higher equilibrium compressive modulus than MSCs similarly maintained. Although properties of both groups increased with respect to starting values, there was no difference in bulk mechanical or biochemical properties with higher seeding density when MSCs were cultured with constant TGF-beta3. Findings also showed that while transient application of TGF-beta3 elicited robust growth from chondrocyte-laden gels, MSCs seeded at the same density failed to respond, although constructs maintained their previously accrued properties and continued to express cartilaginous genes after TGF-beta3 removal. Conversely, MSCs seeded at 60 million cells/mL exhibited a strong anabolic response with transient TGF-beta3 exposure, achieving an equilibrium modulus of approximately 200 kPa. Although this represents the highest modulus we have been able to achieve with MSC-seeded constructs using our culture system, further work remains to optimize MSC chondrogenesis for cartilage tissue engineering, particularly in terms of collagen content and dynamic mechanical properties.

Contrast Enhanced Computed Tomography can predict the glycosaminoglycan content and biomechanical properties of articular cartilage

An early hallmark of osteoarthritis (OA) is the progressive loss of glycosaminoglycans (GAGs), the extracellular matrix (ECM) component of articular cartilage that confers it with compressive stiffness. Our aim in this work is to establish the feasibility of using Contrast Enhanced Computed Tomography (CECT) with an anionic iodinated contrast agent - Cysto Conray II - as a minimally invasive tool to measure the changes in the GAG content as well as the compressive stiffness of articular cartilage. The GAG content of mated osteochondral plugs excised from bovine patello-femoral joints was progressively degraded using chondroitinase ABC. The mated plugs were then immersed in an anionic, tri-iodinated contrast agent, imaged using peripheral quantitative computed tomography (pQCT), subjected to an unconfined compressive stress relaxation test and the GAG content measured using 1,9-dimethylmethylene blue (DMMB) assay. Partial correlation analysis was performed to compare the variation in X-ray attenuation measured by pQCT to the variation in GAG content and in equilibrium compressive modulus. The X-ray attenuation of cartilage exposed to an anionic, tri-iodinated, contrast agent measured by quantitative computed tomography (QCT) accounted for 83% of the variation in GAG content (r(2)=0.83, P<0.0001) and 93% of the variation in the equilibrium compressive modulus (r(2)=0.93, P<0.0001). Using a mated osteochondral plug model to evaluate the biochemical composition and biomechanical properties of cartilage, this study demonstrates the interrelationships between X-ray attenuation, GAG content, and equilibrium compressive modulus, and that CECT can be used to monitor and quantify changes in the GAG content and biomechanical properties of articular cartilage.

Macromer density influences mesenchymal stem cell chondrogenesis and maturation in photocrosslinked hyaluronic acid hydrogels

Engineering cartilage requires that a clinically relevant cell type be situated within a 3D environment that supports cell viability, the production and retention of cartilage-specific extracellular matrix (ECM), and eventually, the establishment of mechanical properties that approach that of the native tissue. In this study, we investigated the ability of bone marrow derived mesenchymal stem cells (MSCs) to undergo chondrogenesis in crosslinked methacrylated hyaluronic acid hydrogels (MeHA) of different macromer concentrations (1, 2, and 5%). Over a 6 week culture period under pro-chondrogenic conditions, we evaluated cartilage-specific gene expression, ECM deposition within constructs and released to the culture media, and mechanical properties in both compression and tension. Further, we examined early matrix assembly and long term histological features of the forming tissues, as well as the ability of macromolecules to diffuse within hydrogels as a function of MeHA macromer concentration. Findings from this study show that variations in macromer density influence MSC chondrogenesis in distinct ways. Increasing HA macromer density promoted chondrogenesis and matrix formation and retention, but yielded functionally inferior constructs due to limited matrix distribution throughout the construct expanse. In 1% MeHA constructs, the equilibrium compressive modulus reached 0.12MPa and s-GAG content reached nearly 3% of the wet weight, values that matched or exceeded those of control agarose constructs and that are 25 and 50% of native tissue levels, respectively. These data provide new insight into how early matrix deposition regulates long term construct development, and defines new parameters for optimizing the formation of functional MSC-based engineered articular cartilage using HA hydrogels.

Changes to articular cartilage following remote application of radiofrequency energy and with or without Cosequin therapy

To determine the short- and long-term changes in the biomechanical properties and metabolic activity of articular cartilage following the remote application of bipolar radiofrequency (bRF) and monopolar radiofrequency (mRF) energy within the rabbit stifle joint. The rabbits were randomly assigned to either Group-1 (normal rabbit food), or they were assigned to Group-2 (2% Cosequin in the diet). Each rabbit underwent bilateral stifle arthroscopy with either bRF or mRF applied to the infrapatellar fat pad for 45 seconds. Cartilage samples were collected at zero, four, and 14 weeks after surgery. Data were analyzed with a mixed model analysis of variance (ANOVA) for chondrocyte death, amount of GAG synthesis, and the equilibrium compressive modulus. A significant increase in histological damage was noted at weeks four and 14 compared to week zero. Most of the chondrocyte death noted with confocal laser microscopy (49 of 56 samples) was noted in the superficial region (outer 25%) of the articular cartilage. GAG synthesis was not significantly different between groups or devices at any time point. A significant difference was not noted in equilibrium compressive modulus throughout the study. Remote application of bRF and mRF energy lead to immediate chondrocyte death. Most of the damage was superficial hence the metabolic activity and biomechanical properties of the extracellular matrix were maintained throughout this study. Treatment with Cosequin did not prevent superficial chondrocyte death caused by the application of radiofrequency (RF) energy with in the joint.