Mechanical properties and structure-function relationships of human chondrocyte-seeded cartilage constructs after in vitro culture.

Autologous Chondrocyte Implantation (ACI) is a widely recognized method for the repair of focal cartilage defects. Despite the accepted use, problems with this technique still exist, including graft hypertrophy, damage to surrounding tissue by sutures, uneven cell distribution, and delamination. Modified ACI techniques overcome these challenges by seeding autologous chondrocytes onto a 3D scaffold and securing the graft into the defect. Many studies on these tissue engineered grafts have identified the compressive properties, but few have examined frictional and shear properties as suggested by FDA guidance. This study is the first to perform three mechanical tests (compressive, frictional, and shear) on human tissue engineered cartilage. The objective was to understand the complex mechanical behavior, function, and changes that occur with time in these constructs grown in vitro using compression, friction, and shear tests. Safranin-O histology and a DMMB assay both revealed increased sulfated glycosaminoglycan (sGAG) content in the scaffolds with increased maturity. Similarly, immunohistochemistry revealed increased lubricin localization on the construct surface. Confined compression and friction tests both revealed improved properties with increased construct maturity. Compressive properties correlated with the sGAG content, while improved friction coefficients were attributed to increased lubricin localization on the construct surfaces. In contrast, shear properties did not improve with increased culture time. This study suggests the various mechanical and biological properties of tissue engineered cartilage improve at different rates, indicating thorough mechanical evaluation of tissue engineered cartilage is critical to understanding the performance of repaired cartilage. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2298-2306, 2017.

Comparison of a Saline-coupled Bipolar Sealer Versus Traditional Electrosurgery in a Porcine Model of Chronic Wound Healing.

OBJECTIVE: This study examines the healing dynamics of in vivo porcine muscle tissue wounds hemostatically treated with a saline-coupled bipolar tissue sealer (SCBS) compared with traditional electrosurgical (ES) coagulation. MATERIALS AND METHODS: Six cutaneous incisions were created on the dorsum of 28 adult male Yorkshire swine. The underlying muscle tissue was incised with a cold scalpel then treated with either SCBS (at 170 W) or traditional ES (at Coag 45 W). Time to hemostasis was recorded. Animals were humanely euthanized at day 2 and weeks 2, 3, or 8; treated tissue was harvested for histopathological evaluation. RESULTS: After 8 weeks, the extent of wound healing was similar between SCBS and ES. Both devices controlled bleeding effectively; however, SCBS-treated wounds exhibited a greater depth of thermal effect over the first 3 weeks despite a shorter treatment time. Wounds treated with SCBS demonstrated fewer inflammatory markers at early time points but healed more slowly, with scores that lagged behind ES for collagen deposition, fibrous tissue maturity, extracellular matrix, and stage of healing. Myofiber regeneration notably increased in SCBS-treated wounds at weeks 2, 3, and 8. By the end of the 8-week recovery period, there were no significant differences in healing parameters between the 2 groups. CONCLUSIONS: Overall, both devices elicited similar progression of healing by 8 weeks. The SCBS produced a deeper thermal effect in a shorter treatment time and improved myofiber regeneration compared with ES and had an equivalent overall course of healing.

Hyaline Articular Matrix Formed by Dynamic Self-Regenerating Cartilage and Hydrogels.

Injuries to the articular cartilage surface are challenging to repair because cartilage possesses a limited capacity for self-repair. The outcomes of current clinical procedures aimed to address these injuries are inconsistent and unsatisfactory. We have developed a novel method for generating hyaline articular cartilage to improve the outcome of joint surface repair. A suspension of 10(7) swine chondrocytes was cultured under reciprocating motion for 14 days. The resulting dynamic self-regenerating cartilage (dSRC) was placed in a cartilage ring and capped with fibrin and collagen gel. A control group consisted of chondrocytes encapsulated in fibrin gel. Constructs were implanted subcutaneously in nude mice and harvested after 6 weeks. Gross, histological, immunohistochemical, biochemical, and biomechanical analyses were performed. In swine patellar groove, dSRC was implanted into osteochondral defects capped with collagen gel and compared to defects filled with osteochondral plugs, collagen gel, or left empty after 6 weeks. In mice, the fibrin- and collagen-capped dSRC constructs showed enhanced contiguous cartilage matrix formation over the control of cells encapsulated in fibrin gel. Biochemically, the fibrin and collagen gel dSRC groups were statistically improved in glycosaminoglycan and hydroxyproline content compared to the control. There was no statistical difference in the biomechanical data between the dSRC groups and the control. The swine model also showed contiguous cartilage matrix in the dSRC group but not in the collagen gel and empty defects. These data demonstrate the survivability and successful matrix formation of dSRC under the mechanical forces experienced by normal hyaline cartilage in the knee joint. The results from this study demonstrate that dSRC capped with hydrogels successfully engineers contiguous articular cartilage matrix in both nonload-bearing and load-bearing environments.

Mechanical properties and structure-function relationships in articular cartilage repaired using IGF-I gene-enhanced chondrocytes.

Several studies have demonstrated the benefits of IGF-I gene therapy in enhancing the histologic and biochemical content of cartilage repaired by chondrocyte transplantation. However, there is little to no data on the mechanical performance of IGF-I augmented cartilage grafts. This study evaluated the compressive properties of full-thickness chondral defects in the equine femur repaired with and without IGF-I gene therapy. Animals were randomly assigned to one of three study cohorts based on chondrocyte treatment provided in each defect: (i) IGF-I gene delivered by recombinant adeno-associated virus (rAAV)-5; (ii) AAV-5 delivering GFP as a reporter; (iii) naïve cells without virus. In each case, the opposite limb was implanted with a fibrin carrier without cells. Samples were prepared for confined compression testing to measure the aggregate modulus and hydraulic permeability. All treatment groups, regardless of cell content or transduction, had mechanical properties inferior to native cartilage. Overexpression of IGF-I increased modulus and lowered permeability relative to other treatments. Investigation of structure-property relationships revealed that Ha and k were linearly correlated with GAG content but logarithmically correlated with collagen content. This provides evidence that IGF-I gene therapy can improve healing of articular cartilage and can greatly increase the mechanical properties of repaired grafts.

Mechanical characterization of matrix-induced autologous chondrocyte implantation (MACI®) grafts in an equine model at 53 weeks.

There has been much interest in using autologous chondrocytes in combination with scaffold materials to aid in cartilage repair. In the present study, a total of 27 animals were used to compare the performance of matrix-assisted chondrocyte implantation (MACI®) using a collagen sponge as a chondrocyte delivery vehicle, the sponge membrane alone, and empty controls. A total of three distinct types of mechanical analyses were performed on repaired cartilage harvested from horses after 53 weeks of implantation: (1) compressive behavior of samples to measure aggregate modulus (HA) and hydraulic permeability (k) in confined compression; (2) local and global shear modulus using confocal strain mapping; and (3) boundary friction coefficient using a custom-built tribometer. Cartilage defects receiving MACI® implants had equilibrium modulus values that were 70% of normal cartilage, and were not statistically different than normal tissue. Defects filled with Maix™ membrane alone or left empty were only 46% and 51-63% of control, respectively. The shear modulus of tissue from all groups of cartilage defects were between 4 and 10 times lower than control tissue, and range from 0.2 to 0.4 MPa. The average values of boundary mode friction coefficients of control tissue from all groups ranged from 0.42 to 0.52. This study represents an extensive characterization of the mechanical performance of the MACI® grafts implant in a large animal model at 53 weeks. Collectively, these data demonstrate a range of implant performance, revealing similar compressive and frictional properties to native tissue, with inferior shear properties.

Effects of enzymatic treatments on the depth-dependent viscoelastic shear properties of articular cartilage.

Osteoarthritis (OA) is a disease that involves the erosion and structural weakening of articular cartilage. OA is characterized by the degradation of collagen and proteoglycans in the extracellular matrix (ECM), particularly at the articular surface by proteinases including matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs).(1) Degradation of collagen and proteoglycans is known to alter shear mechanical properties of cartilage, but study of this phenomenon has been focused on bulk tissue properties. The purpose of this study was to assess microscale cartilage damage induced by trypsin or collagenase using a technique to measure the local shear viscoelastic properties. Safranin-O histology revealed a decrease in proteoglycans near the articular surface after collagenase and trypsin digestions, with proteoglycan depletion increasing in time. Similarly, confocal reflectance micrographs showed increasing collagen degradation in collagenase treated samples, although the collagen network remained intact after trypsin treatment. Both treatments induced changes in shear modulus that were confined to a narrow range (∼400µm) near tissue surface. In addition, collagenase altered the total energy dissipation distribution by up to a factor of 100, with longer digestion times corresponding to higher energy dissipation. The ability to detect local mechanical signatures in tissue composition and mechanics is an important tool for understanding the spatially non-uniform changes that occur in articular cartilage diseases such as OA.