Andreas M. Seitz

TSG-6 Released from Intradermally Injected Mesenchymal Stem Cells Accelerates Wound Healing and Reduces Tissue Fibrosis in Murine Full-Thickness Skin Wounds

Proper activation of macrophages (Mφ) in the inflammatory phase of acute wound healing is essential for physiological tissue repair. However, there is a strong indication that robust Mφ inflammatory responses may be causal for the fibrotic response always accompanying adult wound healing. Using a complementary approach of in vitro and in vivo studies, we here addressed the question of whether mesenchymal stem cells (MSCs)-due to their anti-inflammatory properties-would control Mφ activation and tissue fibrosis in a murine model of full-thickness skin wounds. We have shown that the tumor necrosis factor-α (TNF-α)-stimulated protein 6 (TSG-6) released from MSCs in co-culture with activated Mφ or following injection into wound margins suppressed the release of TNF-α from activated Mφ and concomitantly induced a switch from a high to an anti-fibrotic low transforming growth factor-β1 (TGF-β1)/TGF-β3 ratio. This study provides insight into what we believe to be a previously undescribed multifaceted role of MSC-released TSG-6 in wound healing. MSC-released TSG-6 was identified to improve wound healing by limiting Mφ activation, inflammation, and fibrosis. TSG-6 and MSC-based therapies may thus qualify as promising strategies to enhance tissue repair and to prevent excessive tissue fibrosis.

Effect of partial meniscectomy at the medial posterior horn on tibiofemoral contact mechanics and meniscal hoop strains in human knees.

We examined the influence of partial meniscectomy of 10 mm width on 10 human cadaveric knee joints, as it is performed during the treatment of radial tears in the posterior horn of the medial meniscus, on maximum contact pressure, contact area (CA), and meniscal hoop strain in the lateral and medial knee compartments. In case of 0° and 30° flexion angle, 20% and 50% partial meniscectomy did not influence maximum contact pressure and area. Only in case of 60° knee flexion, 50% partial resection increased medial maximum contact pressure and decreased the medial CA statistically significant. However, 100% partial resection increased maximum contact pressure and decreased CA significantly in the meniscectomized medial knee compartment in all tested knee positions. No significant differences were noted for meniscal hoop strain. From a biomechanical point of view, our in vitro study suggests that the medial joint compartment is not in danger of accelerated cartilage degeneration up to a resection limit of 20% meniscal depth and 10 mm width. Contact mechanics are likely to be more sensitive to partial meniscectomy at higher flexion angles, which has to be further investigated.

Processed xenogenic cartilage as innovative biomatrix for cartilage tissue engineering: effects on chondrocyte differentiation and function

One key point in the development of new bioimplant matrices for the reconstruction and replacement of cartilage defects is to provide an adequate microenvironment to ensure chondrocyte migration and de novo synthesis of cartilage‐specific extracellular matrix (ECM). A recently developed decellularization and sterilization process maintains the three‐dimensional (3D) collagen structure of native septal cartilage while increasing matrix porosity, which is considered to be crucial for cartilage tissue engineering. Human primary nasal septal chondrocytes were amplified in monolayer culture and 3D‐cultured on processed porcine nasal septal cartilage scaffolds. The influence of chondrogenic growth factors on neosynthesis of ECM proteins was examined at the protein and gene expression levels. Seeding experiments demonstrated that processed xenogenic cartilage matrices provide excellent environmental properties for human nasal septal chondrocytes with respect to cell adhesion, migration into the matrix and neosynthesis of cartilage‐specific ECM proteins, such as collagen type II and aggrecan. Matrix biomechanical stability indicated that the constructs retrieve full stability and function during 3D culture for up to 42 days, proportional to collagen type II and GAG production. Thus, processed xenogenic cartilage offers a suitable environment for human nasal chondrocytes and has promising potential for cartilage tissue engineering in the head and neck region. Copyright

Stress-relaxation response of human menisci under confined compression conditions

The objective of this study was to determine the viscoelastic properties of human meniscal tissue during stress-relaxation under confined compression conditions. Lateral and medial longitudinal meniscus plugs of 25 donor knees (ntotal=150) were exposed to stress-relaxation tests under confined compression conditions at three compression levels (ε=0.1; 0.15; 0.2). Mathematical modelling using an exponential 1D-diffusion equation was used to predict the viscoelastic properties. Subsequently, finite element (FE) models were created using identical geometry, properties and test conditions as used for the in-vitro tests. Two constitutively different underlying mathematical formulations were applied to the FE models to reveal possible differences in their predictions for the relaxation response. While the first FE model mimicked the analytical model (FE1), the second FE model used a different biphasic, non-linear approach (FE2). Regression analyses showed promising coefficients of determination (R(2)>0.73) between the experimental data and the predictions obtained from the diffusion equation and the two FE models. Mean aggregate modulus, predicted with the diffusion equation (HA=64.0 kPa) was lower than those obtained with the two FE analyses (HA,FE1=91.9 kPa; HA,FE2=81.5 kPa). Mean hydraulic permeability (kFE2=1.5×10(-15)m(4)/Ns) of the second FE2 approach was statistically lower (p<0.01) than the other permeability values (k=3.9×10(-15)m(4)/Ns; kFE1=3.4×10(-15)m(4)/Ns). These differences are mainly due to the different underlying mathematical models used. However, when compared with corresponding literature, the results of the present study indicated good agreement. The results of the present study contribute to a better understanding of the complex nature of meniscal tissue and might also have an impact on the design of future meniscal substitutes.

Osteoarthritic cartilage explants affect extracellular matrix production and composition in cocultured bone marrow-derived mesenchymal stem cells and articular chondrocytes.

IntroductionIn the present study, we established a novel in vitro coculture model to evaluate the influence of osteoarthritis (OA) cartilage explants on the composition of newly produced matrix and chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs) and the phenotype of OA chondrocytes. In addition, we included a “tri-culture” model, whereby a mixture of BMSCs and chondrocytes was cultured on the surface of OA cartilage explants.MethodsGene expression analysis, protein and glycosaminoglycan (GAG) assays, dot-blot, immunofluorescence, and biomechanical tests were used to characterize the properties of newly generated extracellular matrix (ECM) from chondrocytes and chondrogenically differentiated BMSCs and a mix thereof. We compared articular cartilage explant cocultures with BMSCs, chondrocytes, and mixed cultures (chondrocytes and BMSCs 1:1) embedded in fibrin gels with fibrin gel-embedded cells cultured without cartilage explants (monocultures).ResultsIn general, co- and tri-cultured cell regimens exhibited reduced mRNA and protein levels of collagens I, II, III, and X in comparison with monocultures, whereas no changes in GAG synthesis were observed. All co- and tri-culture regimens tended to exhibit lower Young’s and equilibrium modulus compared with monocultures. In contrast, aggregate modulus and hydraulic permeability seemed to be higher in co- and tri-cultures. Supernatants of cocultures contained significant higher levels of interleukin-1 beta (IL-1β), IL-6, and IL-8. Stimulation of monocultures with IL-1β and IL-6 reduced collagen gene expression in BMSCs and mixed cultures in general but was often upregulated in chondrocytes at late culture time points. IL-8 stimulation affected BMSCs only.ConclusionsOur results suggest an inhibitory effect of OA cartilage on the production of collagens. This indicates a distinct modulatory influence that affects the collagen composition of the de novo-produced ECM from co- and tri-cultured cells and leads to impaired mechanical and biochemical properties of the matrix because of an altered fibrillar network. We suggest that soluble factors, including IL-1β and IL-6, released from OA cartilage partly mediate these effects. Thus, neighbored OA cartilage provides inhibitory signals with respect to BMSCs’ chondrogenic differentiation and matrix composition, which need to be accounted for in future cell-based OA treatment strategies.

Subchondral bone influences chondrogenic differentiation and collagen production of human bone marrow-derived mesenchymal stem cells and articular chondrocytes.

IntroductionOsteoarthritis (OA) is characterized by an imbalance in cartilage and underlying subchondral bone homeostasis. We hypothesized that signals from the subchondral bone may modulate production of matrix components, alter chondrogenic differentiation potential of cocultured bone marrow-derived mesenchymal stem cells (BMSC) and induce a phenotypic shift in differentiated OA chondrocytes.MethodsWe established a novel coculture model between BMSC, mixed cultures (BMSC and chondrocytes) and chondrocytes embedded in fibrin gel with OA and normal subchondral bone explants (OAB and NB). Tissues and cells were either derived from OA or trauma patients. In addition, we used adipose-derived stem cells (ASC) from liposuction. With gene expression analysis, biochemical assays, immunofluorescence and biomechanical tests we characterized the properties of newly generated extracellular matrix (ECM) from chondrocytes and chondrogenically differentiating BMSC cocultured with OAB or NB in comparison with monocultures (cultures without bone explants).ResultsOverall, gene expression of collagens of OAB and NB cocultured cells was reduced compared to monocultures. Concomitantly, we observed significantly lower collagen I, II and III and glycosaminoglycan (GAG) production in OAB cocultured cell lysates. In parallel, we detected increased concentrations of soluble GAGs and basic fibroblast growth factor (bFGF), interleukin (IL)-6 and IL-8 in supernatants of OAB and NB cocultures mainly at early time points. IL-1ß concentration was increased in supernatants of OAB cocultures, but not in NB cocultures. Cell-free NB or OAB explants released different amounts of IL-1ß, bFGF and soluble GAG into cell culture supernatants. In comparison to cocultures, monocultures exhibited higher Young’s modulus and equilibrium modulus. Stimulation of monocultures with IL-1ß led to a downregulation of aggrecan (ACAN) gene expression and in general to induced matrix metalloprotease (MMP)2, MMP3 and MMP-13 gene expression while IL-6 and IL-8 stimulation partly reduced ACAN, MMP3 and MMP-13 gene expression.ConclusionsOur results suggest an alteration of molecular composition and mechanical properties of the newly formed ECM in subchondral bone cocultures. We suggest that soluble factors, that is interleukins and bFGF, released in cocultures exert inhibitory effects on collagen and temporary effects on proteoglycan production, which finally results in a reduction of mechanical strength of newly formed fibrillar networks.

Medial meniscal displacement and strain in three dimensions under compressive loads: MR assessment

To investigate the 3D displacement and the local strain of the medial meniscus and its attachments under compressive loading.

Anterior Knee Laxity Increases Gapping of Posterior Horn Medial Meniscal Tears

Background Meniscal tears often occur in association with anterior cruciate ligament (ACL) lesions or in chronically lax knees. It is also known that meniscal repairs are less likely to heal in ACL-deficient knees. Purpose To test the effect of different knee joint motion and loading conditions on the gapping behavior of longitudinal posterior horn meniscal tears in stable and ACL-deficient knee joints. Study Design Controlled laboratory study. Methods Longitudinal tears of 3 cm were set in the posterior horn of the medial menisci in 10 human cadaveric joints. The medial plateau of the knees was replaced by a transparent replica, and an arthroscope was placed underneath to observe the gapping phenomenon of the meniscal tears. The maximum gap width occurring during flexion-extension under various motion and loading situations was registered in intact and ACL-deficient joints before and after meniscal repair with FasT-Fix suture anchors. Results Longitudinal meniscal tears gapped significantly wider after ACL transection under 30-N axial joint load (P < .05). Increasing the axial load to 200 N or applying external moments to the knee did not lead to further alterations in the gap size. Gapping was significantly reduced after meniscal repair (P < .01). However, after meniscal repair, gapping under 30-N and 200-N axial joint load was still increased significantly after ACL transection compared with the ACL-intact state (P < .05). Conclusion Anterior knee laxity increases gapping across both unrepaired and repaired vertical peripheral medial meniscal posterior horn tears. Clinical Relevance Repairing such meniscal tears without reconstructing the ACL may affect meniscus healing rates or increase the risk of retears. Moderate rehabilitation regimens can be recommended, allowing at least for partial weightbearing and knee motion from extension to 120° of flexion in a stable knee. However, caution should be recommended if meniscal repair is performed in a knee joint with persistent anterior laxity due to ACL deficiency.

Increasing posterior tibial slope does not raise anterior cruciate ligament strain but decreases tibial rotation ability.

BACKGROUND It was investigated whether the strain of the anterior cruciate ligament and tibial kinematics are affected by increasing posterior tibial slope. METHODS 9 human cadaveric knee joints were passively moved between full extension and 120° flexion in a motion and loading simulator under various loading conditions and at 0°, 5°, 10° and 15° posterior tibial slope angles. The anterior cruciate ligament strain and the tibial rotation angle were registered. To assess the influence of posterior tibial slope on the anterior cruciate ligament strain at a fixed flexion angle the anterior cruciate ligament strain was recorded at three different flexion angles of 0°, 30° and 90° while continuously increasing the osteotomy angle from 5° to 15°. FINDINGS The anterior cruciate ligament strain was either not affected by the posterior tibial slope angle or, in some load cases, was decreased for increasing posterior tibial slope (P<0.05). There was a significant decrease of tibial rotation when the posterior tibial slope was increased to 15° for many of the load cases tested (P<0.05). The mean maximum decrease was from 17.4° (SD 5.7°) to 11.2° (SD 4.7°) observed for flexion-extension motion under 30N axial load in combination with an internal rotation moment. INTERPRETATION The hypothesis that increasing posterior tibial slope results in higher anterior cruciate ligament strain was not confirmed. However, knee kinematics were affected in terms of a reduced tibial rotation. From a biomechanical point of view the data do not support the efficacy of sagittal osteotomies as performed to stabilize anterior cruciate ligament deficient knees.

Mechanical properties and morphological analysis of the transitional zone between meniscal body and ligamentous meniscal attachments

In recent years, an increasing number of studies reporting on meniscal root tears have been published. While the meniscus and its ligamentous meniscal attachments have been studied before, little is known about the transitional zone between these two structures. Therefore, the aim of this study was to mechanically and morphologically characterize the transitional zone between meniscus and its meniscal attachments. Dumbbell-shaped specimens were obtained from the transitional zone between meniscus and its meniscal attachments of 6 knee joints. Samples were divided into tibial and central layers of the anterior lateral (AL), anterior medial (AM), posterior lateral (PL) and posterior medial (PM) transitional region. Testing was performed to obtain the dissipated energy during hysteresis as well as the linear modulus (Elin), the maximum strain (εmax), the maximum engineering stress (σmax,eng) and location of rupture during tensile test to failure. Two additional knee joints were used to investigate morphological differences between meniscus, transitional zone and meniscal attachments in 8µm transverse slices. The central layer of the AL, AM and PL dissipated up to 48% less energy than the tibial layer. Elin was highest in the tibial layer of the PM with 107.4±61.1MPa and lowest in the central layer of the PL with 56.0±20.5MPa. The maximum strain was higher in the central layer than in the tibial layer at the AL, AM, and PL locations. The average σmax,eng was 12.7±9.9MPa over all location and layers. 78% of the samples ruptured during tensile test to failure in the transitional zone. The morphological evaluation showed a smooth transitional zone with a transitional curve which was either linear or bell-shaped. The strength found in the transitional zone was lower than in the meniscus and the meniscal attachments, which corresponds well to clinical findings.