Mechano-regulated tenascin-C orchestrates muscle repair

Tenascin-C (TNC) is a mechano-regulated, morphogenic, extracellular matrix protein that is associated with tissue remodeling. The physiological role of TNC remains unclear because transgenic mice engineered for a TNC deficiency, via a defect in TNC secretion, show no major pathologies. We hypothesized that TNC-deficient mice would demonstrate defects in the repair of damaged leg muscles, which would be of functional significance because this tissue is subjected to frequent cycles of mechanical damage and regeneration. TNC-deficient mice demonstrated a blunted expression of the large TNC isoform and a selective atrophy of fast-muscle fibers associated with a defective, fast myogenic expression response to a damaging mechanical challenge. Transcript profiling mapped a set of de-adhesion, angiogenesis, and wound healing regulators as TNC expression targets in striated muscle. Expression of these regulators correlated with the residual expression of a damage-related 200-kDa protein, which resembled the small TNC isoform. Somatic knockin of TNC in fast-muscle fibers confirmed the activation of a complex expression program of interstitial and slow myofiber repair by myofiber-derived TNC. The results presented here show that a TNC-orchestrated molecular pathway integrates muscle repair into the load-dependent control of the striated muscle phenotype.

Ability of dGEMRIC and T2 mapping to evaluate cartilage repair after microfracture: a goat study

OBJECTIVE: To investigate the ability of delayed gadolinium-enhanced magnetic resonance (MR) imaging of cartilage (dGEMRIC) and T2 mapping to evaluate the quality of repair tissue after microfracture. DESIGN: Twelve knees from 12 goats were studied. An osteochondral defect (diameter, 6mm; depth, 3mm) with microfracture was created in the weight-bearing aspect of both the medial and lateral femoral condyles. Goats were euthanized at 24 weeks (n=6) and 48 weeks (n=6) postsurgery. Pre-contrast R1 (R1pre) and post-contrast R1 (R1post) measurements for dGEMRIC and a pre-contrast T2 measurement for T2 mapping were performed with a 3T MR imaging system. MR imaging findings were compared with histological and biochemical assessments. RESULTS: In native cartilage, significant correlations were observed between the R1post and the glycosaminoglycan (GAG) concentration, as well as DeltaR1 (difference between the R1pre and R1post) and the GAG concentration (P<0.05). In repair tissue, a significant correlation was observed between DeltaR1 and the GAG concentration (P<0.05), but not between the R1post and the GAG concentration. In both repair tissue and native cartilage, no correlation was observed between T2 and the water concentration or between T2 and the hydroxyproline (HP) concentration. A zonal variation of T2 and a clear dependence of T2 on the angles relative to B0 were observed in native cartilage, but not in repair tissue. CONCLUSION: dGEMRIC with DeltaR1 measurement might be useful for the evaluation of the GAG concentration in repair tissue after microfracture. T2 mapping might be useful for the differentiation of repair tissue after microfracture from native cartilage; however, its potential to assess the specific biochemical markers in native cartilage as well as repair tissue may be limited.

Structural characterization and reliable biomechanical assessment of integrative cartilage repair

Structural and functional characterization of integrative cartilage repair in controlled model systems can play a key role in the development of innovative strategies to improve the long-term outcome of many cartilage repair procedures. In this work, we first developed a method to reproducibly generate geometrically defined disk/ring cartilage composites and to remove outgrown fibrous layers which can encapsulate cartilaginous tissues during culture. We then used the model system to test the hypothesis that such fibrous layers lead to an overestimation of biomechanical parameters of integration at the disk/ring interface. Transmission electron microscopy images of the composites after 6 weeks of culture indicated that collagen fibrils in the fibrous tissue layer were well integrated into the collagen network of the cartilage disk and ring, whereas molecular bridging between opposing disk/ring cartilage surfaces was less pronounced and restricted to regions with narrow interfacial regions (< 2 microm). Stress-strain profiles generated from mechanical push-out tests for composites with the layers removed displayed a single and distinct peak, whereas profiles for composites with the layers left intact consisted of multiple superimposed peaks. As compared to composites with removed layers, composites with intact layers had significantly higher adhesive strengths (161+/-9 vs. 71+/-11 kPa) and adhesion energies (15.0+/-0.7 vs. 2.7+/-0.4 mJ/mm2). By combining structural and functional analyses, we demonstrated that the outgrowing tissue formed during in vitro culture of cartilaginous specimens should be eliminated in order to reliably quantify biomechanical parameters related to integrative cartilage repair.

Bovine primary chondrocyte culture in synthetic matrix metalloproteinase-sensitive poly(ethylene glycol)-based hydrogels as a scaffold for cartilage repair

A poly(ethylene glycol) (PEG)-based hydrogel was used as a scaffold for chondrocyte culture. Branched PEG-vinylsulfone macromers were end-linked with thiol-bearing matrix metalloproteinase (MMP)-sensitive peptides (GCRDGPQGIWGQDRCG) to form a three-dimensional network in situ under physiologic conditions. Both four- and eight-armed PEG macromer building blocks were examined. Increasing the number of PEG arms increased the elastic modulus of the hydrogels from 4.5 to 13.5 kPa. PEG-dithiol was used to prepare hydrogels that were not sensitive to degradation by cell-derived MMPs. Primary bovine calf chondrocytes were cultured in both MMP-sensitive and MMP-insensitive hydrogels, formed from either four- or eight-armed PEG. Most (>90%) of the cells inside the gels were viable after 1 month of culture and formed cell clusters. Gel matrices with lower elastic modulus and sensitivity to MMP-based matrix remodeling demonstrated larger clusters and more diffuse, less cell surface-constrained cell-derived matrix in the chondron, as determined by light and electron microscopy. Gene expression experiments by real-time RT-PCR showed that the expression of type II collagen and aggrecan was increased in the MMP-sensitive hydrogels, whereas the expression level of MMP-13 was increased in the MMP-insensitive hydrogels. These results indicate that cellular activity can be modulated by the composition of the hydrogel. This study represents one of the first examples of chondrocyte culture in a bioactive synthetic material that can be remodeled by cellular protease activity.

Challenges and strategies in the repair of ruptured annulus fibrosus

Lumbar discectomy is the surgical procedure most frequently performed for patients suffering from low back pain and sciatica. Disc herniation as a consequence of degenerative or traumatic processes is commonly encountered as the underlying cause for the painful condition. While discectomy provides favourable outcome in a majority of cases, there are conditions where unmet requirements exist in terms of treatment, such as large disc protrusions with minimal disc degeneration; in these cases, the high rate of recurrent disc herniation after discectomy is a prevalent problem. An effective biological annular repair could improve the surgical outcome in patients with contained disc herniations but otherwise minor degenerative changes. An attractive approach is a tissue-engineered implant that will enable/stimulate the repair of the ruptured annulus. The strategy is to develop three-dimensional scaffolds and activate them by seeding cells or by incorporating molecular signals that enable new matrix synthesis at the defect site, while the biomaterial provides immediate closure of the defect and maintains the mechanical properties of the disc. This review is structured into (1) introduction, (2) clinical problems, current treatment options and needs, (3) biomechanical demands, (4) cellular and extracellular components, (5) biomaterials for delivery, scaffolding and support, (6) pre-clinical models for evaluation of newly developed cell- and material-based therapies, and (7) conclusions. This article highlights that an interdisciplinary approach is necessary for successful development of new clinical methods for annulus fibrosus repair. This will benefit from a close collaboration between research groups with expertise in all areas addressed in this review.

The medial based bi- or trilobed flap for repair of distal alar defects

BACKGROUND Reconstruction of defects of the lateral nasal ala might be challenging. Reconstruction with a bi- or trilobed flap is common. The laterally based bi- or trilobed flap for defects of the distal ala or lateral tip of the nose produces mostly tissue protrusion in the nasal groove which is aesthetically unpleasant. Why not use more the medially based bi- or trilobed flap? OBJECTIVE To describe the utility of bilobed and trilobed flaps for alar defects insisting on the design of medially based flaps. METHODS To show the technique and practical application for this kind of reconstruction. RESULTS The bi- and trilobed flaps are useful for defect repair between the lateral nasal tip and the distal ala. We observed that in most cases the flap based medially respects anatomical subunits better than the laterally based flap for medium-sized defects of the distal ala of the nose. CONCLUSION I suggest that the bi- and trilobed flaps for repair of the lateral tip/distal ala should more often be medially based. This flap has a specific indication and precise advantage compared to other reconstructions, especially to the laterally based multilobed flaps in this specific indication.

Loss of Cdx2 Expression in Primary Tumors and Lymph Node Metastases is Specific for Mismatch Repair-Deficiency in Colorectal Cancer

Background: Approximately 20% of all colorectal cancers are hypothesized to arise from the "serrated pathway" characterized by mutation in BRAF, high-level CpG Island Methylator Phenotype, and microsatellite instability/mismatch repair (MMR)-deficiency. MMR-deficient cancers show frequent losses of Cdx2, a homeodomain transcription factor. Here, we determine the predictive value of Cdx2 expression for MMR-deficiency and investigate changes in expression between primary cancers and matched lymph node metastases. Methods: Immunohistochemistry for Cdx2, Mlh1, Msh2, Msh6, and Pms2 was performed on whole tissue sections from 201 patients with primary colorectal cancer and 59 cases of matched lymph node metastases. Receiver operating characteristic curve analysis and Area under the Curve (AUC) were investigated; association of Cdx2 with clinicopathological features and patient survival was carried out. Results: Loss of Cdx2 expression was associated with higher tumor grade (p = 0.0002), advanced pT (p = 0.0166), and perineural invasion (p = 0.0228). Cdx2 loss was an unfavorable prognostic factor in univariate (p = 0.0145) and multivariate [p = 0.0427; HR (95% CI): 0.58 (0.34-0.98)] analysis. The accuracy (AUC) for discriminating MMR-proficient and - deficient cancers was 87% [OR (95% CI): 0.96 (0.95-0.98); p < 0.0001]. Specificity and negative predictive value for MMR-deficiency was 99.1 and 96.3%. One hundred and seventy-four patients had MMR-proficient cancers, of which 60 (34.5%) showed Cdx2 loss. Cdx2 loss in metastases was related to MMR-deficiency (p < 0.0001). There was no difference in expression between primary tumors and matched metastases. Conclusion: Loss of Cdx2 is a sensitive and specific predictor of MMR-deficiency, but is not limited to these tumors, suggesting that events "upstream" of the development of microsatellite instability may impact Cdx2 expression.

Possible role of Cdx2 in the serrated pathway of colorectal cancer characterized by BRAF mutation, high-level CpG Island methylator phenotype and mismatch repair-deficiency

Colorectal cancer is a heterogeneous disease at the histomorphological, clinical and molecular level. Approximately 20% of cases may progress through the "serrated" pathway characterized by BRAF mutation and high-level CpG Island Methylator Phenotype (CIMP). A large subgroup are additionally microsatellite instable (MSI) and demonstrate significant loss of tumor suppressor Cdx2. The aim of this study is to determine the specificity of Cdx2 protein expression and CpG promoter hypermethylation for BRAF(V600E) and high-level CIMP in colorectal cancer. Cdx2, Mlh1, Msh2, Msh6, and Pms2 were analyzed by immunohistochemistry using a multi-punch tissue microarray (TMA; n = 220 patients). KRAS and BRAF(V600E) mutation analysis, CDX2 methylation and CIMP were investigated. Loss of Cdx2 was correlated with larger tumor size (P = 0.0154), right-sided location (P = 0.0014), higher tumor grade (P < 0.0001), more advanced pT (P = 0.0234) and lymphatic invasion (P = 0.0351). Specificity was 100% for mismatch repair (MMR)-deficiency (P < 0.0001), 92.2% (P < 0.0001) for BRAF(V600E) and 91.8% for CIMP-high. Combined analysis of BRAF(V600E) /CIMP identified Cdx2 loss as sensitive (80%) and specific (91.5%) for mutation/high status. These results were validated on eight well-established colorectal cancer cell lines. CDX2 methylation correlated with BRAF(V600E) (P = 0.0184) and with Cdx2 protein loss (P = 0.0028). These results seem to indicate that Cdx2 may play a role in the serrated pathway to colorectal cancer as underlined by strong relationships with BRAF(V600E) , CIMP-high and MMR-deficiency. Whether this protein can only be used as a "surrogate" marker, or is functionally involved in the progression of these tumors remains to be elucidated.

Suitability of Common Collagen Scaffolds for Anterior Cruciate Ligament Repair

Introduction: Anterior cruciate ligament (ACL) injuries are very common; in Germany incidence of ACL ruptures is estimated at 32 per 100 000 in the general population and in the sports community this rate more than doubles. Current gold standard for anterior cruciate lig- ament repair is reconstruction using an autograft [1]. However, this approach has shown some limitations. A new method has been her- alded by the Knee Team at the Bern University Hospital (Inselspital) and the Sonnenhof clinic called Dynamic Intraligamentary Stabilization (DIS), which keeps ACL remnants in place in order to promote biologi- cal healing and makes use of a dynamic screw system [2]. The aim of this study was to investigate the cytocompatibility of collagen patches in combination with DIS to support regeneration of the ACL. The spe- cific hypothesis we tested was whether MSCs would differentiate towards TCs in co-culture. Materials and methods: Primary Tenocytes (TCs) and human bone marrow derived mesenchymal stem cells (MSCs) were harvested from ACL removed during knee prothesis or from bone marrow aspirations (Ethical Permit 187/10). Cells were seeded on two types of three dimensional carriers currently approved for cartilage repair, Novocart (NC, B. Brown) and Chondro-Gide (CG, Geistlich). These scaffolds comprise collagen structures with interconnecting pores originally developed for seeding of chondrocytes in the case of CG. ~40k cells were seeded on punched zylindrical cores of 8 mm in Ø and cultured on CG or NC patches for up to 7 days. The cells were either cultured as TC only, MSC only or co-cultured in a 1:1 mix on the scaffolds and on both sides of culture inserts (PET, high density pore Ø 0.4 mm, BD, Fal- con) with cell-cell contact. We monitored DNA content, GAG and HOP-content, tracked the cells using DIL and DIO fluorescent dyes (Molecular Probes, Life technologies) and confocal laser scanning and SEM microscopy as well as RT-PCR of tenocyte specific markers (i.e. col 1 and 3, TNC, TNMD, SCXA&B, and markers of dedifferentiation ACAN, col2, MMP3, MMP13). Finally, H&E stain was interpreted on cryosections and SEM images of cells on the scaffold were taken. Results: ThecLSMimagesshowedcellproliferationoverthe7dayson both matrices, however, on CG there were much fewer MSCs attached than on NC. SEM images showed a roundish chondrocyte-like pheno- type of cells on CG whereas on NC the phenotype was more teno- cyte-like (Fig. 1). Gene expression of both, MSC and TC seem to confirm a more favorable environment in 3D for both patches rather than monolayer control.

Cell therapy for intervertebral disc repair: advancing cell therapy from bench to clinics

Intervertebral disc (IVD) degeneration is a major cause of pain and disability; yet therapeutic options are limited and treatment often remains unsatisfactory. In recent years, research activities have intensified in tissue engineering and regenerative medicine, and pre-clinical studies have demonstrated encouraging results. Nonetheless, the translation of new biological therapies into clinical practice faces substantial barriers. During the symposium "Where Science meets Clinics", sponsored by the AO Foundation and held in Davos, Switzerland, from September 5-7, 2013, hurdles for translation were outlined, and ways to overcome them were discussed. With respect to cell therapy for IVD repair, it is obvious that regenerative treatment is indicated at early stages of disc degeneration, before structural changes have occurred. It is envisaged that in the near future, screening techniques and non-invasive imaging methods will be available to detect early degenerative changes. The promises of cell therapy include a sustained effect on matrix synthesis, inflammation control, and prevention of angio- and neuro-genesis. Discogenic pain, originating from "black discs" or annular injury, prevention of adjacent segment disease, and prevention of post-discectomy syndrome were identified as prospective indications for cell therapy. Before such therapy can safely and effectively be introduced into clinics, the identification of the patient population and proper standardisation of diagnostic parameters and outcome measurements are indispensable. Furthermore, open questions regarding the optimal cell type and delivery method need to be resolved in order to overcome the safety concerns implied with certain procedures. Finally, appropriate large animal models and well-designed clinical studies will be required, particularly addressing safety aspects.

An educational review of cartilage repair: precepts & practice - myths & misconceptions - progress & prospects.

OBJECTIVE The repair of cartilaginous lesions within synovial joints is still an unresolved and weighty clinical problem. Although research activity in this area has been indefatigably sustained, no significant progress has been made during the past decade. The aim of this educational review is to heighten the awareness amongst students and scientists of the basic issues that must be tackled and resolved before we can hope to escape from the whirlpool of stagnation into which we have fallen: cartilage repair redivivus! DESIGN Articular-cartilage lesions may be induced traumatically (e.g., by sports injuries and occupational accidents) or pathologically during the course of a degenerative disease (e.g., osteoarthritis). This review addresses the biological basis of cartilage repair and surveys current trends in treatment strategies, focussing on those that are most widely adopted by orthopaedic surgeons [viz., abrasive chondroplasty, microfracturing/microdrilling, osteochondral grafting and autologous-chondrocyte implantation (ACI)]. Also described are current research activities in the field of cartilage-tissue engineering, which, as a therapeutic principle, holds more promise for success than any other experimental approach. RESULTS AND CONCLUSIONS Tissue engineering aims to reconstitute a tissue both structurally and functionally. This process can be conducted entirely in vitro, initially in vitro and then in vivo (in situ), or entirely in vivo. Three key constituents usually form the building blocks of such an approach: a matrix scaffold, cells, and signalling molecules. Of the proposed approaches, none have yet advanced beyond the phase of experimental development to the level of clinical induction. The hurdles that need to be surmounted for ultimate success are discussed.

Only Neochordoplasty Achieves Physiological Trans-valvular Pressure Gradients After Repair Of Acute Posterior Leaflet Prolapse In Porcine Mitral Valves

Objective: Minimizing resection and preserving leaflet tissue has been previously shown to be beneficial for mitral valve function and leaflet kinematics after repair of acute posterior leaflet prolapse in porcine valves. We examined the effects of different additional methods of mitral valve repair (neochordoplasty, ring annuloplasty, edge-to-edge repair and triangular resection) on hemodynamics at different heart rates in an experimental model. Methods: Severe acute P2 prolapse was created in eight porcine mitral valves by resecting the posterior marginal chordae. Valve hemodynamics was quantified under pulsatile conditions in an in vitro heart simulator before and after surgical manipulation. Mitral regurgitation was corrected using four different methods of repair on the same valve: neochordoplasty with expanded polytetrafluoroethylene sutures alone and together with ring annuloplasty, edge-to-edge repair and triangular resection, both with non-restrictive annuloplasty. Residual mitral valve leak, trans-valvular pressure gradients, flow and cardiac output were measured at 60 and 80 beats/min. A validated statistical linear mixed model was used to analyze the effect of treatment. The p values were calculated using a two-sided Wald test. Results: Only neochordoplasty with expanded polytetrafluoroethylene sutures but without ring annuloplasty achieved similar hemodynamics compared to those of the native mitral valve (p range 0.071-0.901). Trans-valvular diastolic pressure gradients were within a physiologic range but significantly higher than those of the native valve following neochordoplasty with ring annuloplasty (p=0.000), triangular resection (p=0.000) and edge-to-edge repair (p=0.000). Neochordoplasty alone was significantly better in terms of hemodynamic than neochordoplasty with a ring annuloplasty (p=0.000). These values were stable regardless of heart rate or ring size. Conclusions: Neochordoplasty without ring annuloplasty is the only repair technique able to achieve almost native physiological hemodynamics after correction of leaflet prolapse in a porcine experimental model of acute chordal rupture.

Intervertebral Disc Repair using Fleece-Membrane Composite Silk Material and Genipin-enhanced Fibrin Hydrogel

Introduction: Treating low back pain (LBP) has become an increasing challenge, as it is one of the main factors causing pain and is accompanied by high costs for the individual and the society. LBP can be caused by trauma of the intervertebral disc (IVD) or IVD degeneration. In the case of disc herniation the inner gelatinous part of the IVD, called nucleus pulposus, is pressed through the fibrous, annulus fibrosus that forms the outer part of the IVD. Today’s gold standard for treatment is extensive surgery as removal of the IVD and fusion of the vertebrae. In order to find a more gentle way to treat LBP and restore the native IVD we use a novel silk fleece-membrane composite from genetically modified silk worms whose silk contains a growth factor (GDF-6) that is associated with pushing stem cells towards a disc like phenotype (1). By combining it with a genipin-enhanced fibrin hydrogel we tested its suitability in organ culture on prior injured bovine IVD in our custom built two-degree of freedom bioreactor to mimic natural loading conditions. Material & Methods: Bovine IVDs of 12-17 months old animals were isolated by first removing all surrounding tissue followed by cutting out the IVDs as previously described (2). Culturing of discs occurred in high glucose Dulbecco's Modified Eagle Medium (HG-DMEM) supplemented with 5% serum as previously described (2). On the next day injury was induced using a 2mm biopsy punch (Polymed, Switzerland). The formed cavity was filled with (0.4%) genipin-enhanced human based fibrin hydrogel (35-55mg/mL human fibrinogen, Baxter, Austria) and sealed with a silk fleece-membrane composite (Spintec Engineering, Germany). Different culture conditions were applied: free swelling, static diurnal load of 0.2MPa for 8h/d and complex loading at 0.2MPa compression combined with ± 2° torsion at 0.2Hz for 8h/d (2). After 14 days of culture cell activity was determined with resazurin assay. Additionally, glycosaminoglycan (dimethyl-methylene blue), DNA (Hoechst) and collagen content (hydroxy- proline) were determined. Finally, real-time qPCR of major IVD marker and inflammation genes was performed to judge integrity of IVDs. Results: The fibrin hydrogel is able to keep the silk seal in place throughout the 14 days of in organ culture under all conditions. Additionally, cell activity showed optimistic results and we could not confirm negative effects of the repaired discs regarding overexpression of inflammation markers. Conclusions: The genipin-enhanced fibrin hydrogel in combination with the silk fleece- membrane composite seems to be a promising approach for IVD repair. Currently we assess the capability of GDF-6 incorporated in our silk composites on human mesenchymal stem cells and later on in organ culture. References 1. Clarke LE, McConnell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA. Growth differentiation factor 6 and transforming growth factor-beta differentially mediate mesenchymal stem cell differentiation, composition and micromechanical properties of nucleus pulposus constructs. Arthritis Res Ther 2014, Mar 12;16(2):R67. 2. Chan SC, Gantenbein-Ritter B. Preparation of intact bovine tail intervertebral discs for organ culture. J Vis Exp 2012, Feb 2;60(60):e3490. Acknowledgements. This work is funded by the Gebert Rüf Foundation, project number GRS-028/13.

Repair of Annulus fibrosus with Genipin-enhanced Fibrin Hydrogel and silk membrane- fleece

Introduction Low back pain is often caused by a trauma causing disc herniation and /or disc degeneration. Although there are some promising approaches for nucleus pulposus repair, the inner tissue of the intervertebral disc (IVD) so far no treatment or repair is available for annulus fibrosus (AF) injuries. Here we aimed to develop a new method to seal and repair AF injuries by using a silk fleece composite and a genipin enhanced hydrogel. Methods Bovine (b) IVDs were harvested under aseptic conditions and kept in free swelling conditions for 24h in high-glucose DMEM containing 5% bovine serum for equilibration (1). A circular 2mm biopsy punch (Polymed Medical Center, Switzerland) was used to form a reproducible defect in the AF. For filling the defect and keeping the silk composite in place a human-derived fibrin gel (Baxter Tisseel, Switzerland) enhanced with 4.2mg/ml of the cross linker genipin (Wako Chemicals GmbH, Germany) was used. The silk composite consists of a mesh- and a membrane side (Spintec Engineering GmbH, Germany); the membrane is facing outwards to form a seal. bIVDs were cultured in vitro for 14 days either under dynamic load in a custom-built bioreactor under physiological conditions (0.2MPa load and ±2° torsion at 0.2Hz for 8h/day) or static diurnal load of 0.2MPa (2). At the end of culture discs were checked for seal failure, disc height, metabolic activity, cell death by necrosis (LDH assay), DNA content and glycosaminoglycan content. Results Silk composite maintained its position throughout the 14 days of culture under loaded conditions. Although repaired discs performed slightly lower in cell activity, DNA and GAG content were in the range of the control. Also LDH resulted in similar values compared to control discs (Fig 1). Height loss in repaired discs was in the same range as for static diurnal loaded control samples. For dynamically loaded samples the decrease was comparable to the injured, unrepaired discs. Fig 1 LDH of repaired discs compared to control disc after 24h in free swelling conditions for equilibration and first three loading cycles. Conclusions Silk-genipin-fibrin reinforced hydrogel is a promising approach to close AF defects as tested by two degree of freedom loading. In further experiments cytocompatibility of genipin has to be investigated. References 1. Chan SC, Gantenbein-Ritter B. Preparation of intact bovine tail intervertebral discs for organ culture. J Vis Exp 2012, Feb 2;60(60):e3490. 2. Walser J, Ferguson SJ, Gantenbein-Ritter B. Design of a mechanical loading device to culture intact bovine caudal motional segments of the spine under twisting motion. In: Davies J, editors. Replacing animal models: a practical guide to creating and using biomimetic alternatives. Chichester, UK: John Wiley & Sons, Ltd.; 2012. p. 89-105. Acknowledgements This project is funded by the Gerbert Rüf Stiftung project # GRS-028/13 and the Swiss National Science Project SNF #310030_153411.