Hydrogels for nucleus replacement—Facing the biomechanical challenge

Hydrogels are considered promising for disc regeneration strategies. However, it is currently unknown whether the destruction of the natural interface between nucleus and surrounding structures caused by nucleotomy and an inadequate annulus closure diminishes the mechanical competence of the disc. This in vitro study aimed to clarify these mechanisms and to evaluate whether hydrogels are able to restore the biomechanical behaviour of the disc. Nucleus pressure in an ovine intervertebral disc was measured in vivo during day and night and adapted to an in vitro axial compressive diurnal (15min) and night (30min) load. Effects of different defects on disc height and nucleus pressure were subsequently measured in vitro using 30 ovine motion segments. Following cases were considered: intact; annulus incision repaired by suture and glue; annulus incision with removal and re-implantation of nucleus tissue; and two different hydrogels repaired by suture and glue. The intradiscal pressure in vivo was 0.75MPa during day and 0.5MPa during night corresponding to an in vitro axial compressive force of 130 and 58N, respectively. The compression test showed that neither the implantation of hydrogels nor the re-implantation of the natural nucleus, assumed as being the ideal implant, was able to restore the mechanical functionality of an intact disc. Results indicate the importance of the natural anchorage of the nucleus with its surrounding structures and the relevance of an appropriate annulus closure. Therefore, hydrogels that are able to mimic the mechanical behaviour of the native nucleus may fail in restoring the mechanical behaviour of the disc.

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.

Dual Role of Mesenchymal Stem Cells Allows for Microvascularized Bone Tissue-Like Environments in PEG Hydrogels.

In vitro engineered tissues which recapitulate functional and morphological properties of bone marrow and bone tissue will be desirable to study bone regeneration under fully controlled conditions. Among the key players in the initial phase of bone regeneration are mesenchymal stem cells (MSCs) and endothelial cells (ECs) that are in close contact in many tissues. Additionally, the generation of tissue constructs for in vivo transplantations has included the use of ECs since insufficient vascularization is one of the bottlenecks in (bone) tissue engineering. Here, 3D cocultures of human bone marrow derived MSCs (hBM-MSCs) and human umbilical vein endothelial cells (HUVECs) in synthetic biomimetic poly(ethylene glycol) (PEG)-based matrices are directed toward vascularized bone mimicking tissue constructs. In this environment, bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor-2 (FGF-2) promotes the formation of vascular networks. However, while osteogenic differentiation is achieved with BMP-2, the treatment with FGF-2 suppressed osteogenic differentiation. Thus, this study shows that cocultures of hBM-MSCs and HUVECs in biological inert PEG matrices can be directed toward bone and bone marrow-like 3D tissue constructs.

Biomaterials for Intervertebral Disc Regeneration

The intervertebral disc (IVD) is a complex avascular organ of viscoelastic properties. The current research focus is to regenerate and to partially restore a degenerated IVD by ‘smart’ biomaterials in combination of cell therapy and/or growth factors. For the two tissues of the IVD, that is, the nucleus pulposus (NP) and the annulus fibrosus (AF), biomaterials of different mechanical properties are needed. The ideal biomaterial to restore the water-rich NP and the tensile-force resistant AF has not been identified yet. The lack of blood vessels and the relative scarcity of specially adapted cells of the IVD organ demand novel concepts of tissue-engineered biological approaches to regenerate or replace the IVD. Injectable biodegradable hydrogels with swelling properties are in focus for NP replacement, whereas electrospun biphasic composites and silk, among other biodegradable polymers, are discussed for AF reinforcement.

Intervertebral disc regeneration or repair with biomaterials and stem cell therapy--feasible or fiction?

The "gold standard" for treatment of intervertebral disc herniations and degenerated discs is still spinal fusion, corresponding to the saying "no disc - no pain". Mechanical prostheses, which are currently implanted, do only have medium outcome success and have relatively high re-operation rates. Here, we discuss some of the biological intervertebral disc replacement approaches, which can be subdivided into at least two classes in accordance to the two different tissue types, the nucleus pulposus (NP) and the annulus fibrosus (AF). On the side of NP replacement hydrogels have been extensively tested in vitro and in vivo. However, these gels are usually a trade-off between cell biocompatibility and load-bearing capacity, hydrogels which fulfill both are still lacking. On the side of AF repair much less is known and the question of the anchoring of implants is still to be addressed. New hope for cell therapy comes from developmental biology investigations on the existence of intervertebral disc progenitor cells, which would be an ideal cell source for cell therapy. Also notochordal cells (remnants of the embryonic notochord) have been recently pushed back into focus since these cells have regenerative potential and can activate disc cells. Growth factor treatment and molecular therapies could be less problematic. The biological solutions for NP and AF replacement are still more fiction than fact. However, tissue engineering just scratched the tip of the iceberg, more satisfying solutions are yet to be added to the biomedical pipeline.

The transpedicular approach as an alternative route for intervertebral disc regeneration

STUDY DESIGN Descriptive anatomical study on ovine and human cadaveric lumbar spinal segments. OBJECTIVE To describe the alternative transpedicular approach to deliver therapeutic agents into intervertebral disc (IVD). SUMMARY OF BACKGROUND DATA The present delivery approach of therapeutic agents (growth factors/cells/hydrogels) within the IVD is through injection, via the annulus fibrosus (AF). However, it has recently been demonstrated that small needle puncture of the AF leads to further degeneration and disc herniation. In addition, the injected material has a high chance to be extruded through the AF injury. METHODS Lumbar ovine and human spinal segments were used. Under fluoroscopy, a 2-mm Kirschner wire was introduced in the caudal vertebra through the pedicle and the inferior endplate to the nucleus pulposus. Gross anatomy analysis and high-resolution peripheral quantitative computed tomography (HR-pQCT) were performed to assess the right position of the wire in pedicles. Discography and nucleotomy were performed using a 14G cannula insertion or a 2-mm arthroscopic shaver blade, respectively. Nucleoplasty was also performed with agarose gel/contrast agent and imaged with HR-pQCT. RESULTS Gross anatomy, fluoroscopy, and HR-pQCT images showed that the nucleus pulposus could be approached through the endplate via the pedicle without affecting the spinal canal and the neural foramina. The contrast agent was delivered into the IVD and nucleus pulposus was removed from the disc and filled with agarose gel. CONCLUSION This study describes how a transpedicular approach can be used as an alternative route to deliver therapeutic agents to the disc without disruption of the AF showing the potential use of this technique in preclinical research and highlighting its clinical relevance for IVD regeneration.

A papain-induced disc degeneration model for the assessment of thermo-reversible hydrogel-cells therapeutic approach

Nucleus pulposus (NP) regeneration by the application of injectable cell-embedded hydrogels is an appealing approach for tissue engineering. We investigated a thermo-reversible hydrogel (TR-HG), based on a modified polysaccharide with a thermo-reversible polyamide [poly(N-isopropylacrylamide), pNIPAM], which is made to behave as a liquid at room temperature and hardens at > 32 °C. In order to test the hydrogel, a papain-induced bovine caudal disc degeneration model (PDDM), creating a cavity in the NP, was employed. Human mesenchymal stem cells (hMSCs) or autologous bovine NP cells (bNPCs) were seeded in TR-HG; hMSCs were additionally preconditioned with rhGDF-5 for 7 days. Then, TR-HG was reversed to a fluid and the cell suspension injected into the PDDM and kept under static loading for 7 days. Experimental design was: (D1) fresh disc control + PBS injection; (D2) PDDM + PBS injection; (D3) PDDM + TR-HG (material control); (D4) PDDM + TR-HG + bNPCs; (D5) PDDM + TR-HG + hMSCs. Magnetic resonance imaging performed before and after loading, on days 9 and 16, allowed imaging of the hydrogel-filled PDDM and assessment of disc height and volume changes. In gel-injected discs the NP region showed a major drop in volume and disc height during culture under static load. The RT–PCR results of injected hMSCs showed significant upregulation of ACAN, COL2A1, VCAN and SOX9 during culture in the disc cavity, whereas the gene expression profile of NP cells remained unchanged. The cell viability of injected cells (NPCs or hMSCs) was maintained at over 86% in 3D culture and dropped to ~72% after organ culture. Our results underline the need for load-bearing hydrogels that are also cyto-compatible.

Minimally invasive photopolymerization in intervertebral disc tissue cavities

Photopolymerized hydrogels are commonly used for a broad range of biomedical applications. As long as the polymer volume is accessible, gels can easily be hardened using light illumination. However, in clinics, especially for minimally invasive surgery, it becomes highly challenging to control photopolymerization. The ratios between polymerization- volume and radiating-surface-area are several orders of magnitude higher than for ex-vivo settings. Also tissue scattering occurs and influences the reaction. We developed a Monte Carlo model for photopolymerization, which takes into account the solid/liquid phase changes, moving solid/liquid-boundaries and refraction on these boundaries as well as tissue scattering in arbitrarily designable tissue cavities. The model provides a tool to tailor both the light probe and the scattering/absorption properties of the photopolymer for applications such as medical implants or tissue replacements. Based on the simulations, we have previously shown that by adding scattering additives to the liquid monomer, the photopolymerized volume was considerably increased. In this study, we have used bovine intervertebral disc cavities, as a model for spinal degeneration, to study photopolymerization in-vitro. The cavity is created by enzyme digestion. Using a custom designed probe, hydrogels were injected and photopolymerized. Magnetic resonance imaging (MRI) and visual inspection tools were employed to investigate the successful photopolymerization outcomes. The results provide insights for the development of novel endoscopic light-scattering polymerization probes paving the way for a new generation of implantable hydrogels.

Mechanical Loading Promoted Discogenic Differentiation of Human Mesenchymal Stem Cells Incorporated in 3D-PEG Scaffolds with RhGDF5 and RGD

Hydrogels have been described as ideal scaffolds for cells of 3D tissue constructs and hold strong promises with respect to in vitro 3D-cell-culture, where cells are isolated from native extracellular matrix (ECM). Synthesized polyethyleneglycol (PEG) hydrogels are appealing with regard to potential for cell therapy or as vehicles for drug delivery or even to regenerate tissue with similar hydrogel-like properties such as the nucleus pulposus of the intervertebral disc (IVD). Here, we tested whether incorporation of RGD motive would hinder discogenic differentiation of primary bone marrow-derived human mesenchymal stem cells (hMSCs) but favor proliferation of undifferentiated hMSCs. HMSCs were embedded in +RGD containing or without RGD PEG hydrogel and pre-conditioned with or without growth and differentiation factor-5 (rhGDF-5) for 13 days. Afterwards, all hMSCs-PEG gels were subsequently cyclically loaded (15% strain, 1Hz) for 5 consecutive days in a bioreactor to generate an IVD-like phenotype. Higher metabolic activity (resazurin assay) was found in groups with rhGDF5 in both gel types with and without RGD. Cell viability and morphology measured by confocal laser microscopy and DNA content showed decreased values (~60%) after 18 days of culture. Real-time RT-PCR of an array of 15 key genes suspected to be distinctive for IVD cells revealed moderate response to rhGDF5 and mechanical loading as also shown by histology staining. Preconditioning and mechanical loading showed relatively moderate responses revealed from both RT-PCR and histology although hMSCs were demonstrated to be potent to differentiate into chondrocyte-progenitor cells in micro- mass and 3D alginate bead culture.

Evaluation of HA-fibrin-based hydrogel for restoration of degenerated intervertebral disc

Introduction: Intervertebral disc degeneration is associated with loss of nucleus pulposus (NP) tissue and reduced disc height[1]. A number of therapies, including synthetic and natural biomaterials, have been developed to restore full disc function and to minimize the pain and disability caused by this disease. Fibrin-based biomaterials are used as a replacement for NP or as a cell carrier for tissue engineering approaches[2]. While the behavior of such gels is well-characterized from a material point of view, little is known about their contribution to intervertebral disc (IVD) restoration under dynamic loads. The aim of the present study is the evaluation of a hyaluronic acid fibrin-based hydrogel (ProCore) used to repair an in vitro model of disc degeneration under dynamic loading. Methods: In vitro model of disc degeneration was induced in intact coccygeal bovine IVD by papain digestion of the NP as previously described[3]. In order to characterize fibrin hydrogels, four experimental groups were considered: 1) intact IVD (control), 2) IVD injected with PBS, 3) injection of hydrogels in degenerative IVD and 4) injection of hydrogels in combination with human bone marrow-derived mesenchymal stem cells (MSC) in degenerative IVD. All of the groups were subjected to dynamic loading protocols consisting of 0.2MPa static compression superimposed with ±2° torsion at 0.2Hz for 8h per day and maintained for 7 days. Additionally, one group consisted of degenerative IVD injected with hydrogel and subjected to static compression. Disc heights were monitored after the duration of the loading and compared to the initial disc height. The macrostructure of the formed tissue and the cellular distribution was evaluated by histological means. Results: After one week of loading, the degenerative IVD filled with hydrogel in combination with MSC (dynamic load), hydrogels (dynamic load) and hydrogels (static load) showed a reduction in height by 30%, 15% and 20%, respectively, as compared to their initial disc height. Histological sections showed that the HA-fibrin gel fully occupied the nucleotomized region of the disc and that fibrin was effective in filling the discontinuities of the cavity region. Furthermore, the cells were homogenously distributed along the fibrin hydrogels after 7 days of loading. Discussion: In this study, we showed that fibrin hydrogels showed a good integration within the papain-induced model of disc degeneration and can withstand the applied loads. Fibrin hydrogels can contribute to disc restoration by possibly maintaining adequate stiffness of the tissue and thus preventing disorganization of the surrounding IVD. References: 1. Jarman, J.P., Arpinar, V.E., Baruah, D., Klein, A.P., Maiman, D.J., and Tugan Muftuler, L. (2014). Intervertebral disc height loss demonstrates the threshold of major pathological changes during degeneration. Eur Spine J . 2. Colombini, A., Ceriani, C., Banfi, G., Brayda-Bruno, M., and Moretti, M. (2014). Fibrin in intervertebral disc tissue engineering. Tissue Eng Part B Rev . 3. Chan, S.C., Bürki, A., Bonél, H.M., Benneker, L.M., and Gantenbein-Ritter, B. (2013). Papain-induced in vitro disc degeneration model for the study of injectable nucleus pulposus therapy. Spine J 13, 273-283. Acknowledgement We thank the Swiss National Science Foundation SNF #310030_153411 for funding.

Genetically engineered silk and genipin-enhanced fibrin hydrogel for annulus fibrosus repair

INTRODUCTION: Around 80% of people are affected by low back pain at least once in their life, often caused by trauma provoking intervertebral disc (IVD) herniation and/or IVD degeneration. Apart from some promising approaches for nucleus pulposus repair, so far no treatment or repair is available for the outer fibrous tissue, annulus fibrosus (AF). We aimed for sealing and repairing an AF injury in a bovine IVD organ culture model in vitro over 14 days under different loading conditions. For this purpose, a silk fleece composite from Bombyx mori silk was combined with genipin-enhanced fibrin hydrogel [1]. METHODS: Bovine IVDs of 12-17 months old animals were isolated by first removing all surrounding tissue, followed by cutting out the IVDs [2]. Culturing of discs occurred in high glucose Dulbecco's Modified Eagle Medium (HG-DMEM) supplemented with 5% serum as previously described. 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. Complex loading was applied by a custom built 2 degree of freedom bioreactor [3]. 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 genes was performed. RESULTS: The silk seal closing the injury site could successfully withstand the forces of all three loading conditions with no misplacement over the two weeks’ culture. Nevertheless, disc height of the repaired discs did not significantly differ from the injured group. The disc phenotype could be maintained as demonstrated by biochemical analysis of gene expression, cell activity, DNA-, collagen- and GAG content. The silk itself was evaluated to be highly biocompatible for hMSC, as revealed by cytotoxicity assays. DISCUSSION & CONCLUSIONS: The silk can be considered a highly-elastic and biocompatible material for AF closure and the genipin-enhanced fibrin hydrogel has also good biomechanical properties. However, the cyto-compatibility of genipin seems rather poor and other hydrogels and/or cross-linkers should be looked into. REFERENCES: 1 C.C. Guterl et al. (2014) Characterization of Mechanics and Cytocompatibility of Fibrin Genipin Annulus Fibrosus Sealant with the Addition of Cell Adhesion Molecules, Tissue Eng Part A 2 S.C. Chan, B. Gantenbein-Ritter (2012) Preparation of intact bovine tail intervertebral discs for organ culture, J Vis Exp 3 B Gantenbein et al. (2015) Organ Culture Bioreactors - Platforms to Study Human Intervertebral Disc Degeneration and Regenerative Therapy, Curr Stem Cell Res Ther [epub ahead of print] ACKNOWLEDGEMENTS: This project is supported by the Gebert Rüf Stiftung project # GRS-028/13.