Intervertebral Disc Regeneration Injection of a Cell-Loaded Collagen Hydrogel in a Sheep Model.

Degenerated intervertebral discs (IVDs) were treated with autologous adipose-derived stem cells (ASC) loaded into an injectable collagen scaffold in a sheep model to investigate the implant's therapeutic potential regarding the progression of degeneration of previously damaged discs. In this study, 18 merino sheep were subjected to a 3-step minimally invasive injury and treatment model, which consisted of surgically induced disc degeneration, treatment of IVDs with an ASC-loaded collagen hydrogel 6 weeks post-operatively, and assessment of the implant's influence on degenerative tissue changes after 6 and 12 months of grazing. Autologous ASCs were extracted from subcutaneous adipose tissue and cultivated in vitro. At the end of the experiment, disc heights were determined by µ-CT measurements and morphological tissue changes were histologically examined.Histological investigations show that, after treatment with the ASC-loaded collagen hydrogel implant, degeneration-specific features were observed less frequently. Quantitative studies of the degree of degeneration did not demonstrate a significant influence on potential tissue regeneration with treatment. Regarding disc height analysis, at both 6 and 12 months after treatment with the ASC-loaded collagen hydrogel implant a stabilization of the disc height can be seen. A complete restoration of the intervertebral disc heights however could not be achieved.The reported injection procedure describes in a preclinical model a translational therapeutic approach for degenerative disc diseases based on adipose-derived stem cells in a collagen hydrogel scaffold. Further investigations are planned with the use of a different injectable scaffold material using the same test model.

Percutaneous posterolateral approach for the simulation of a far-lateral disc herniation in an ovine model.

PURPOSE: This work describes a minimally invasive damage model for ovine lumbar discs via partial nucleotomy using a posterolateral approach. METHODS: Two cadavers were dissected to analyze the percutaneous corridor. Subsequently, 28 ovine had their annulus fibrosus punctured via awl penetration under fluoroscopic control and nucleus pulposus tissue removed via rongeur. Efficacy was assessed by animal morbidity, ease of access to T12-S1 disc spaces, and production of a mechanical injury as verified by discography, radiography, and histology. RESULTS: T12-S1 were accessible with minimal nerve damage morbidity. Scar tissue sealed the disc puncture site in all animals within 6 weeks, withstanding 1 MP of intradiscal pressure. Partial nucleotomy led to a significant reduction in intervertebral disk height and an increased histological degeneration score. CONCLUSION: Inducing a reproducible injury pattern of disc degeneration required minimal time, effort, and equipment. The posterolateral approach allows operation on several discs within a single surgery and multiple animal surgeries within a single day.

Micro-CT evaluation of asymmetrical ovine intervertebral disc height loss from surgical approach.

PURPOSE: The primary goal of this study is to clearly define and evaluate new intervertebral disc height parameters in analysing the morphological pathology of disc degeneration for application in damage model and regeneration therapy development, as well as applying traditional variables to 3-D characterization methods. METHODS: A posterolateral surgical approach was used to induce disc degeneration in an ovine model. At 12-months post-operation, sheep vertebral segments were removed and characterized using micro-CT to evaluate disc height parameters in regard to injury localization. RESULTS: Statistically significant differences between the disc height loss of the left and right side of the disc, consistent with the lateral surgical approach used were seen using the modified average disc height method by Dabbs et al. However, convexity index and the newly proposed Cross Tilt Index did not conclusively demonstrate a difference. CONCLUSION: Two-dimensional morphological evaluations can be applied in 3-D to provide a more complete picture of disc height loss for injury models. New 3-D parameters that are tailored to the type of surgical approach used should be investigated, with the 9-point system described herein providing a useful basis for derived values. Additionally, the surgical approach chosen when artificially injuring the disc can result in asymmetrical degeneration, as indicated by uneven disc height loss.

Long-term pre-clinical evaluation of an injectable chitosan nanocellulose hydrogel with encapsulated adipose-derived stem cells in an ovine model for IVD regeneration.

The potential therapeutic benefit of adipose-derived stem cells (ASCs) encapsulated in an injectable hydrogel for stimulating intervertebral disc (IVD) regeneration has been assessed by a number of translational and preclinical studies. However, previous work has been primarily limited to small animal models and short-term outcomes of only a few weeks. Long-term studies in representative large animal models are crucial for translation into clinical success, especially for permanent stabilization of major defects such as disc herniation. An injectable chitosan carboxymethyl cellulose hydrogel scaffold loaded with ASCs was evaluated regarding its intraoperative handling, crosslinking kinetics, cell viability, fully-crosslinked viscoelasticity, and long-term therapeutic effects in an ovine model. Three IVDs per animal were damaged in 10 sheep. Subcutaneous adipose tissue was the source for autologous ASCs. Six weeks after IVD damage, two of the damaged IVDs were treated via ASC-loaded hydrogel injection. After 12 months following the implantation, IVD disc height and histological and cellular changes were assessed. This system was reliable and easy to handle intraoperatively. Over 12 months, IVD height was stabilized and degeneration progression significantly mitigated compared to untreated, damaged IVDs. Here we show for the first time in a large animal model that an injectable chitosan carboxymethyl cellulose hydrogel system with encapsulated ASCs is able to affect long-term stabilization of an injured IVD and significantly decrease degeneration processes as compared to controls.

Long-Term Pathology of Ovine Lumbar Spine Degeneration Following Injury Via Percutaneous Minimally Invasive Partial Nucleotomy.

The focus of this work is to assess the long-term progression of degeneration in the ovine lumbar spine following a minimally invasive model injury comparable to the damage of an intervertebral disc (IVD) herniation. A partial nucleotomy was performed on 18 sheep via the percutaneous dorsolateral approach. The animals were culled at 6 and 12 months to evaluate the damaged and neighboring functional spine units (FSUs) for degenerative characteristics via µ-CT and histology. Both quantitative µ-CT and histology investigations demonstrated statistically significant differences between the native and damaged FSUs investigated. Qualitative analysis of µ-CT revealed numerous pathological markers consistent with intervertebral disc degeneration (IDD), with differences in frequency and severity between the native and damaged FSUs. The annulus fibrosus reforms a pressure seal within 6 weeks, but the extent of the trauma is significant enough to initiate IVD degeneration, which is already clearly visible at 6 months and especially so 12 months post-op. IDD pathology consistent with signs of a herniation was seen in both the 6- and 12-month groups. This technique provides a useful model injury for the preclinical evaluation of IDD in large animal models, especially in regards to simulating disc herniation as well as for testing the efficacy of associated therapies in the future. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2376-2388, 2019.

Risperidone-Loaded PLGA-Lipid Particles with Improved Release Kinetics: Manufacturing and Detailed Characterization by Electron Microscopy and Nano-CT.

For parenteral controlled drug release, the desired zero order release profile with no lag time is often difficult to achieve. To overcome the undesired lag time of the current commercial risperidone controlled release formulation, we developed PLGA-lipid microcapsules (MCs) and PLGA-lipid microgels (MGs). The lipid phase was composed of middle chain triglycerides (MCT) or isopropylmyristate (IPM). Hydroxystearic acid was used as an oleogelator. The three-dimensional inner structure of Risperidone-loaded MCs and MGs was assessed by using the invasive method of electron microscopy with focused ion beam cutting (FIB-SEM) and the noninvasive method of high-resolution nanoscale X-ray computed tomography (nano-CT). FIB-SEM and nano-CT measurements revealed the presence of highly dispersed spherical structures around two micrometres in size. Drug release kinetics did strongly depend on the used lipid phase and the presence or absence of hydroxystearic acid. We achieved a nearly zero order release without a lag time over 60 days with the MC-MCT formulation. In conclusion, the developed lipid-PLGA microparticles are attractive alternatives to pure PLGA-based particles. The advantages include improved release profiles, which can be easily tuned by the lipid composition.

Microstructure analysis method for evaluating degenerated intervertebral disc tissue.

Degeneration of intervertebral disc (IVD) tissue is characterized by several structural changes that result in variations in disc physiology and loss of biomechanical function. The complex process of degeneration exhibits highly intercorrelated biomechanical, biochemical, and cellular interactions. There is currently some understanding of the cellular changes in degenerated intervertebral disc tissue, but microstructural changes and deterioration of the tissue matrix has previously been rarely explored. In this work, sequestered IVD tissue was successfully characterized using histology, light microscopy, and scanning electron microscopy (SEM) to quantitatively evaluate parameters of interest for intervertebral disc degeneration (IDD) such as delamination of the collagenous matrix, cell density, cell size, and extra cellular matrix (ECM) thickness. Additional qualitative parameters investigated included matrix fibration and irregularity, neovascularization of the IVD, granular inclusions in the matrix, and cell cluster formation. The results of this study corroborated several previously published findings, including those positively correlating female gender and IVD cell density, age and cell size, and female gender and ECM thickness. Additionally, an array of quantitative and qualitative investigations of IVD degeneration could be successfully evaluated using the given methodology, resin-embedded SEM in particular. SEM is especially practical for studying micromorphological changes in tissue, as other microscopy methods can cause artificial tissue damage due to the preparation method. Investigation of the microstructural changes occurring in degenerated tissue provides a greater understanding of the complex process of disc degeneration as a whole. Developing a more complete picture of the degenerative changes taking place in the intervertebral disc is crucial for the advancement and application of regenerative therapies based on the pathology of intervertebral disc degeneration.

Morphological Characterization of the Self-Assembly of Virus Movement Proteins into Nanotubes in the Absence of Virus Particles.

One infection mechanism of plant viruses is the generation of nanotubes by viral movement proteins, allowing cell-to-cell virus particle transport. Previously, it was assumed that viral nanotubes extend directly from the host-cell plasma membrane. In virus-infected plants, these nanotubes reach an extraordinary diameter:length ratio (≈100 nm:µm or mm range). Here, viral nanotubes are produced in a transient protoplast system; the coding sequence for alfalfa mosaic virus movement protein is translationally fused to green fluorescent protein. The maximum extension of viral nanotubes into the culture medium is achieved 24-48 h posttransfection, with lengths in the micro- and millimeter ranges. Scanning electron microscopy and transmission electron microscopy show that strong inhomogeneous viral nanotubes are formed compared to particle-filled systems. The nanotubes have similar length, but fluctuating wall thickness and diameter and are susceptible to entanglement and recombination. Indirect methods demonstrate that movement proteins assemble independently at the top of the nanotube. These viral nanotubes grow distinctly from previously known natural particle-filled systems and are a unique biological tubular nanomaterial that has the potential for micro- or nanoapplications as a mechanically stable structural component.

Micro-computed tomography, scanning electron microscopy and energy X-ray spectroscopy studies of facet joint degeneration: A comparison to clinical imaging.

Segmental degeneration in the human lumbar spine affects both the intervertebral discs and facet joints. Facet joint degeneration not only affects the cartilage surface, but also alters the cellular properties of the cartilage tissue and the structure of the subchondral bone. The primary focus of this study is the investigation of these microstructural changes that are caused by facet joint degeneration. Microstructural analyses of degenerated facet joint samples, obtained from patients following operative lumbar interbody fusion, have not previously been extensively investigated. This study analyzes human facet joint samples from the inferior articular process using scanning electron microscopy, micro-computed tomography, and energy dispersive X-ray spectroscopy to evaluate parameters of interest in facet joint degeneration such as elemental composition, cartilage layer thickness and cell density, calcification zone thickness, subchondral bone portion, and trabecular bone porosity. These microstructural analyses demonstrate fragmentation, cracking, and destruction of the cartilage layer, a thickened calcification zone, localized calcification areas, and cell cluster formation as pathological manifestations of facet joint degeneration. The detailed description of these microstructural changes is critical for a comprehensive understanding of the pathology of facet joint degeneration, as well as the subsequent development and efficacy analysis of regenerative treatment strategies.

Self-supporting nanoporous alumina membranes as substrates for hepatic cell cultures.

Membranes made from nanoporous alumina exhibit interesting properties for their use in biomedical research. They show high porosity and the pore diameters can be easily adjusted in a reproducible manner. Nanoporous alumina membranes are thus ideal substrates for the cultivation of polar cells (e.g., hepatocytes) or the establishment of indirect co-cultures. The porous nature of the material allows supply of nutrients to both sides of adherent cells and the exchange of molecules across the membrane. However, it is well-known that surface features in the nanometer range affect cellular behavior. In this study, the response of HepG2 cells to nanoporous alumina membranes with three different pore diameters, ranging from 50 to 250 nm, has been evaluated. The cellular interactions with the nanoporous materials were assessed by investigating cell adhesion, morphology, and proliferation. Cell functionality was measured by means of albumin production. The membranes supported good cell adhesion and spreading. Compared to tissue culture plastic, the cells on the porous substrates developed distinct focal adhesion sites and actin stress fibers. Additionally, electron microscopical investigations revealed the penetration of cellular extensions into pores with diameters bigger than 200 nm. Furthermore, cell proliferation significantly increased with an increase in pore diameter, whereas the albumin production followed a reverse trend. Thus, it seems to be possible to direct cellular behavior of HepG2 cells growing on nanoporous alumina by changing the pore diameter of the material. Hence, nanoporous alumina membranes can be useful culture substrates to develop new approaches in the field of liver tissue engineering.

Investigation of cell-substrate interactions by focused ion beam preparation and scanning electron microscopy.

Cell-substrate interactions, which are an important issue in tissue engineering, have been studied using focused ion beam (FIB) milling and scanning electron microscopy (SEM). Sample cross-sections were generated at predefined positions (target preparation) to investigate the interdependency of growing cells and the substrate material. The experiments focus on two cell culturing systems, hepatocytes (HepG2) on nanoporous aluminum oxide (alumina) membranes and mouse fibroblasts (L929) and primary nerve cells on silicon chips comprised of microneedles. Cross-sections of these soft/hard hybrid systems cannot be prepared by conventional techniques like microtomy. Morphological investigations of hepatocytes growing on nanoporous alumina membranes demonstrate that there is in-growth of microvilli from the cell surface into porous membranes having pore diameters larger than 200 nm. Furthermore, for various cell cultures on microneedle arrays contact between the cells and the microneedles can be observed at high resolution. Based on FIB milled cross-sections and SEM micrographs cells which are only in contact with microneedles and cells which are penetrated by microneedles can be clearly distinguished. Target preparation of biological samples by the FIB technique especially offers the possibility of preparing not only soft materials but also hybrid samples (soft/hard materials). Followed by high resolution imaging by SEM, new insights into cell surface interactions can be obtained.