Svenja Illien-Jünger

The combined effects of limited nutrition and high-frequency loading on intervertebral discs with endplates.

Study Design. Whole ovine caudal intervertebral discs were cultured under simulated-physiologic or high-frequency loading and either sufficient or limited nutrition for 7 days. Objective. To study the effect of high-frequency loading under sufficient or limited glucose conditions and to investigate the additive effects of load and nutrition on cell survival, gene expression, and cell activity after 7 days of culture. Summary of Background Data. Limited nutrition and certain mechanical stimuli are generally believed to be etiologic factors for disc degeneration. Although these effects and their interactions have been demonstrated in cell culture, no investigations have been reported in entire discs. Methods. Discs were maintained in a whole organ culture bioreactor system under simulated-physiologic (0.2 Hz) or high-frequency (10 Hz) loading, in media with either limited (2 g/L) or sufficient (4.5 g/L) glucose concentration. After 7 days, cell viability, relative gene expression, newly synthesized chondroitin sulfate content, glycosaminoglycan synthesis rate, and disc morphology were assessed after culture and compared with fresh tissue. Results. Culture under either limited glucose or high-frequency loading conditions led to a significant drop in cell viability. Combined treatment with limited glucose and high-frequency loading resulted in an additive increase in cell death in both the anulus fibrosus and nucleus pulposus and in an increase in MMP13 gene expression. Conclusion. Supporting in vivo studies and cell culture experiments, high-frequency loading simulating vibration conditions shows detrimental effects on intervertebral disc cells in whole organ culture. The effect on cell viability was exacerbated by limited nutrition culture. However, neither frequency nor limited glucose affected cell metabolism, measured by glycosaminoglycan synthesis rate. Longer culture periods may be required to detect changes at the extracellular matrix level.

Combined Anti-Inflammatory and Anti-AGE Drug Treatments Have a Protective Effect on Intervertebral Discs in Mice with Diabetes

Objective Diabetes and low back pain are debilitating diseases and modern epidemics. Diabetes and obesity are also highly correlated with intervertebral disc (IVD) degeneration and back pain. Advanced-glycation-end-products (AGEs) increase reactive-oxygen-species (ROS) and inflammation, and are one cause for early development of diabetes mellitus. We hypothesize that diabetes results in accumulation of AGEs in spines and associated spinal pathology via increased catabolism. We present a mouse model showing that: 1) diabetes induces pathological changes to structure and composition of IVDs and vertebrae; 2) diabetes is associated with accumulation of AGEs, TNFα, and increased catabolism spinal structures; and 3) oral-treatments with a combination of anti-inflammatory and anti-AGE drugs mitigate these diabetes-induced degenerative changes to the spine. Methods Three age-matched groups of ROP-Os mice were compared: non-diabetic, diabetic (streptozotocin (STZ)-induced), or diabetic mice treated with pentosan-polysulfate (anti-inflammatory) and pyridoxamine (AGE-inhibitor). Mice were euthanized and vertebra-IVD segments were analyzed by μCT, histology and Immunohistochemistry. Results Diabetic mice exhibited several pathological changes including loss in IVD height, decreased vertebral bone mass, decreased glycosaminoglycan content and morphologically altered IVDs with focal deposition of tissues highly expressing TNFα, MMP-13 and ADAMTS-5. Accumulation of larger amounts of methylglyoxal suggested that AGE accumulation was associated with these diabetic degenerative changes. However, treatment prevented or reduced these pathological effects on vertebrae and IVD. Conclusion This is the first study to demonstrate specific degenerative changes to nucleus pulposus (NP) morphology and their association with AGE accumulation in a diabetic mouse model. Furthermore, this is the first study to demonstrate that oral-treatments can inhibit AGE-induced ROS and inflammation in spinal structures and provide a potential treatment to slow progression of degenerative spine changes in diabetes. Since diabetes, IVD degeneration, and accumulation of AGEs are frequent consequences of aging, early treatments to reduce AGE-induced ROS and Inflammation may have broad public-health implications.

Homing of Mesenchymal Stem Cells in Induced Degenerative Intervertebral Discs in a Whole Organ Culture System

Study Design. Homing of human bone marrow–derived mesenchymal stem cells (BMSCs) was studied using ex vivo cultured bovine caudal intervertebral discs (IVDs). Objective. To investigate in a whole organ culture whether metabolic and mechanical challenges can induce BMSC recruitment into the IVD. Summary of Background Data. Cells from injured tissues release cytokines and mediators that enable the recruitment of progenitor cells. BMSCs have the ability to survive within the IVD. Methods. Bovine IVDs with or without endplates were cultured for 1 week under simulated physiological or degenerative conditions; disc cells were analyzed for cell viability and gene expression, whereas media was analyzed for nitric oxide production and chemotaxis. Homing of BMSCs was investigated by supplying PKH-labeled human BMSCs onto cultured IVDs (1 × 106 cells/disc on d 8, 10, and 12 of culture); on day 14, the number of homed BMSCs was microscopically assessed. Moreover, a comparative study was performed between transduced BMSCs (transduced with an adenovirus encoding for insulin-like growth factor 1 [IGF-1]) and nontransduced BMSCs. Disc proteoglycan synthesis rate was quantified via 35S incorporation. The secretion of IGF-1 was evaluated by enzyme-linked immunosorbent assay on both simulated physiological and degenerative discs. Results. Discs cultured under degenerative conditions showed reduced cell viability, upregulation of matrix degrading enzymes, and increased nitric oxide production compared with simulated physiological discs. Greater homing occurred under degenerative compared with physiological conditions with or without endplate. Media of degenerative discs demonstrated a chemoattractive activity toward BMSCs. Finally, discs homed with IGF-1–transduced BMSCs showed increased IGF-1 secretion and significantly higher proteoglycan synthesis rate than discs supplied with nontransduced BMSCs. Conclusion. We have demonstrated for the first time that degenerative conditions induce the release of factors promoting BMSC recruitment in an ex vivo organ culture. Moreover, IGF-1 transduction of BMSCs strongly increases the rate of proteoglycan synthesis within degenerative discs. This finding offers a new delivery system for BMSCs and treatment strategy for IVD regeneration.

Development and validation of a bioreactor system for dynamic loading and mechanical characterization of whole human intervertebral discs in organ culture.

Intervertebral disc (IVD) degeneration is a common cause of back pain, and attempts to develop therapies are frustrated by lack of model systems that mimic the human condition. Human IVD organ culture models can address this gap, yet current models are limited since vertebral endplates are removed to maintain cell viability, physiological loading is not applied, and mechanical behaviors are not measured. This study aimed to (i) establish a method for isolating human IVDs from autopsy with intact vertebral endplates, and (ii) develop and validate an organ culture loading system for human or bovine IVDs. Human IVDs with intact endplates were isolated from cadavers within 48h of death and cultured for up to 21 days. IVDs remained viable with ~80% cell viability in nucleus and annulus regions. A dynamic loading system was designed and built with the capacity to culture 9 bovine or 6 human IVDs simultaneously while applying simulated physiologic loads (maximum force: 4kN) and measuring IVD mechanical behaviors. The loading system accurately applied dynamic loading regimes (RMS error <2.5N and total harmonic distortion <2.45%), and precisely evaluated mechanical behavior of rubber and bovine IVDs. Bovine IVDs maintained their mechanical behavior and retained >85% viable cells throughout the 3 week culture period. This organ culture loading system can closely mimic physiological conditions and be used to investigate response of living human and bovine IVDs to mechanical and chemical challenges and to screen therapeutic repair techniques.

Development of a bovine decellularized extracellular matrix-biomaterial for nucleus pulposus regeneration.

Painful intervertebral disc (IVD) degeneration is a common cause for spinal surgery. There is a clinical need to develop injectable biomaterials capable of promoting IVD regeneration, yet many available biomaterials do not mimic the native extracellular matrix (ECM) or promote matrix production. This study aimed to develop a decellularized injectable bovine ECM material that maintains structural and compositional features of native tissue and promotes nucleus pulposus (NP) cell (NPC) and mesenchymal stem cell (MSC) adaption. Injectable decellularized ECM constructs were created using 3 NP tissue decellularization methods (con.A: sodium deoxycholate, con.B: sodium deoxycholate & sodium dodecyl sulfate, con.C: sodium deoxycholate, sodium dodecyl sulfate & TritonX‐100) and evaluated for protein, microstructure, and for cell adaptation in 21 day human NPC and MSC culture experiments. Con.A was most efficient at DNA depletion, preserved best collagen microstructure and content, and maintained the highest glycosaminoglycan (GAG) content. NPCs in decellularized constructs of con.A&B demonstrated newly synthesized GAG production, which was apparent from “halos” of GAG staining surrounding seeded NPCs. Con.A also promoted MSC adaption with high cell viability and ECM production. The injectable decellularized NP biomaterial that used sodium deoxycholate without additional decellularization steps maintained native NP tissue structure and composition closest to natural ECM and promoted cellular adaptation of NP cells and MSCs. This natural decellularized biomaterial warrants further investigation for its potential as an injectable cell seeded supplement to augment NP replacement biomaterials and deliver NPCs or MSCs.

Chronic Ingestion of Advanced Glycation End Products Induces Degenerative Spinal Changes and Hypertrophy in Aging Pre-Diabetic Mice

Intervertebral disc (IVD) degeneration and pathological spinal changes are major causes of back pain, which is the top cause of global disability. Obese and diabetic individuals are at increased risk for back pain and musculoskeletal complications. Modern diets contain high levels of advanced glycation end products (AGEs), cyto-toxic components which are known contributors to obesity, diabetes and accelerated aging pathologies. There is little information about potential effects of AGE rich diet on spinal pathology, which may be a contributing cause for back pain which is common in obese and diabetic individuals. This study investigated the role of specific AGE precursors (e.g. methylglyoxal-derivatives (MG)) on IVD and vertebral pathologies in aging C57BL6 mice that were fed isocaloric diets with standard (dMG+) or reduced amounts of MG derivatives (dMG-; containing 60-70% less dMG). dMG+ mice exhibited a pre-diabetic phenotype, as they were insulin resistant but not hyperglycemic. Vertebrae of dMG+ mice displayed increased cortical-thickness and cortical-area, greater MG-AGE accumulation and ectopic calcification in vertebral endplates. IVD morphology of dMG+ mice exhibited ectopic calcification, hypertrophic differentiation and glycosaminoglycan loss relative to dMG- mice. Overall, chronic exposure to dietary AGEs promoted age-accelerated IVD degeneration and vertebral alterations involving ectopic calcification which occurred in parallel with insulin resistance, and which were prevented with dMG- diet. This study described a new mouse model for diet-induced spinal degeneration, and results were in support of the hypothesis that chronic AGE ingestion could be a factor contributing to a pre-diabetic state, ectopic calcifications in spinal tissues, and musculoskeletal complications that are more generally known to occur with chronic diabetic conditions.

TNFα Transport Induced by Dynamic Loading Alters Biomechanics of Intact Intervertebral Discs

Objective Intervertebral disc (IVD) degeneration is an important contributor to the development of back pain, and a key factor relating pain and degeneration are the presence of pro-inflammatory cytokines and IVD motion. There is surprisingly limited understanding of how mechanics and inflammation interact in the IVD. This study investigated interactions between mechanical loading and pro-inflammatory cytokines in a large animal organ culture model to address fundamental questions regarding (i.) how inflammatory mediators arise within the IVD, (ii.) how long inflammatory mediators persist, and (iii.) how inflammatory mediators influence IVD biomechanics. Methods Bovine caudal IVDs were cultured for 6 or 20-days under static & dynamic loading with or without exogenous TNFα in the culture medium, simulating a consequence of inflammation of the surrounding spinal tissues. TNFα transport within the IVD was assessed via immunohistochemistry. Changes in IVD structural integrity (dimensions, histology & aggrecan degradation), biomechanical behavior (Creep, Recovery & Dynamic stiffness) and pro-inflammatory cytokines in the culture medium (ELISA) were assessed. Results TNFα was able to penetrate intact IVDs when subjected to dynamic loading but not static loading. Once transported within the IVD, pro-inflammatory mediators persisted for 4–8 days after TNFα removal. TNFα exposure induced changes in IVD biomechanics (reduced diurnal displacements & increased dynamic stiffness). Discussion This study demonstrated that exposure to TNFα, as might occur from injured surrounding tissues, can penetrate healthy intact IVDs, induce expression of additional pro-inflammatory cytokines and alter IVD mechanical behavior. We conclude that exposure to pro-inflammatory cytokine may be an initiating event in the progression of IVD degeneration in addition to being a consequence of disease.

Detrimental effects of discectomy on intervertebral disc biology can be decelerated by growth factor treatment during surgery: a large animal organ culture model

BACKGROUND CONTEXT Lumbar discectomies are common surgical interventions that treat radiculopathy by removing herniated and loose intervertebral disc (IVD) tissues. However, remaining IVD tissue can continue to degenerate resulting in long-term clinical problems. Little information is available on the effects of discectomy on IVD biology. Currently, no treatments exist that can suspend or reverse the degeneration of the remaining IVD. PURPOSE To improve the knowledge on how discectomy procedures influence IVD physiology and to assess the potential of growth factor treatment as an augmentation during surgery. STUDY DESIGN To determine effects of discectomy on IVDs with and without transforming growth factor beta 3 (TGFβ3) augmentation using bovine IVD organ culture. METHODS This study determined effects of discectomy with and without TGFβ3 injection using 1-, 6-, and 19-day organ culture experiments. Treated IVDs were injected with 0.2 μg TGFβ3 in 20 μL phosphate-buffered saline+bovine serum albumin into several locations of the discectomy site. Cell viability, gene expression, nitric oxide (NO) release, IVD height, aggrecan degradation, and proteoglycan content were determined. RESULTS Discectomy significantly increased cell death, aggrecan degradation, and NO release in healthy IVDs. Transforming growth factor beta 3 injection treatment prevented or mitigated these effects for the 19-day culture period. CONCLUSIONS Discectomy procedures induced cell death, catabolism, and NO production in healthy IVDs, and we conclude that post-discectomy degeneration is likely to be associated with cell death and matrix degradation. Transforming growth factor beta 3 injection augmented discectomy procedures by acting to protect IVD tissues by maintaining cell viability, limiting matrix degradation, and suppressing NO. We conclude that discectomy procedures can be improved with injectable therapies at the time of surgery although further in vivo and human studies are required.

In vitro and biomechanical screening of polyethylene glycol and poly(trimethylene carbonate) block copolymers for annulus fibrosus repair

Herniated intervertebral discs (IVDs) are a common cause of back and neck pain. There is an unmet clinical need to seal annulus fibrosus (AF) defects, as discectomy surgeries address acute pain but are complicated by reherniation and recurrent pain. Copolymers of polyethylene glycol with trimethylene carbonate (TMC) and hexamethylene diisocyanate (HDI) end‐groups were formulated as AF sealants as the HDI form covalent bonds with native AF tissue. TMC adhesives were evaluated and optimized using the design criteria: stable size, strong adherence to AF tissue, high cytocompatibility, restoration of IVD biomechanics to intact levels following in situ repair, and low extrusion risk. TMC adhesives had high adhesion strength as assessed with a pushout test (150 kPa), and low degradation rates over 3 weeks in vitro. Both TMC adhesives had shear moduli (220 and 490 kPa) similar to, but somewhat higher than, AF tissue. The adhesive with three TMC moieties per branch (TMC3) was selected for additional in situ testing because it best matched AF shear properties. TMC3 restored torsional stiffness, torsional hysteresis area and axial range of motion to intact states. However, in a failure test of compressive deformation under fixed 5 ° flexion, some herniation risk was observed with failure strength of 5.9 MPa compared with 13.5 MPa for intact samples; TMC3 herniated under cyclic organ culture testing. These TMC adhesives performed well during in vitro and in situ testing, but additional optimization to enhance failure strength is required to further this material to advanced screening tests, such as long‐term degradation. Copyright

Confocal Imaging Protocols for Live/Dead Staining in Three-Dimensional Carriers

In tissue engineering, a variety of methods are commonly used to evaluate survival of cells inside tissues or three-dimensional (3D) carriers. Among these methods confocal laser scanning microscopy opened accessibility of 3D tissue using live cell imaging into the tissue or 3D scaffolds. However, although this technique is ideally applied to 3D tissue or scaffolds with thickness up to several millimetres, this application is surprisingly rare and scans are often done on slices with thickness <20 μm. Here, we present novel protocols for the staining of 3D tissue (e.g. intervertebral disc tissue) and scaffolds, such as fibrin gels or alginate beads.