Embedding of human vertebral bodies leads to higher ultimate load and altered damage localisation under axial compression

Computer tomography (CT)-based finite element (FE) models of vertebral bodies assess fracture load in vitro better than dual energy X-ray absorptiometry, but boundary conditions affect stress distribution under the endplates that may influence ultimate load and damage localisation under post-yield strains. Therefore, HRpQCT-based homogenised FE models of 12 vertebral bodies were subjected to axial compression with two distinct boundary conditions: embedding in polymethylmethalcrylate (PMMA) and bonding to a healthy intervertebral disc (IVD) with distinct hyperelastic properties for nucleus and annulus. Bone volume fraction and fabric assessed from HRpQCT data were used to determine the elastic, plastic and damage behaviour of bone. Ultimate forces obtained with PMMA were 22% higher than with IVD but correlated highly (R2 = 0.99). At ultimate force, distinct fractions of damage were computed in the endplates (PMMA: 6%, IVD: 70%), cortex and trabecular sub-regions, which confirms previous observations that in contrast to PMMA embedding, failure initiated underneath the nuclei in healthy IVDs. In conclusion, axial loading of vertebral bodies via PMMA embedding versus healthy IVD overestimates ultimate load and leads to distinct damage localisation and failure pattern.

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.

Does normalized signal intensity of cervical discs on T2 weighted MRI images change in whiplash patients?

PURPOSE We tested the hypothesis that whiplash trauma leads to changes of the signal intensity of cervical discs in T2-weighted images. METHODS AND MATERIALS 50 whiplash patients (18-65 years) were examined within 48h after motor vehicle accident, and again after 3 and 6 months and compared to 50 age- and sex-matched controls. Signal intensity in ROI's of the discs at the levels C2/3 to C7/T1 and the adjacent vertebral bodies were measured on sagittal T2 weighted MR images and normalized using the average of ROI's in fat tissue. The contrast between discs and both adjacent vertebrae was calculated and disc degeneration was graded by the Pfirrmann-grading system. RESULTS Whiplash trauma did not have a significant effect on the normalized signals from discs and vertebrae, on the contrast between discs and adjacent vertebrae, or on the Pfirrmann grading. However, the contrast between discs and adjacent vertebrae and the Pfirrmann grading showed a strong correlation. In healthy volunteers, the contrast between discs and adjacent vertebrae and Pfirrmann grading increased with age and was dependent on the disc level. CONCLUSION We could not find any trauma related changes of cervical disc signal intensities. Normalized signals of discs and Pfirrmann grading changed with age and varied between disc levels with the used MR sequence.

Sensory innervation of the dorsal longitudinal ligament and the meninges in the lumbar spine of the dog

Although intervertebral disc herniation is a well-known disease in dogs, pain management for this condition has remained a challenge. The goal of the present study is to address the lack of information regarding the innervation of anatomical structures within the canine vertebral canal. Immunolabeling was performed with antibodies against protein gene product 9.5, Tuj-1 (neuron-specific class III β-tubulin), calcitonin gene-related peptide, and neuropeptide Y in combination with the lectin from Lycopersicon esculentum as a marker for blood vessels. Staining was indicative of both sensory and sympathetic fibers. Innervation density was the highest in lateral areas, intermediate in dorsal areas, and the lowest in ventral areas. In the dorsal longitudinal ligament (DLL), the highest innervation density was observed in the lateral regions. Innervation was lower at mid-vertebral levels than at intervertebral levels. The presence of sensory and sympathetic fibers in the canine dura and DLL suggests that pain may originate from both these structures. Due to these regional differences in sensory innervation patterns, trauma to intervertebral DLL and lateral dura is expected to be particularly painful. The results ought to provide a better basis for the assessment of medicinal and surgical procedures.

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.

Longitudinal extension of myelomalacia by intramedullary and subdural hemorrhage in a canine model of spinal cord injury

BACKGROUND CONTEXT In canine intervertebral disc (IVD) extrusion, a spontaneous animal model of spinal cord injury, hemorrhage is a consistent finding. In rodent models, hemorrhage might be involved in secondary tissue destruction by biochemical mechanisms. PURPOSE This study aimed to investigate a causal association between the extents of intramedullary, subdural and epidural hemorrhage and the severity of spinal cord damage following IVD extrusion in dogs. STUDY DESIGN/SETTING A retrospective study using histologic spinal cord sections from 83 dogs euthanized following IVD extrusion was carried out. METHODS The degree of hemorrhage (intramedullary, subdural, epidural), the degree of spinal cord damage in the epicenter (white and gray matter), and the longitudinal extent of myelomalacia were graded. Associations between the extent of hemorrhage and the degree of spinal cord damage were evaluated statistically. RESULTS Intramedullary and subdural hemorrhages were significantly associated with the degree of white (p<.001/ p=.004) and gray (both p<.001) matter damage, and with the longitudinal extension of myelomalacia (p<.001/p=.005). Intriguingly, accumulation of hemorrhagic cord debris inside or dorsal to a distended and ruptured central canal in segments distant to the epicenter of the lesion was observed exhibiting a wave-like pattern on longitudinal assessment. The occurrence of this debris accumulation was associated with high degrees of tissue destruction (all p<.001). CONCLUSIONS Tissue liquefaction and increased intramedullary pressure associated with hemorrhage are involved in the progression of spinal cord destruction in a canine model of spinal cord injury and ascending or descending myelomalacia. Functional and dynamic studies are needed to investigate this concept further.

In vitro analysis of osteogenic inhibition in non-union spinal fusion caused by nucleus pulposus cells.

Discectomy and spinal fusion is the gold standard for spinal surgery to relieve pain. However, fusion can be hindered for yet unknown reasons that lead to non-fusions with pseudo-arthrosis. Clinical observations indicate that presence of residual intervertebral disc (IVD) tissue might hinder the ossification. We hypothesize that BMP-antagonists are constantly secreted by IVD cells and potentially prevent the ossification process. Furthermore, L51P, the engineered BMP2 variant, stimulates osseo-induction of bone marrow-derived mesenchymal stem cells (MSC) by antagonizing BMP-inhibitors. Human MSCs, primary nucleus pulposus (NPC) and annulus pulposus cells (AFC) were isolated and expanded in monolayer cultures up to passage 3. IVD cells were seeded in 1.2% alginate beads (4Mio/mL) and separated by culture inserts from MSCs. MSCs were kept in 1:control medium, 2:osteogenic medium±alginate beads, 3:osteogenic medium+NPC (±L51P) and 4:osteogenic medium+AFC (±L51P) for 21 days. Relative gene expression of bone-related genes, alkaline phosphatase assay and histological staining were performed. Osteogenesis of MSCs was hindered as shown by reduced alizarin red staining in the presence of NPC. No such inhibition was observed if co-cultured with alginate only or in the presence of AFC. The results were confirmed on the RNA and protein level. Addition of L51Pto the co- cultures, however, induced mineralization of MSCs in presence of NPC. We demonstrated that NPC secrete BMP-antagonists that prevent osteogenesis of MSCs and L51P can antagonize BMP-antagonists and induce bone formation.

CT-guided infiltration saves surgical intervention and fastens return to work compared to anatomical landmark-guided infiltration in patients with lumbosciatica

BACKGROUND Infiltration procedures are a common treatment of lumbar radiculopathy. There is a wide variety of infiltration techniques without an established gold standard. Therefore, we compared the effectiveness of CT-guided transforaminal infiltrations versus anatomical landmark-guided transforaminal infiltrations at the lower lumbar spine in case of acute sciatica at L3-L5. METHODS A retrospective chart review was conducted of 107 outpatients treated between 2009 and 2011. All patients were diagnosed with lumbar radiculopathic pain secondary to disc herniation in L3-L5. A total of 52 patients received CT-guided transforaminal infiltrations; 55 patients received non-imaging-guided nerve root infiltrations. The therapeutic success was evaluated regarding number of physician contacts, duration of treatment, type of analgesics used and loss of work days. Defined endpoint was surgery at the lower lumbar spine. RESULTS In the CT group, patients needed significantly less oral analgesics (p < 0.001). Overall treatment duration and physician contacts were significantly lower in the CT group (p < 0.001 and 0.002) either. In the CT group, patients lost significant fewer work days due to incapacity (p < 0.001). Surgery had to be performed in 18.2 % of the non-imaging group patients (CT group: 1.9 %; p = 0.008). CONCLUSION This study shows that CT-guided periradicular infiltration in lumbosciatica caused by intervertebral disc herniation is significantly superior to non-imaging, anatomical landmark-guided infiltration, regarding the parameters investigated. The high number of treatment failures in the non-imaging group underlines the inferiority of this treatment concept.

The use of an electronic von Frey device for evaluation of sensory threshold in neurologically normal dogs and those with acute spinal cord injury

The utility and inter-session repeatability of sensory threshold measurements using an electronic von Frey anesthesiometer (VFA) were assessed in a group of six neurologically normal dogs. Sensory threshold values obtained in neurologically normal dogs were compared to those of dogs with acute spinal cord injury (SCI) caused by intervertebral disc extrusion (n=6) and to a group of neurologically normal dogs with cranial cruciate ligament rupture (CCLR; n=6). Sensory threshold values in neurologically normal dogs were 155.8 ± 37.7 g and 154.7 ± 67.2 g for the left and right pelvic limbs, respectively. The difference in mean sensory threshold values obtained for the group when two distinct testing sessions were compared was not statistically significant (P>0.05). Mean sensory threshold values for the group with SCI were significantly higher than those for neurologically normal dogs at 351.1 ± 116.5 g and 420.3 ± 157.7 g for the left and right pelvic limbs, respectively (P=0.01). A comparison of sensory threshold values for the group with CCLR and neurologically normal dogs was not statistically significant (P>0.05). The modified dorsal technique for VFA described here represents a reliable method to assess sensory threshold in neurologically normal dogs and in those with SCI.

A photopolymerized composite hydrogel and surgical implanting tool for a nucleus pulposus replacement

Nucleus pulposus replacements have been subjected to highly controversial discussions over the last 40 years. Their use has not yet resulted in a positive outcome to treat herniated disc or degenerated disc disease. The main reason is that not a single implant or tissue replacement was able to withstand the loads within an intervertebral disc. Here, we report on the development of a photo-polymerizable poly(ethylene glycol)dimethacrylate nano-fibrillated cellulose composite hydrogel which was tuned according to native tissue properties. Using a customized minimally-invasive medical device to inject and photopolymerize the hydrogel insitu, samples were implanted through an incision of 1 mm into an intervertebral disc of a bovine organ model to evaluate their long-term performance. When implanted into the bovine disc model, the composite hydrogel implant was able to significantly re-establish disc height after surgery (p < 0.0025). The height was maintained after 0.5 million loading cycles (p < 0.025). The mechanical resistance of the novel composite hydrogel material combined with the minimally invasive implantation procedure into a bovine disc resulted in a promising functional orthopedic implant for the replacement of the nucleus pulposus.

Angiopoietin-1 receptor Tie2 distinguishes multipotent differentiation capability in bovine coccygeal nucleus pulposus cells.

BACKGROUND The intervertebral disc (IVD) has limited self-healing potential and disc repair strategies require an appropriate cell source such as progenitor cells that could regenerate the damaged cells and tissues. The objective of this study was to identify nucleus pulposus-derived progenitor cells (NPPC) and examine their potential in regenerative medicine in vitro. METHODS Nucleus pulposus cells (NPC) were obtained from 1-year-old bovine coccygeal discs by enzymatic digestion and were sorted for the angiopoietin-1 receptor Tie2. The obtained Tie2- and Tie2+ fractions of cells were differentiated into osteogenic, adipogenic, and chondrogenic lineages in vitro. Colony-forming units were prepared from both cell populations and the colonies formed were analyzed and quantified after 8 days of culture. In order to improve the preservation of the Tie2+ phenotype of NPPC in monolayer cultures, we tested a selection of growth factors known to have stimulating effects, cocultured NPPC with IVD tissue, and exposed them to hypoxic conditions (2 % O2). RESULTS After 3 weeks of differentiation culture, only the NPC that were positive for Tie2 were able to differentiate into osteocytes, adipocytes, and chondrocytes as characterized by calcium deposition (p < 0.0001), fat droplet formation (p < 0.0001), and glycosaminoglycan content (p = 0.0095 vs. Tie2- NPC), respectively. Sorted Tie2- and Tie2+ subpopulations of cells both formed colonies; however, the colonies formed from Tie2+ cells were spheroid in shape, whereas those from Tie2- cells were spread and fibroblastic. In addition, Tie2+ cells formed more colonies in 3D culture (p = 0.011) than Tie2- cells. During expansion, a fast decline in the fraction of Tie2+ cells was observed (p < 0.0001), which was partially reversed by low oxygen concentration (p = 0.0068) and supplementation of the culture with fibroblast growth factor 2 (FGF2) (p < 0.0001). CONCLUSIONS Our results showed that the bovine nucleus pulposus contains NPPC that are Tie2+. These cells fulfilled formally progenitor criteria that were maintained in subsequent monolayer culture for up to 7 days by addition of FGF2 or hypoxic conditions. We propose that the nucleus pulposus represents a niche of precursor cells for regeneration of the IVD.

Core stability exercise in chronic low back pain

Exercise is commonly used in the management of chronic musculoskeletal conditions, including chronic low back pain (CLBP). The focus of exercise is varied and may include parameters ranging from strength and endurance training, to specific training of muscle coordination and control. The assumption underpinning these approaches is that improved neuromuscular function will restore or augment the control and support of the spine and pelvis. In a biomechanical model of CLBP, which assumes that pain recurrence is caused by repeated mechanical irritation of pain sensitive structures [1], it is proposed that this improved control and stability would reduce mechanical irritation and lead to pain relief [1]. Although this model provides explanation for the chronicity of LBP, perpetuation of pain is more complex, and contemporary neuroscience holds the view that chronic pain is mediated by a range of changes including both peripheral (eg, peripheral sensitization) and central neuroplastic changes [2]. Although this does not exclude the role of improved control of the lumbar spine and pelvis in management of CLBP, particularly when there is peripheral sensitization, it highlights the need to look beyond outdated simplistic models. One factor that this information highlights is that the refinement of control and coordination may be more important than simple strength and endurance training for the trunk muscles. The objective of this article is to discuss the rationale for core stability exercise in the management of CLBP, to consider critical factors for its implementation, and to review evidence for efficacy of the approach.

Design and synthesis of proteoglycan analogues for tissue repair and regeneration

This thesis is concerned with the design and synthesis of a novel, injectable proteoglycan analogue for tissue repair. This is of particular relevance to the restoration of disc height to a degraded nucleus pulposus of the intervertebral disc. The focus is on the use of sulfonate monomers as proteoglycan analogues, in particular sodium 2-acrylamido-2-methylpropane sulfonic acid and the potassium salt of 3-sulfopropyl acrylate. For most biomedical applications, synthetic hydrogels need to show dimensional stability to changes in pH, osmolarity, and temperature. This is readily achieved by neutral structures however ionic sulfonate containing hydrogels are responsive to environmental change which renders them difficult to manage in most tissue replacement applications. In this case osmotic responsiveness rather than stability is desirable. Therefore sulfonate based materials possess advantageous properties. This is a result of the sulfonate becoming an ideal surrogate for the sulfate group present within the structure of natural proteoglycans. This thesis reports polymerisation studies based on the production of a redox initiated copolymer system capable of polymerising in situ within a timescale of circa. 5-7 minutes. The rheological properties, osmotic drive, and residual monomer content of successful compositions is analysed. Properties are adapted to mimic those of the target natural tissue. The adaptation of the material for use as an injectable intra-ocular lens, with hyaluronic acid as an interpenetrate is reported. The synthesis of a radiopaque macromer to allow visibility of the repair system once in situ is investigated and discussed. The results presented in this thesis describe a suitable proteoglycan tissue analogue which is injectable, biomimetic, osmotically responsive and mechanically stable in its desired application.

Proteoglycan analogues for ophthalmic and orthopaedic applications

The successful development of biomaterials is increasingly influenced by biomimesis essentially the use of biological structures as design templates. This approach has been used here in the design of injectable systems for the nucleus of the intervertebral disc, corneal inlays and intraocular lenses (IOLs). The strategy is based on the use of C-linked sulphonates to mimic the O-linked sulphate groups that are the hydration drivers in proteoglycans. The elastic modulus of the materials can be tailored for specific applications.

Injectable hydrogels with high fixed charge density and swelling pressure for nucleus pulposus repair:biomimetic glycosaminoglycan analogues

The load-bearing biomechanical role of the intervertebral disc is governed by the composition and organization of its major macromolecular components, collagen and aggrecan. The major function of aggrecan is to maintain tissue hydration, and hence disc height, under the high loads imposed by muscle activity and body weight. Key to this role is the high negative fixed charge of its glycosaminoglycan side chains, which impart a high osmotic pressure to the tissue, thus regulating and maintaining tissue hydration and hence disc height under load. In degenerate discs, aggrecan degrades and is lost from the disc, particularly centrally from the nucleus pulposus. This loss of fixed charge results in reduced hydration and loss of disc height; such changes are closely associated with low back pain. The present authors developed biomimetic glycosaminoglycan analogues based on sulphonate-containing polymers. These biomimetics are deliverable via injection into the disc where they polymerize in situ, forming a non-degradable, nuclear "implant" aimed at restoring disc height to degenerate discs, thereby relieving back pain. In vitro, these glycosaminoglycan analogues possess appropriate fixed charge density, hydration and osmotic responsiveness, thereby displaying the capacity to restore disc height and function. Preliminary biomechanical tests using a degenerate explant model showed that the implant adapts to the space into which it is injected and restores stiffness. These hydrogels mimic the role taken by glycosaminoglycans in vivo and, unlike other hydrogels, provide an intrinsic swelling pressure, which can maintain disc hydration and height under the high and variable compressive loads encountered in vivo. © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.