Intact Intervertebral Disc Research Articles

Cryopreserving the intact intervertebral disc without compromising viability.

Tissue cryopreservation requires saturation of the structure with cryoprotectants (CPAs) that are also toxic to cells within a short timeframe unless frozen. The race between CPA delivery and cell death is the main barrier to realizing transplantation banks that can indefinitely preserve tissues and organs. Unrealistic cost and urgency leaves less life-threatening ailments unable to capitalize on traditional organ transplantation systems that immediately match and transport unfrozen organs. For instance, human intervertebral discs (IVD) could be transplanted to treat back pain or used as ex vivo models for studying regenerative therapies, but both face logistical hurdles in organ acquisition and transport. Here we aimed to overcome those challenges by cryopreserving intact IVDs using compressive loading and swelling to accelerate CPA delivery. CPAs were tested on bovine nucleus pulposus cells to determine the least cytotoxic solution. Capitalizing on our CPAs Computed Tomography (CT) contrast enhancement, we imaged and quantified saturation time in intact bovine IVDs under different conditions in a bioreactor. Finally, the entire protocol was tested, including 1 week of frozen storage, to confirm tissue viability in multiple IVD regions after thawing. Results showed cryopreserving medium containing dimethyl sulfoxide and ethylene glycol gave over 7.5 h before cytotoxicity. While non-loaded IVDs required over 3 days to fully saturate, a dynamic loading protocol followed by CPA addition and free-swelling decreased saturation time to <5 h. After cryopreserving IVDs for 1 week with the optimized CPA and permeation method, all IVD regions had 85% cell viability, not significantly different from fresh unfrozen controls. This study created a novel solution to a roadblock in IVD research and development. Using post-compression swelling CPA can be delivered to an intact IVD over 20× more quickly than previous methods, enabling cryopreservation of the IVD with no detectable loss in cell viability.

Gene Expression Profiles Perturbed by Injury to the Mouse Intervertebral Disc.

Back pain subsequent to intervertebral disc (IVD) injury is a common clinical problem. Previous work examining early molecular changes post injury mainly used a candidate marker approach. In this study, gene expression in the injured and intact mouse tail IVDs was determined with a nonbiased whole transcriptome approach. Mouse tail IVD injury was induced by a needle puncture. Whole murine transcriptome was determined by RNASeq. Transcriptomes of injured IVDs were compared with those of intact controls by bioinformatic methods. Among the 18,078 murine genes examined, 592 genes were differentially expressed (P.adj < 0.01). Novel genes upregulated in injured compared with intact IVDs included Chl1, Lum, etc. Ontology study of upregulated genes revealed that leukocyte migration was the most enriched biological process, and network analysis showed that Tnfa had the most protein-protein interactions. Novel downregulated genes in the injured IVDs included 4833412C05Rik, Myoc, etc. The most enriched downregulated pathways were related to cytoskeletal organization. Novel genes highly regulated post disc injury were identified with an unbiased approach; they may serve as biomarkers of injury and response to treatments in future experiments. Enriched biological pathways and molecules with high numbers of connections may be targets for treatments post injury.

Clinical biomechanics of the spine in three unsolved problems. A brief analytical review

Chronic pathology of the spine, especially its forms, such as degenerative disc disease (DDD), is one of the most common in the human population and a marker for a person. Even though this pathology lacks the burden of mortality, its existence and consequences worsen the quality of life. Hypotheses of the high prevalence of DDD often appeal to a person's upright gait and the function of the spine as a movable vertical support, which means a permanent significant axial load of the intervertebral discs (IVDs). Therefore, finding out the magnitude of such a load, its dependence on the body's position in space, and types of motor activity is an essential practical task of the biomechanics of the spine as a separate interdisciplinary direction of biomedical research. Despite all the efforts and significant activity during the 70s and 80s of the last century, the central questions of clinical biomechanics of the spine still need to be explored. It is visible from the state of development of three "legendary" problems ‒ elucidation of intradiscal pressure against the background of usual types of physical activity, the role of sitting in the promotion of DDD of the lumbar region, and determination of the role of intra-abdominal pressure in reducing the axial load of this region of the spine. For example, the results of the investigations can state that assessment of intradiscal pressure against the background of human behavioral activity has so far been the focus of a disproportionately small number of works, which, due to the weakness of the accompanying visualization and the technical unreliability of the sensors did not obtain a sufficient empirical base for statistically significant conclusions. Therefore, the urgent task of the future is developing and using a more accurate, reliable, miniature, and durable intradiscal pressure monitoring technique, which would make it possible to evaluate this parameter on large samples of volunteers with conditionally intact IVD and against the background of pathology. In this regard, the assumptions about the role of sitting in the development of DDD of the lumbar spine remain unverified.Similarly, the research on the phenomenon of intra-abdominal pressure needs to determine under what conditions and mechanisms this factor can affect the magnitude of the axial load on the lumbar spine. Also, constructing more insightful models of the biomechanics of the spine is only possible with expanding ideas about the composition, vascularization, and innervation of the IVD, biology, and pathology of IVD cells. The practical outcome of all these studies is delineation of the most dangerous types of motor activity in the promotion of DDD, which will bring us closer to understanding the drivers of DDD and thus improving the means of preventing and treating this ubiquitous pathology.

Cadaveric biomechanical studies of ADDISC total lumbar disc prosthesis

BackgroundMost total disc replacements provide excessive mobility and not reproduce spinal kinematics, inducing zygapophyseal joint arthritic changes and chronic back pain. In cadaveric lumbosacral spines, we studied if a new lumbar disc prosthesis kinematics mimics the intact intervertebral disc. MethodsIn eight cold preserved cadaveric lumbosacral spines, we registered the movement ranges in flexion, extension, right and left lateral bending, and rotation in the intact status, post-discectomy, and after our prosthesis implantation, comparing them for each specimen. FindingsComparing the intact lumbosacral spine with the L4-L5 prosthesis implanted specimens, we saw statistically significant differences in lateral bending and right rotation but not in the full range of rotation. Analyzing segments, we also noticed statistically significant differences at L4-L5 in flexion-extension and rotation. On the other hand, the L4-L5 discectomy, compared to the baseline spine condition, showed a statistically significant mobility increase in flexion, extension, lateral bending, and axial rotation, with an abnormal instantaneous center of rotation, which destabilizes the segment partly due to anterior annulus surgical removal. Disc prosthesis implantation reversed these changes in instantaneous center of rotation, but the prosthesis failed to restore the initial range of motion due to the destabilization of the ligaments in the operated disc. InterpretationThe ADDISC total disc replacement reproduces the intact disc kinematics and Instantaneous Center of Rotation, but the prosthesis fails to restore the initial range of motion due to ligament destabilization. More studies will be necessary to define a technique that restores the damaged ligaments when implanting the prosthesis.

Chemonucleolysis combined with dynamic loading for inducing degeneration in bovine caudal intervertebral discs.

Chemonucleolysis has become an established method of producing whole organ culture models of intervertebral disc (IVD) degeneration. However, the field needs more side-by-side comparisons of the degenerative effects of the major enzymes used in chemonucleolysis towards gaining a greater understanding of how these organ culture models mimic the wide spectrum of characteristics observed in human degeneration. In the current work we induced chemonucleolysis in bovine coccygeal IVDs with 100µL of papain (65U/mL), chondroitinase ABC (chABC, 5U/mL), or collagenase II (col'ase, 0.5U/mL). Each enzyme was applied in a concentration projected to produce moderate levels of degeneration. After 7days of culture with daily dynamic physiological loading (0.02-0.2MPa, 0.2Hz, 2h), the cellular, biochemical and histological properties of the IVDs were evaluated in comparison to a PBS-injected control. Papain and collagenase, but not chABC, produced macroscopic voids in the tissues. Compared to day 0 intact IVDs, papain induced the greatest magnitude glycosaminoglycan (GAG) loss compared to chABC and col'ase. Papain also induced the greatest height loss (3%), compared to 0.7%, 1.2% and 0.4% for chABC, col'ase, and PBS, respectively. Cell viability in the region adjacent to papain and PBS-injection remained at nearly 100% over the 7-day culture period, whereas it was reduced to 60%-70% by chABC and col'ase. Generally, enzyme treatment tended to downregulate gene expression for major ECM markers, type I collagen (COL1), type II collagen (COL2), and aggrecan (ACAN) in the tissue adjacent to injection. However, chABC treatment induced an increase in COL2 gene expression, which was significant compared to the papain treated group. In general, papain and col'ase treatment tended to recapitulate aspects of advanced IVD degeneration, whereas chABC treatment captured aspects of early-stage degeneration. Chemonucleolysis of whole bovine IVDs is a useful tool providing researchers with a robust spectrum of degenerative changes and can be utilized for examination of therapeutic interventions.

Bionate® nucleus disc replacement: bench testing comparing two different designs

BackgroundIntervertebral disc nucleus degeneration initiates a degenerative cascade and can induce chronic low back pain. Nucleus replacement aims to replace the nucleus while the annulus is still intact. Over time, several designs have been introduced, but the definitive solution continues to be elusive. Therefore, we aimed to create a new nucleus replacement that replicates intact intervertebral disc biomechanics, and thus has the potential for clinical applications.Materials and methodsTwo implants with an outer ring and one (D2) with an additional midline strut were compared. Static and fatigue tests were performed with an INSTRON 8874 following the American Society for Testing and Materials F2267-04, F2346-05, 2077-03, D2990-01, and WK4863. Implant stiffness was analyzed at 0–300 N, 500–2000 N, and 2000–6000 N and implant compression at 300 N, 1000 N, 2000 N, and 6000 N. Wear tests were performed following ISO 18192-1:2008 and 18192-2:2010. GNU Octave software was used to calculate movement angles and parameters. The statistical analysis package R was used with the Deducer user interface. Statistically significant differences between the two designs were analyzed with ANOVA, followed by a post hoc analysis.ResultsD1 had better behavior in unconfined compression tests, while D2 showed a “jump.” D2 deformed 1 mm more than D1. Sterilized implants were more rigid and deformed less. Both designs showed similar behavior under confined compression and when adding shear. A silicone annulus minimized differences between the designs. Wear under compression fatigue was negligible for D1 but permanent for D2. D1 suffered permanent height deformation but kept its width. D2 suffered less height loss than D1 but underwent a permanent width deformation. Both designs showed excellent responses to compression fatigue with no breaks, cracks, or delamination. At 10 million cycles, D2 showed 3-times higher wear than D1. D1 had better and more homogeneous behavior, and its wear was relatively low. It showed good mechanical endurance under dynamic loading conditions, with excellent response to axial compression fatigue loading without functional failure after long-term testing.ConclusionD1 performed better than D2. Further studies in cadaveric specimens, and eventually in a clinical setting, are recommended.Level of evidence 2c.

Biomechanical Response of Four Roussouly's Sagittal Alignment Lumbar to Degeneration of Different Parts of Intervertebral Disc: Finite Element Model Analysis

BackgroundSpinal degeneration with age is commonly accompanied by excessive kyphosis and low-back pain, however, little is known about the connection between lumbar sagittal morphology and its degenerative biomechanics. This study investigates the biomechanical response of four Roussouly's sagittal alignment lumbar to degeneration of various parts of the intervertebral disc (IVD) based on threedimensional finite element (FE) models. MethodsUsing Roussouly's type parametric FE models, material properties of the degenerate nucleus populous (NP), annulus fibrosis matrix (AFM), and collagen fibers were assumed to be half of the intact IVD. A follower preload and vertical force were applied to simulate physical standing posture. Resultsthe reduced strength of the NP and AFM led to the increase of lumbar anteflexion, while the fiber mechanical properties have little effect on it. When facing IVD degeneration, Type 1 lumbar showed increased intradiscal pressures (IDPs) and fiber stress at the L1-2 and L4-S1 segments. Type 2 lumbar exhibited the highest lumbar anteflexion and pelvic rearward rotation, as well as increased IDPs among the models. Type 3 lumbar had the best biomechanical stability. Type 4 lumbar showed the higher AFM stress but the lower IDPs among the four types. ConclusionsIVD degeneration generated sagittal imbalance by increasing lumbar anteflexion movement (i.e., loss lordosis) and pelvic rearback rotation. The biomechanical response of the four Roussouly's lumbar types differed in intervertebral rotation and stress distribution.

Biomechanical evaluation of a novel intervertebral disc repair technique for large box-shaped ruptures.

Objective: The purpose of this study was to analyze the feasibility of repairing a ruptured intervertebral disc using a patch secured to the inner surface of the annulus fibrosus (AF). Different material properties and geometries for the patch were evaluated. Methods: Using finite element analysis, this study created a large box-shaped rupture in the posterior-lateral region of the AF and then repaired it with a circular and square inner patch. The elastic modulus of the patches ranged from 1 to 50MPa to determine the effect on the nucleus pulposus (NP) pressure, vertical displacement, disc bulge, AF stress, segmental range of motion (ROM), patch stress, and suture stress. The results were compared against the intact spine to determine the most suitable shape and properties for the repair patch. Results: The intervertebral height and ROM of the repaired lumbar spine was similar to the intact spine and was independent of the patch material properties and geometry. The patches with a modulus of 2-3MPa resulted in an NP pressure and AF stresses closest to the healthy disc, and produced minimal contact pressure on the cleft surfaces and minimal stress on the suture and patch of all models. Circular patches caused lower NP pressure, AF stress and patch stress than the square patch, but also caused greater stress on the suture. Conclusion: A circular patch with an elastic modulus of 2-3MPa secured to the inner region of the ruptured annulus fibrosus was able to immediately close the rupture and maintain an NP pressure and AF stress similar to the intact intervertebral disc. This patch had the lowest risk of complications and produced the greatest restorative effect of all patches simulated in this study.

ADDISC lumbar disc prosthesis: Analytical and FEA testing of novel implants

The intact intervertebral disc is a six-freedom degree elastic deformation structure with shock absorption. “Ball-and-socket” TDR do not reproduce these properties inducing zygapophyseal joint overload. Elastomeric TDRs reproduce better normal disc kinematics, but repeated core deformation causes its degeneration. We aimed to create a new TDR (ADDISC) reproducing healthy disc features. We designed TDR, analyzed (Finite Element Analysis), and measured every 500,000 cycles for 10 million cycles of the flexion-extension, lateral bending, and axial rotation cyclic compression bench-testing. In the inlay case, we weighted it and measured its deformation.ADDISC has two semi-spherical articular surfaces, one rotation centre for flexion, another for extension, the third for lateral bending, and a polycarbonate urethane inlay providing shock absorption. The first contact is between PCU and metal surfaces. There is no metal-metal contact up to 2000 N, and CoCr28Mo6 absorbs the load. After 10 million cycles at 1.2–2.0 kN loads, wear 140.96 mg (35.50 mm3), but no implant failures. Our TDR has a physiological motion range due to its articular surfaces' shape and the PCU inlay bumpers, minimizing the facet joint overload. ADDISC mimics healthy disc biomechanics and Instantaneous Rotation Center, absorbs shock, reduces wear, and has excellent long-term endurance.

Regional variations in discrete collagen fibre mechanics within intact intervertebral disc resolved using synchrotron computed tomography and digital volume correlation

Many soft tissues, such as the intervertebral disc (IVD), have a hierarchical fibrous composite structure which suffers from regional damage. We hypothesise that these tissue regions have distinct, inherent fibre structure and structural response upon loading. Here we used synchrotron computed tomography (sCT) to resolve collagen fibre bundles (∼5μm width) in 3D throughout an intact native rat lumbar IVD under increasing compressive load. Using intact samples meant that tissue boundaries (such as endplate-disc or nucleus-annulus) and residual strain were preserved; this is vital for characterising both the inherent structure and structural changes upon loading in tissue regions functioning in a near-native environment. Nano-scale displacement measurements along >10,000 individual fibres were tracked, and fibre orientation, curvature and strain changes were compared between the posterior-lateral region and the anterior region. These methods can be widely applied to other soft tissues, to identify fibre structures which cause tissue regions to be more susceptible to injury and degeneration. Our results demonstrate for the first time that highly-localised changes in fibre orientation, curvature and strain indicate differences in regional strain transfer and mechanical function (e.g. tissue compliance). This included decreased fibre reorientation at higher loads, specific tissue morphology which reduced capacity for flexibility and high strain at the disc-endplate boundary. Statement of significanceThe analyses presented here are applicable to many collagenous soft tissues which suffer from regional damage. We aimed to investigate regional intervertebral disc (IVD) structural and functional differences by characterising collagen fibre architecture and linking specific fibre- and tissue-level deformation behaviours. Synchrotron CT provided the first demonstration of tracking discrete fibres in 3D within an intact IVD. Detailed analysis of regions was performed using over 200k points, spaced every 8 μm along 10k individual fibres. Such comprehensive structural characterisation is significant in informing future computational models. Morphological indicators of tissue compliance (change in fibre curvature and orientation) and fibre strain measurements revealed localised and regional differences in tissue behaviour.

Preparation of Hard Tissues Like Bone or Cartilage for Shotgun Mass Spectrometry Analysis of the Proteome.

Proteomic analyses of intervertebral discs (IVDs) reveal information for understanding the fundamentals of biological processes and pathogenesis but also provide insights for novel pharmaceutical development. Sensitive mass spectrometry techniques and bioinformatics have advanced the detection and identification of proteins from any sample. Due to the challenges of catastrophic sample-loss artifacts during hard-tissue extraction, however, many researchers have omitted the cartilage endplates of IVDs for protein extraction, analyzing only the cellular components of the annulus fibrosus and/or nucleus pulposus. The full proteomic picture of IVDs is compromised without extracting proteins from intact IVDs. Here, we describe a novel preparation method using snap-freeze grinding, which allows for mechanical disruption and customized chemical lysis of hard tissues such as bone or cartilage. This method replaces the time-consuming and insufficient conventional tissue homogenization methods. Sample loss and contamination could be minimized during proteolysis by using an in-solution protein digestion and desalting procedure. We demonstrate excellent proteome coverage with intact mouse IVDs by analyzing samples in a hybrid quadrupole time-of-flight tandem mass spectrometer. © 2021 Wiley Periodicals LLC.

P8. Correlations between sagittal plane disc dimensions and principal surface strains across the L3-4 intact IVD during in vitro multidirectional loading

<h3>BACKGROUND CONTEXT</h3> Understanding the biomechanical behavior of the intact intervertebral disc (IVD) is of great importance when studying disc disease. Experimental data describing the relationship between disc size and sub-regional principal strains of the intact lumbar intervertebral disc during multi-directional loading are lacking. <h3>PURPOSE</h3> The purpose of this study was to investigate possible relationships between lateral view disc dimensions and principal surface strains across different regions of an intact L3-4 disc assessed using digital image correlation (DIC) during multi-directional loading. <h3>STUDY DESIGN/SETTING</h3> Correlation analysis of dimensional data obtained from lateral X-rays of intact L3-4 lumbar discs at rest and principal surface strains of the same discs obtained from digital image correlation analysis during in vitro loading. <h3>PATIENT SAMPLE</h3> There were 14 cadaveric spine segments including 6F/8M, mean(±SD) age: 45±13years; BMD: 0.916±0.120 g/cm2. <h3>OUTCOME MEASURES</h3> Correlation coefficients (and p-values) between disc dimensions (sagittal view disc height and area) and principal surface strains during multi-directional loading, in sub-regions of intact L3-4 IVD. <h3>METHODS</h3> The mean height and depth of the L3-4 IVD of 14 seemingly healthy human spine segments at rest were determined using lateral view X-rays and ImageJ. The IVDs were part of L3-S1 spine segments that were tested using non-destructive loading (7.5 Nm) in flexion (FL), extension (EX), right lateral bending (RLB) right/left axial rotation (RAR/LAR), followed by compression (Comp, 400 N). Mean principal strains (Pmax and Pmin) in four similarly sized quarters of the L3-4 IVD were assessed using 3D DIC (Vic-3D) during loading with cameras positioned directly lateral on the specimen's left side. Relationships between sagittal view disc dimensions at rest and mean principal strains per lateral view disc quarter [Q1(ant)-Q4(post)] during loading were studied using Pearson Correlation analysis (SigmaPlot v14 with p<0.05). <h3>RESULTS</h3> Disc height correlated significantly and positively with Pmax (tensile/stretching strain) mid-anteriorly (Q2) during Comp (R=0.545, p=0.044). There were no other significant correlations between disc height and principal strains in any other region of the disc during any direction of loading (p>0.05). In the mid-posterior disc region (Q3), Pmax correlated significantly and negatively with sagittal plane disc area during FL (R=-0.538, p=0.047) and RLB (R=-0.592, p=0.033, Fig.), showing that a larger disc (taller and deeper) bulges/stretches less during flexion and right lateral bending than a smaller disc (narrow disc height combined with shorter vertebral depth, anterior-posteriorly), mid-posteriorly. Pmin (compressive strain) correlated significantly and positively with sagittal plane disc area in the mid-posterior disc region during Comp (Q3: R=0.650, p=0.012; and Q4: R=0.619, p=0.018), indicating that a larger disc (taller and deeper) shrinks less vertically than a small disc posteriorly during compression. <h3>CONCLUSIONS</h3> Significant relationships were found between disc dimensions and mean principal strains in sagittal view sub-regions of the L3-4 intervertebral disc, during different directions of loading. These relationships aid in understanding intact disc biomechanical behavior during loading and will help improve the quality of finite element models of lumbar spine segments. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

P10. Sub-regional analysis of principal strains across the intact L3-4 disc during in vitro multi-directional loading using 3D digital image correlation (DIC)

P10. Sub-regional analysis of principal strains across the intact L3-4 disc during in vitro multi-directional loading using 3D digital image correlation (DIC)

Can UVA-light-activated riboflavin-induced collagen crosslinking be transferred from ophthalmology to spine surgery? A feasibility study on bovine intervertebral disc.

BackgroundCollagen cross-links contribute to the mechanical resilience of the intervertebral disc (IVD). UVA-light-activated riboflavin-induced collagen crosslinking (UVA-CXL) is a well-established and effective ophthalmological intervention that increases the mechanical rigidity of the collagen-rich corneal matrix in Keratoconus. This study explores the feasibility, safety and efficacy of translating this intervention in reinforcing the IVD.MethodsAnnulus fibrosus (AF) cells were isolated from bovine IVDs and treated with different combinations of riboflavin (RF) concentrations (0.05–8 mM) and UVA light intensities (0.3–4 mW/cm2). Metabolic activity (resazurin assay), cell viability (TUNEL assay), and gene expression of apoptosis regulators C-FOS and PT5 were assessed immediately and 24 hours after treatment. Biomechanical effects of UVA-CXL on IVDs were measured by indentation analysis of changes in the instantaneous modulus and by peel-force delamination strength analysis of the AF prior and after treatment.ResultsDifferent intensities of UVA did not impair the metabolic activity of AF cells. However, RF affected metabolic activity (p < 0.001). PT53 expression was similar in all RF conditions tested while C-FOS expression decreased 24 hours after treatment. Twenty-four hours after treatment, no apoptotic cells were observed in any condition tested. Biomechanical characterizations showed a significant increase in the annular peel strength of the UVA-CXL group, when compared to controls of UVA and RF alone (p < 0.05). UVA-CXL treated IVDs showed up to 152% higher (p < 0.001) instantaneous modulus values compared to the untreated control.ConclusionThis is the first study on UVA-CXL treatment of IVD. It induced significantly increased delamination strength and instantaneous modulus indentation values in intact IVD samples in a structure–function relationship. RF concentrations and UVA intensities utilized in ophthalmological clinical protocols were well tolerated by the AF cells. Our findings suggest that UVA-CXL may be a promising tool to reinforce the IVD matrix.

The effects of rivaroxaban, an oral anticoagulant, on human IVD primary cultures.

IntroductionThe present study aimed to investigate the potential effects of rivaroxaban, an oral anticoagulant that inhibits the effects of factor Xa, on intact intervertebral disc tissue cells and the extracellular matrix (ECM).Material and methodsRivaroxaban was applied to primary human cell cultures prepared from tissues of the intervertebral disc. Comparative molecular analyses were performed on non-drug-treated control group samples. Descriptive statistics were presented as the mean ± standard deviation. An analysis of variance test was performed to determine whether there were significant differences in the mean across the groups. When differences across groups were observed, Tukey’s honestly significant difference post-hoc test was used for multiple pairwise comparisons. The significance of the obtained data was determined statistically. The α significance value was < 0.05.ResultsThe cells in the control group and in the rivaroxaban-treated group were viable, healthy, and proliferated (p < 0.05). However, the expression levels of the chondroadherin gene (CHAD), cartilage oligo matrix protein (COMP), matrix metalloproteinase (MMP)-13, and MMP-19 genes were changed (p < 0.05).ConclusionsAlthough rivaroxaban does not suppress cell proliferation due to morphological, biological, and biochemical changes in the intervertebral disc tissue, it may change the expression of genes that are related to ECM maintenance.

Elevated inflammatory gene expression in intervertebral disc tissues in mice with ADAM8 inactivated

We found ADAM8 enzymatic activity elevated in degenerative human intervertebral disc (IVD). Here, we examined the discs in ADAM8-inactivation mice that carry a mutation preventing self-activation of the enzyme. Surprisingly, elevated gene expression for inflammatory markers (Cxcl1, IL6) was observed in injured discs of ADAM8 mutant mice, along with elevated expression of type 2 collagen gene (Col2a1), compared with wild type controls. Injured annulus fibrosus of mutant and wild type mice contained a higher proportion of large collagen fibers compared with intact discs, as documented by microscopic examination under circular polarized light. In the intact IVDs, Adam8EQ mouse AF contained lower proportion of yellow (intermediate) fiber than WT mice. This suggests that ADAM8 may regulate inflammation and collagen fiber assembly. The seemingly contradictory findings of elevated inflammatory markers in mutant mice and excessive ADAM8 activity in human degenerative discs suggest that ADAM8 may interact with other enzymatic and pro-inflammatory processes needed for tissue maintenance and repair. As a future therapeutic intervention to retard intervertebral disc degeneration, partial inhibition of ADAM8 proteolysis may be more desirable than complete inactivation of this enzyme.

Toxicity of the acetyl-para-aminophenol group of medicines to intact intervertebral disc tissue cells.

The present study aimed to investigate the effects of paracetamol, an analgesic and antipyretic that is used in emergency departments and neurosurgery departments for postoperative pain management on intervertebral disc tissue. Paracetamol-treated human primary cell cultures and untreated cell cultures were compared using molecular analyses. Cell proliferation and gene expression were statistically analyzed. Cell proliferation was suppressed on days 10 (P=0.05) and 20 (P<0.05) in the paracetamol-treated groups. Gene expression of chondroadherin, matrix metalloproteinase (MMP)-7, MMP-13 and MMP-19 was higher in the paracetamol-treated samples while gene expression of Cartilage Oligomeric Matrix Protein and interleukin-1β was lower (P<0.05). Paracetamol, which appears innocuous compared with many analgesics, may increase the expression of MMPs, which serve a significant role in catabolic reactions and suppress the proliferation of intact intervertebral disc tissue cells.

Tactics of surgical treatment of patients with lumbar spondylolysis

The bilateral defect of the pars interarticularis (spondylolysis) is often the cause of low back pain syndrome and can lead to development of spondylolistesis. In some cases inefficiency of conservative treatment of this condition forces orthopedists to use surgical technologies. At the same time, in young patients with intact intervertebral discs, the rigid segmental fixation of the spine should be avoided. Where no neural decompression is needed, selective osteosynthesis of the pars defect is an optimal technique. The authors present the results of surgical treatment of 15 patients with single and two-level lumbar spondylolysis, 4 of which revealed minimal I degree lytic spondylolystesis of the L5 vertebrae. Localization of the pathological process in all patients was noted on both sides. In two patients spondylolysis defects of two vertebrae were detected (in one- adjacent L4 and L5, in the other - L2 and L4 vertebrae with sacralization of L5). The average period from the onset of symptoms ranged from 6 months to 2 years (an average of 14 months). All patients undergone bone autoplasty with iliac crest bone graft, and osteosynthesis of vertebral arches by a combined laminar-transpedicular system of the screw - rod - hook type. All patients had excellent and good anatomic and functional results. The used method of surgical treatment of patients of this category should be considered pathogenetically justified, as it is aimed at repair of spondylolytic defects as the main cause of segmental instability and forward displacement of vertebrae. The possibility to avoid fixation of intact segments of spine allows to categorize this operation as organ-preserving.

In Vitro Characterization of Multilamellar Fibers with Uniaxially Oriented Electrospun Type I Collagen Scaffolds

Fabrication of an appropriate scaffold is critical in order to recapitulate the architecture and functionality of the native tissue. In this study, we attempted to create favorable collagen fiber alignment and multilamellar with uniaxially oriented layers, using a disc collector by turning mats 90 degrees horizontally at specific times. Different concentrations of rat tail‐derived type I collagen (3, 6, 8% w/v) in 1,1,1,3,3,3‐hexafluoroisopropanol (HFIP) are used for electrospinning affairs. The 6% w/v collagen at an applied voltage of 20 kV and collector rotation of 2500 rpm was selected to exhibit bead‐free homogeneous nanofiber with fiber thickness of 0.14 ± 0.4 µm, maximum thickness of 0.5 ± 0.08 µm, and 60% porosity. Also, scanning electron microscope images of electrospun fibers showed 3D multilamellar scaffold with the goodness of 96.5% ± 0.8 in each aligned uniaxially oriented fiber layer. Cross‐linking of collagen fibers with N‐(3‐dimethylaminopropyl)‐N0‐ethylcarbodiimide hydrochloride (EDC)/N‐hydroxysuccinimide (NHS) reduced the fiber degradation rate and preserved the fiber morphology and alignment. The multilamellar mat showed significant increase in tensile strength and average breaking elongation in comparison with unilamellar mat. In vitro cell culture, using human adipose tissue‐derived mesenchymal stem cells (hAT‐MSCs) on cross‐linked scaffold, showed improvement in cell proliferation, attachment, migration, and intercellular junction with a flattened morphology. Raman spectra revealed the preservation of collagen structure. In addition, Raman spectra of the cell containing scaffold were the same as those of an intact intervertebral disc as a sample to be used in engineering tissues. In conclusion, our results showed that the 3D multilamellar collagen nanofibrous scaffold is more appropriate for the tissues that have multilamellar structure.

Evaluation of the expression and proliferation of degenerative markers in primary cell cultures obtained from human intervertebral disc tissue

Aim: A major cause of low back pain is disc degeneration. Nevertheless, no specific and reliable markers of the degeneration of the nucleus pulposus (NP) are available. This presented study aimed to examine changes in the expressions of genes in primary cell cultures isolated from intact and degenerated tissues to give insights into the biopathogenesis of intervertebral disc (IVD) tissue.Material and Methods: Tissues of eight patients (n = 8; average age: 41.74 ± 9.86 years) were resected through microdiscectomy, and primary cell cultures were prepared using degenerated disc tissue. The cultured degenerated tissues served as the study group. The samples in the control group comprised the intact tissues of patients (n = 8; average age: 38.68 ± 7.91 years) resected following a trauma. Morphology of the cell surface were evaluated using an inverted light/fluorescent microscopy at 0 and 24 h on days 10 and 21. The expressions of the chondroadherin (CHAD), cartilage oligomeric matrix protein (COMP), interleukin-1 beta (IL-1 beta), and matrix metalloproteinase (MMP)-7 and MMP-19 genes were evaluated using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The data obtained were statistically analyzed.Results: The four genes investigated, except COMP (P > 0.05), changed significantly in primary cell cultures isolated from degenerative IVD tissues. This result was statistically significant (P 0.05). The gene expressions in the samples derived from intact IVD tissues changed markedly and these changes were associated with proliferation (P 0.05).Conclusion: Analyzing the changes in gene expression levels associated with IVD should contribute to future studies on the prevention and treatment of such pathologies. The data obtained from the present study will shed light on cellular-based personal targeted therapies through which genetic information can be transmitted to cells.