Zorica Buser

Structured coculture of stem cells and disc cells prevent disc degeneration in a rat model

BACKGROUND CONTEXT Harnessing the potential of stem cells is an important strategy for regenerative medicine. This study explores the use of bilaminar coculture pellets (BCPs) of mesenchymal stem cells (MSCs) and nucleus pulposus cells (NPCs) as a cell-based therapy for intervertebral disc regeneration. Prior in vitro experiments have shown that BCP can help differentiate MSCs and substantially improve new matrix deposition. PURPOSE To evaluate the clinical relevance of BCPs by testing the system in vivo. STUDY DESIGN/SETTING We have designed a novel spherical BCP where MSCs are enclosed in a shell of NPCs. The pellets were tested in vivo in a rat tail model of disc degeneration. METHODS Rat caudal intervertebral discs were denucleated and treated with BCP in a fibrin sealant (FS) carrier (controls were MSCs suspended in FS; NPCs suspended in FS; MSCs and NPCs suspended in FS; FS only; and surgery only). At 14 and 35 days after implantation, the animals were euthanized and discs were evaluated for proteoglycan content, enzyme-linked immunosorbent assay for inflammatory cytokines, cell retention using polymerase chain reaction, disc height, histology, and disc grade based on a blinded scoring system. RESULTS The proteoglycan and cytokine levels were not significantly different among groups. The BCP group had higher cell retention than controls. Disc height and disc grade increased over time only in the BCP group. Bilaminar coculture pellets were the only treatment to show proteoglycan staining in the nucleus space at 35 days. CONCLUSIONS This study shows that BCPs may prevent postnucleotomy disc degeneration in vivo. Larger animals and longer time points will be necessary to further judge potential clinical impact. As opposed to strategies that require growth factor supplements, predifferentiation, or genetic manipulations, BCPs are a self-sustaining and targeted method for tissue regeneration in situ.

Biological and biomechanical effects of fibrin injection into porcine intervertebral discs.

Study Design. Surgically denucleated porcine intervertebral discs (IVD) were injected with BIOSTAT BIOLOGX Fibrin Sealant (FS), and the in vivo effects were assessed over time by histological, biochemical, and mechanical criteria. Objective. The objectives were to test whether the intradiscal injection of FS stimulates disc healing. Summary of Background Data. Disc avascularity prevents the deposition of a provisional fibrin scaffold that typically facilitates soft tissue repair. Poor disc wound healing leads to disc damage accumulation and chronic inflammation characterized by overproduction of proinflammatory cytokines and proteolytic enzymes. Methods. Four lumbar IVDs from each of 31 Yucatan minipigs were randomized to untreated controls; degenerative injury (nucleotomy); and nucleotomy plus FS injection. Animals were killed at 1, 2, 3, 6, and 12 weeks postsurgery. IVDs were harvested to quantify (1) architecture using morphological and histological grading; (2) proteoglycan composition using DMMB assay; (3) cytokine content using ELISA; and (4) mechanical properties using quantitative pressure/volume testing. Results. There was progressive invasion of annular tissue into the nucleus of nucleotomy discs and concomitant reduction in proteoglycan content. By contrast, FS supplementation inhibited nuclear fibrosis and facilitated proteoglycan content recovery over time. FS discs synthesized significantly less TNF-&agr; than degenerate discs (66% vs. 226%, P < 0.05) and had upregulation of IL-4 (310% vs. 166%) and TGF-&bgr; (400% vs. 117%) at 2 to 3 weeks posttreatment. At the third week postsurgery, the denucleated discs were less stiff than controls (pressure modulus 779.9 psi vs. 2754.8 psi; P < 0.05) and failed at lower pressures (250.5 psi vs. 492.5 psi; P < 0.05). The stiffness and leakage pressure of the FS-treated discs recovered to control values after 6 and 12 weeks, respectively. Conclusion. FS facilitated structural, compositional, and mechanical repair of the surgically damaged IVD. These FS-derived benefits are likely due to its conductive scaffold properties and metabolically active constituents such as thrombin, factor XIII, and aprotinin acetate.

Mechanical stimulation alters pleiotrophin and aggrecan expression by human intervertebral disc cells and influences their capacity to stimulate endothelial migration

Study Design. The influence of mechanical load on pleiotrophin (PTM) and aggrecan expression by intervertebral disc (IVD) cells, and the effects of disc cell conditioned medium on endothelial cell migration was investigated. Objective. To examine possible interactions of mechanical loads and known pro- and antiangiogenic factors, which may regulate disc angiogenesis during degeneration. Summary of Background Data. Pleiotrophin expression can be influenced by mechanical stimulation and has been associated with disc vascularization. Disc aggrecan inhibits endothelial cell migration, suggesting an antiangiogenic role. A possible interplay between these factors is unknown. Methods. The influence of the respective predominant load (cyclic strain for anulus fibrosus and hydrostatic pressure for nucleus pulposus cells) on PTN and aggrecan expression by IVD cells was determined by real-time RT-PCR and Western blotting (PTN only). The effects of IVD cell conditioned medium on endothelial cell migration were analyzed in a bioassay using human microvascular endothelial (HMEC-1) cells. Results. Application of both mechanical loads resulted in significant alterations of gene expression of PTN (+67%, P = 0.004 in anulus cells; +29%, P = 0.03 in nucleus cells) and aggrecan (+42%, P = 0.03 in anulus cells, −25%, P = 0.03 in nucleus cells). These effects depended on the cell type, the applied load, and timescale. Conditioned media of nucleus pulposus cells enhanced HMEC-1 migration, but this effect was diminished after 2.5 MPa hydrostatic pressure, when aggrecan expression was diminished, but not 0.25 MPa, when expression levels were unchanged. Conclusion. Mechanical loading influences PTN expression by human IVD cells. Conditioned media from nucleus pulposus cell cultures stimulated HMEC-1 endothelial cell migration. This study demonstrates that the influence of mechanical loads on vascularization of the human IVD is likely to be complex and does not correlate simply with altered expression of known pro- and antiangiogenic factors.

Inflammatory response of intervertebral disc cells is reduced by fibrin sealant scaffold in vitro

Intervertebral disc (IVD) degeneration is a complex process characterized by elevated concentrations of proinflammatory cytokines and proteolytic enzymes. Because of pro‐healing constituents, we hypothesized that fibrin sealant (FS) can reduce inflammation and augment soft tissue healing within the damaged or degenerative IVD. To test this, human and porcine nucleus pulposus (NP) and annulus fibrosus (AF) cells were extracted from tissues and encapsulated into alginate beads (NP cells) and type I collagen sponges (AF cells). Half of the alginate and collagen scaffolds were embedded in FS. To provoke inflammatory behaviours, the constructs were cultured with and without continuous IL‐1α (10 ng/ml) for 4, 7 and 14 days. ELISA was used to quantify the cellular synthesis (ng/µg DNA) of clinically relevant cytokines, proteolytic enzymes and growth factors. In NP cell constructs with IL‐1α, the syntheses of TNFα, IL‐1β, IL‐6, IL‐8 was elevated at all culture durations. In the presence of FS, secretion of several pro‐inflammatory cytokines were significantly reduced [IL‐6 and IL‐8 (porcine); and TNFα, IL‐1β, IL‐6, IL‐8 (human)]. Consistent with these reductions, human NP cultures exposed to FS and FS + IL‐1α synthesized significantly reduced amounts of MMP‐1 and −3 compared to constructs with IL‐1α. For porcine and human AF cells, there were no significant differences in the synthesis of the inflammatory or proteolytic cytokines relative to controls (without IL‐1α) at any culture duration. However, the porcine AF cells exposed to FS synthesized elevated amounts of the anti‐inflammatory cytokine IL‐4. The results suggest that FS may have beneficial effects for patients with degenerated intervertebral discs. Copyright

Influence of T1 Slope on the Cervical Sagittal Balance in Degenerative Cervical Spine: An Analysis Using Kinematic MRI.

Study Design. A retrospective kinematic magnetic resonance imaging (kMRI) study. Objective. To evaluate the utility of kMRI in determining the relationship between cervical sagittal balance and TI alignment. Summary of Background Data. Thoracic inlet parameters play an important role in cervical spine sagittal balance. However, most of the literature is based on lower resolution cervical X-rays or CT scans in the supine position. Methods. Cervical spine kMRI of 83 patients with degenerative cervical spine conditions (20–68 yr of age) was analyzed for: (1) cervical spine parameters: C2–C7 angle, C2–C7 sagittal vertical axis (SVA), cranial tilt, and cervical tilt; and (2) T1 parameters: thoracic inlet angle (TIA), T1 slope, and neck tilt (NT). Multiple logistic regression analysis and Pearson correlation coefficients were performed. Results. The mean TIA, T1 slope, and NT were 78.0, 33.2, and 44.8°, respectively. The mean C2–7 angle, SVA of C2–C7, cervical tilt, and cranial tilt were −15.4°, 22.0 mm, 18.1°, and 15.1°, respectively. The ratio of cervical:cranial tilt was maintained as 55:45%. A significant correlation was found between the C2–C7 angle and T1 slope (r = 0.731), TIA and C2–C7 angle (r = 0.406), cervical tilt with C2–C7 angle (r = 0.671), T1 slope with TIA (r = 0.429), TIA with neck tilt (r = 0.733), TIA with cervical tilt (r = 0.377), SVA C2–C7 with cervical tilt (r = −0.480), SVA C2–C7 with cranial tilt (r = 0.912), and C2–7 SVA with the ratio of cranial tilt to cervical tilt (r = 0.694). Conclusion. An individual with a large T1 slope required large cervical lordosis to preserve physiologic sagittal balance of the cervical spine. Cranial tilt was the cervical parameter most strongly correlated with SVA C2–C7, and thus may be a good parameter to assess decompensation of cervical sagittal balance. Level of Evidence: 3

Synthetic bone graft versus autograft or allograft for spinal fusion: a systematic review

The purpose of this review was to compare the efficacy and safety of synthetic bone graft substitutes versus autograft or allograft for the treatment of lumbar and cervical spinal degenerative diseases. Multiple major medical reference databases were searched for studies that evaluated spinal fusion using synthetic bone graft substitutes (either alone or with an autograft or allograft) compared with autograft and allograft. Randomized controlled trials (RCT) and cohort studies with more than 10 patients were included. Radiographic fusion, patient-reported outcomes, and functional outcomes were the primary outcomes of interest. The search yielded 214 citations with 27 studies that met the inclusion criteria. For the patients with lumbar spinal degenerative disease, data from 19 comparative studies were included: 3 RCTs, 12 prospective, and 4 retrospective studies. Hydroxyapatite (HA), HA+collagen, β-tricalcium phosphate (β-TCP), calcium sulfate, or polymethylmethacrylate (PMMA) were used. Overall, there were no differences between the treatment groups in terms of fusion, functional outcomes, or complications, except in 1 study that found higher rates of HA graft absorption. For the patients with cervical degenerative conditions, data from 8 comparative studies were included: 4 RCTs and 4 cohort studies (1 prospective and 3 retrospective studies). Synthetic grafts included HA, β-TCP/HA, PMMA, and biocompatible osteoconductive polymer (BOP). The PMMA and BOP grafts led to lower fusion rates, and PMMA, HA, and BOP had greater risks of graft fragmentation, settling, and instrumentation problems compared with iliac crest bone graft. The overall quality of evidence evaluating the potential use and superiority of the synthetic biological materials for lumbar and cervical fusion in this systematic review was low or insufficient, largely due to the high potential for bias and small sample sizes. Thus, definitive conclusions or recommendations regarding the use of these synthetic materials should be made cautiously and within the context of the limitations of the evidence.

Reoperation Rates Following Single-Level Lumbar Discectomy.

Study Design. Retrospective analysis of national insurance billing database. Objective. To examine trends in reoperation after single-level lumbar discectomy. Summary of Background Data. Lumbar discectomy is the most commonly performed procedure for treatment of radiculopathy caused by disc herniation. Randomized clinical trials have demonstrated the advantage of discectomy over nonsurgical treatment options, allowing for a more rapid reduction in symptoms. However, population-level data regarding reoperation after single level discectomy is limited. Methods. Data were collected using the commercially available PearlDiver software for patients billed with the Current Procedural Terminology code for our index procedure, hemilaminotomy and removal of disc material, between January 2007 and September 2014. The index group was then followed for up to 4 years for recurrent lumbar surgery, including spinal fusion, laminectomy, and additional discectomy. Results. Analysis of data obtained from 13,654 patient records revealed a rate of additional lumbar surgeries after single-level discectomy of 3.95% (539/13654) within 3 months and 12.2% (766/6274) within 4 years of the index procedure. Lumbar spinal fusion was performed on 5.9% (370/6274) of patients within 4 years. Patients who received a re-exploration discectomy within 2 years of the index procedure went on to receive lumbar fusion at a rate of 38.4% (48/125) within the 4 years after the re-exploration discectomy. The average additional cost of lumbar reoperation, as measured by insurance reimbursement, was approximately

C5 Palsy After Cervical Spine Surgery: A Multicenter Retrospective Review of 59 Cases.

11,161 per-patient per year. Conclusion. We report an overall 4-year reoperation rate of 12.2% after single-level discectomy. In addition, we report a rate of progression to lumbar fusion following re-exploration discectomy of 38.4% within 4 years of reoperation. Further studies are needed regarding the best treatment algorithm in patients with reherniation or iatrogenic instability after lumbar discectomy. This study should enhance the shared decision making process by providing surgeons and patients with valuable data regarding the frequency and nature of reoperations after discectomy. Level of Evidence: 3

Kinematic relationship between missed ligamentum flavum bulge and degenerative factors in the cervical spine

Study Design: A multicenter, retrospective review of C5 palsy after cervical spine surgery. Objective: Postoperative C5 palsy is a known complication of cervical decompressive spinal surgery. The goal of this study was to review the incidence, patient characteristics, and outcome of C5 palsy in patients undergoing cervical spine surgery. Methods: We conducted a multicenter, retrospective review of 13 946 patients across 21 centers who received cervical spine surgery (levels C2 to C7) between January 1, 2005, and December 31, 2011, inclusive. P values were calculated using 2-sample t test for continuous variables and χ2 tests or Fisher exact tests for categorical variables. Results: Of the 13 946 cases reviewed, 59 patients experienced a postoperative C5 palsy. The incidence rate across the 21 sites ranged from 0% to 2.5%. At most recent follow-up, 32 patients reported complete resolution of symptoms (54.2%), 15 had symptoms resolve with residual effects (25.4%), 10 patients did not recover (17.0%), and 2 were lost to follow-up (3.4%). Conclusion: C5 palsy occurred in all surgical approaches and across a variety of diagnoses. The majority of patients had full recovery or recovery with residual effects. This study represents the largest series of North American patients reviewed to date.

The Clinical Correlations between Diabetes, Cigarette Smoking and Obesity on Intervertebral Degenerative Disc Disease of the Lumbar Spine

BACKGROUND CONTEXT Bulging of ligamentum flavum can happen with the aging process and can lead to compression of the spinal cord and nerves. However, the distribution and the risk factors associated with a missed ligamentum flavum bulge (LFB) are unknown. PURPOSE The aim was to evaluate the distribution and risk factors associated with missed LFB in the cervical spine. STUDY DESIGN This was a retrospective analysis of kinematic magnetic resonance images (kMRI). PATIENT SAMPLE Patients diagnosed with symptomatic neck pain or radiculopathy between March 2011 and October 2012 were included. OUTCOME MEASURES The outcome measures were missed LFB and degenerative factors. METHODS A total of 200 patients (1,000 cervical segments) underwent upright kMRI in neutral, flexion, and extension postures. The LFB, sagittal cervical angles, disc herniation, disc degeneration, disc height, angular motion, translational motion, age, and gender were recorded. After excluding segments with LFB in neutral and flexion position, Pearson and Spearman correlation coefficients were used to evaluate the relation between the risk factors and missed LFB in the extension position. RESULTS The average depth of LFB was 0.24±0.71 mm at C2-C3, 1.02±1.42 mm at C3-C4, 1.65±1.48 mm at C4-C5, 2.13±1.37 mm at C5-C6, and 1.05±1.54 mm at C6-C7. The distribution of LFB was the most frequent at C5-C6 level (76.58%) followed by C4-C5 (63.06%). Disc herniation, disc degeneration, angular variation, and translational motion were significantly correlated with missed LFB at C4-C5 andC5-C6. Disc degeneration was the only factor significantly correlated with missed LFB at all cervical segments. CONCLUSIONS Occurrence and depth of missed LFB was the highest at C4-C5 and C5-C6 compared with other cervical levels. Disc degeneration, disc herniation, angular variation, and translational motion could play a role in the development of LFB at C4-C5 andC5-C6.