Local FK506 delivery induces osteogenesis in rat bone defect and rabbit spine fusion models.

Bone grafting procedures are commonly used for the repair, regeneration, and fusion of bones in a wide range of orthopaedic surgeries, including large bone defects and spine fusion procedures. Autografts are the clinical gold standard, though recombinant human bone morphogenetic proteins (rhBMPs) are often used, particularly in difficult clinical situations. However, treatment with rhBMPs can have off-target effects and increase surgical costs, adding to patients' already high economic and mental burden. Recent studies have identified that FDA-approved immunosuppressant drug, FK506 (Tacrolimus), can also activate the BMP pathway by binding to its inhibitors. This study tested the hypothesis that FK506, as a standalone treatment, could induce osteogenic differentiation of human mesenchymal stromal cells (hMSCs), as well as functional bone formation in a rat segmental bone defect model and rabbit spinal fusion model. FK506 enhanced osteogenic differentiation and mineralization of hMSCs in vitro. Standalone treatment with FK506 delivered on a collagen sponge produced consistent bone bridging of a critically sized rat femoral defect with functional mechanical properties comparable to naïve bone. In a rabbit single level posterolateral spine fusion model, treatment with FK506 delivered on a collagen sponge successfully fused the L5-L6 vertebrae at rates comparable to rhBMP-2 treatment. These data demonstrate the ability of FK506 to induce bone formation in human cells and two challenging in vivo models, and indicate FK506 can be utilized to treat a variety of spine disorders.

Local FK506 delivery induces osteogenesis in in vivo rat bone defect and rabbit spine fusion models.

Bone grafting procedures are commonly used for the repair, regeneration, and fusion of bones in in a wide range of orthopaedic surgeries, including large bone defects and spine fusion procedures. Autografts are the clinical gold standard, though recombinant human bone morphogenetic proteins (rhBMPs) are often used, particularly in difficult clinical situations. However, treatment with rhBMPs can have off-target effects and significantly increase surgical costs, adding to patients' already high economic and mental burden. Recent studies have identified that FDA-approved immunosuppressant drug, FK506 (Tacrolimus), can also activate the BMP pathway by binding to its inhibitors. This study tested the hypothesis that FK506, as a standalone treatment, could induce osteogenic differentiation of human mesenchymal stromal cells (hMSCs), as well as functional bone formation in a rat segmental bone defect model and rabbit spinal fusion model. FK506 potentiated the effect of low dose BMP-2 to enhance osteogenic differentiation and mineralization of hMSCs in vitro. Standalone treatment with FK506 delivered on a collagen sponge, produced consistent bone bridging of a rat critically-sized femoral defect with functional mechanical properties comparable to naïve bone. In a rabbit single level posterolateral spine fusion model, treatment with FK506 delivered on a collagen sponge successfully fused the L5-L6 vertebrae at rates comparable to rhBMP-2 treatment. These data demonstrate the ability of FK506 to induce bone formation in human cells and two challenging in vivo models, and indicate FK506 can be utilized either as a standalone treatment or in conjunction with rhBMP to treat a variety of spine disorders.

Comparative Complications Associated With BMP Use In Patients Undergoing ACDF for Degenerative Spinal Conditions: Systematic Review and Meta-Analysis.

STUDY DESIGN: Systematic Review and Meta-Analysis. OBJECTIVES: To compare complication incidence in patients with or without the use of recombinant human Bone Morphogenic Protein-2 (BMP2) undergoing anterior cervical discectomy and fusion (ACDF) for degenerative conditions. METHODS: A systematic search of eight online databases was conducted using PRISMA guidelines. Inclusion criteria included English language studies with a minimum of 10 adult patients undergoing instrumented ACDF surgery for a degenerative spinal condition in which BMP2 was used in all patients or one of the treatment arms. Studies with patients undergoing circumferential fusions, with non-degenerative indications, or which did not report post-operative complication data were excluded. Patients with and without BMP2 were compared in terms of the incidence of dysphagia/dysphonia, anterior soft tissue complications (hematoma, seroma, infection, dysphagia/dysphonia), nonunion, medical complications, and new neurologic deficits. RESULTS: Of 1832 preliminary search results, 27 manuscripts were included. Meta-analysis revealed the relative risk of dysphagia or dysphonia (RR = 1.39, CI 95% 1.18 - 1.64, P = <.001), anterior soft tissue complications (RR = 1.43, CI 95% 1.25-1.64, P = <.001), and medical complications (RR = 1.32, CI 95% 1.06-1.66, P = .013) were statistically significant in the BMP2 group while the relative risk of non-union (RR = .5, CI 95% .23 - 1.13, P = .09) trended lower in the BMP2 group. Neurological deficit (RR = 1.06, CI 95% .82-1.37, P = .66), and additional medical complications (RR = 1.53, CI 95% .98-2.38, P = .06) were not found to be statistically different between the groups. CONCLUSIONS: This meta-analysis identified a high rate of arthrodesis when BMP2 was used in ACDF, but confirmed increased rates of dysphagia and anterior soft tissue complications. Surgeons may consider reserving BMP2 implementation for cases with a high risk of non-union, and should be aware of the risk of airway compromise.

Sclerostin small-molecule inhibitors promote osteogenesis by activating canonical Wnt and BMP pathways.

Background: The clinical healing environment after a posterior spinal arthrodesis surgery is one of the most clinically challenging bone-healing environments across all orthopedic interventions due to the absence of a contained space and the need to form de novo bone. Our group has previously reported that sclerostin in expressed locally at high levels throughout a developing spinal fusion. However, the role of sclerostin in controlling bone fusion remains to be established. Methods: We computationally identified two FDA-approved drugs, as well as a single novel small-molecule drug, for their ability to disrupt the interaction between sclerostin and its receptor, LRP5/6. The drugs were tested in several in vitro biochemical assays using murine MC3T3 and MSCs, assessing their ability to (1) enhance canonical Wnt signaling, (2) promote the accumulation of the active (non-phosphorylated) form of ß-catenin, and (3) enhance the intensity and signaling duration of BMP signaling. These drugs were then tested subcutaneously in rats as standalone osteoinductive agents on plain collagen sponges. Finally, the top drug candidates (called VA1 and C07) were tested in a rabbit posterolateral spine fusion model for their ability to achieve a successful fusion at 6 wk. Results: We show that by controlling GSK3b phosphorylation our three small-molecule inhibitors (SMIs) simultaneously enhance canonical Wnt signaling and potentiate canonical BMP signaling intensity and duration. We also demonstrate that the SMIs produce dose-dependent ectopic mineralization in vivo in rats as well as significantly increase posterolateral spine fusion rates in rabbits in vivo, both as standalone osteogenic drugs and in combination with autologous iliac crest bone graft. Conclusions: Few if any osteogenic small molecules possess the osteoinductive potency of BMP itself - that is, the ability to form de novo ectopic bone as a standalone agent. Herein, we describe two such SMIs that have this unique ability and were shown to induce de novo bone in a stringent in vivo environment. These SMIs may have the potential to be used in novel, cost-effective bone graft substitutes for either achieving spinal fusion or in the healing of critical-sized fracture defects. Funding: This work was supported by a Veteran Affairs Career Development Award (IK2-BX003845).

Harmonization and standardization of nucleus pulposus cell extraction and culture methods.

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods: The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated. Results: Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture. Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60-100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide.

AAV6 as an effective gene delivery vector for prolonged transgene expression in intervertebral disc cells in vivo.

Intervertebral disc degeneration is the main contributor to low back pain, now the leading cause of disability worldwide. Gene transfer, either in a therapeutic attempt or in basic research to understand the mechanisms of disc degeneration, is a fascinating and promising tool to manipulate the complex physiology of the disc. Viral vectors based on the adeno-associated virus (AAV) have emerged as powerful transgene delivery vehicles yet a systematic investigation into their respective tropism, transduction efficiency, and relative toxicity have not yet been performed in the disc in vivo. Herein, we used in vivo bioluminescence imaging to systematically compare multiple AAV serotypes, injection volumes, titers, promoters, and luciferase reporters to determine which result in high transduction efficiency of murine nucleus pulposus (NP) cells in vivo. We find that AAV6 using a CAG promoter to drive transgene expression, delivered into the NP of murine caudal discs at a titer of 1011 GC/mL, provides excellent transduction efficiency/kinetics and low toxicity in vivo. We also show, for the first time, that the transduction of NP cells can be significantly boosted in vivo by the use of small cell permeabilization peptides. Finally, to our knowledge, we are the first to demonstrate the use of optical tissue clearing and three-dimensional lightsheet microscopy in the disc, which was used to visualize fine details of tissue and cell architecture in whole intact discs following AAV6 delivery. Taken together, these data will contribute to the success of using AAV-mediated gene delivery for basic and translational studies of the IVD.

Development of a standardized histopathology scoring system for intervertebral disc degeneration in rat models: An initiative of the ORS spine section.

BACKGROUND: Rats are a widely accepted preclinical model for evaluating intervertebral disc (IVD) degeneration and regeneration. IVD morphology is commonly assessed using histology, which forms the foundation for quantifying the state of IVD degeneration. IVD degeneration severity is evaluated using different grading systems that focus on distinct degenerative features. A standard grading system would facilitate more accurate comparison across laboratories and more robust comparisons of different models and interventions. AIMS: This study aimed to develop a histology grading system to quantify IVD degeneration for different rat models. MATERIALS & METHODS: This study involved a literature review, a survey of experts in the field, and a validation study using 25 slides that were scored by 15 graders from different international institutes to determine inter- and intra-rater reliability. RESULTS: A new IVD degeneration grading system was established and it consists of eight significant degenerative features, including nucleus pulposus (NP) shape, NP area, NP cell number, NP cell morphology, annulus fibrosus (AF) lamellar organization, AF tears/fissures/disruptions, NP-AF border appearance, as well as endplate disruptions/microfractures and osteophyte/ossification. The validation study indicated this system was easily adopted, and able to discern different severities of degenerative changes from different rat IVD degeneration models with high reproducibility for both experienced and inexperienced graders. In addition, a widely-accepted protocol for histological preparation of rat IVD samples based on the survey findings include paraffin embedding, sagittal orientation, section thickness < 10 µm, and staining using H&E and/or SO/FG to facilitate comparison across laboratories. CONCLUSION: The proposed histological preparation protocol and grading system provide a platform for more precise comparisons and more robust evaluation of rat IVD degeneration models and interventions across laboratories.

The temporal and spatial expression of sclerostin and Wnt signaling factors during the maturation of posterolateral lumbar spine fusions.

The bone healing environment in the posterolateral spine following arthrodesis surgery is one of the most challenging in all of orthopedics and our understanding of the molecular signaling pathways mediating osteogenesis during spinal fusion is limited. In this study, the spatial and temporal expression pattern of Wnt signaling factors and inhibitors during spinal fusion was assessed for the first time. Bilateral posterolateral spine arthrodesis with autologous iliac crest bone graft was performed on 21 New Zealand White rabbits. At 1-, 2-, 3-, 4-, and 6-weeks, the expression of sclerostin and a variety of canonical and noncanonical Wnts signaling factors was measured by qRT-PCR from tissue separately collected from the transverse processes, the Outer and Inner Zones of the fusion mass, and the adjancent paraspinal muscle. Immunohistochemistry for sclerostin protein was also performed. Sclerostin and many Wnt factors, especially Wnt3a and Wnt5a, were found to have distinct spatial and temporal expression patterns. For example, harvesting ICBG caused a significant increase in sclerostin expression. Furthermore, the paraspinal muscle immediately adjacent to the transplanted ICBG also had significant increases in sclerostin expression at 3 weeks, suggesting new potential mechanisms for pseudarthroses following spinal arthrodesis. The presented work is the first description of the spatial and temporal expression of sclerostin and Wnt signaling factors in the developing spine fusion, filling an important knowledge gap in the basic biology of spinal fusion and potentially aiding in the development of novel biologics to increase spinal fusion rates.

Correction: Sangadala, S. et al. FK506 Induces Ligand-Independent Activation of the Bone Morphogenetic Protein Pathway and Osteogenesis. Int. J. Mol. Sci. 2019, 20, 1900.

The authors wish to make the following corrections to this paper [...].

Single-cell RNA-seq identifies unique transcriptional landscapes of human nucleus pulposus and annulus fibrosus cells.

Intervertebral disc (IVD) disease (IDD) is a complex, multifactorial disease. While various aspects of IDD progression have been reported, the underlying molecular pathways and transcriptional networks that govern the maintenance of healthy nucleus pulposus (NP) and annulus fibrosus (AF) have not been fully elucidated. We defined the transcriptome map of healthy human IVD by performing single-cell RNA-sequencing (scRNA-seq) in primary AF and NP cells isolated from non-degenerated lumbar disc. Our systematic and comprehensive analyses revealed distinct genetic architecture of human NP and AF compartments and identified 2,196 differentially expressed genes. Gene enrichment analysis showed that SFRP1, BIRC5, CYTL1, ESM1 and CCNB2 genes were highly expressed in the AF cells; whereas, COL2A1, DSC3, COL9A3, COL11A1, and ANGPTL7 were mostly expressed in the NP cells. Further, functional annotation clustering analysis revealed the enrichment of receptor signaling pathways genes in AF cells, while NP cells showed high expression of genes related to the protein synthesis machinery. Subsequent interaction network analysis revealed a structured network of extracellular matrix genes in NP compartments. Our regulatory network analysis identified FOXM1 and KDM4E as signature transcription factor of AF and NP respectively, which might be involved in the regulation of core genes of AF and NP transcriptome.

Network Analysis Identifies Gene Regulatory Network Indicating the Role of RUNX1 in Human Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration (IDD) is a multifactorial physiological process which is often associated with lower back pain. Previous studies have identified some molecular markers associated with disc degeneration, which despite their significant contributions, have provided limited insight into the etiology of IDD. In this study, we utilized a network medicine approach to uncover potential molecular mediators of IDD. Our systematic analyses of IDD associated with 284 genes included functional annotation clustering, interaction networks, network cluster analysis and Transcription factors (TFs)-target gene network analysis. The functional enrichment and protein-protein interaction network analysis highlighted the role of inflammatory genes and cytokine/chemokine signaling in IDD. Moreover, sub-network analysis identified significant clusters possessing organized networks of 24 cytokine and chemokine genes, which may be considered as key modulators for IDD. The expression of these genes was validated in independent microarray datasets. In addition, the regulatory network analysis identified the role of multiple transcription factors, with RUNX1 being a master regulator in the pathogenesis of IDD. Our analyses highlighted the role of cytokine genes and interacting pathways in IDD and further improved our understanding of the genetic mechanisms underlying IDD.

Rigid-Plating and Cortico-Cancellous Allograft Are Effective for 3-Level Anterior Cervical Discectomy and Fusion: Radiographic and Clinical Outcomes.

OBJECTIVE: To determine the risk factors associated with radiographic changes and clinical outcomes following 3-level anterior cervical discectomy and fusion (ACDF) using rigidplate constructs and cortico-cancellous allograft. ACDF has demonstrated efficacy for treatment of multilevel degenerative cervical conditions, but current data exists in small heterogeneous forms. METHODS: A retrospective review included 98 patients with primary 3-level ACDF surgery at one institution from 2008 to 2013 with minimum 1-year follow-up. Cervical sagittal vertical axis (SVA), segmental height, fusion, and lordosis radiographs were measured preoperatively and at 2 postoperative periods. RESULTS: Rates of asymptomatic pseudarthroses and total reoperations were 18% and 4%, respectively. Results demonstrated immediate improvements in cervical lordosis (5.5°, p < 0.01) and segmental height (5.0-mm increase, p < 0.01) with little changes in the cervical SVA (3.2-mm increase, p < 0.01). The segmental height decreased from immediate postoperative period to final follow-up (1.7-mm decrease, p < 0.01). Older age was protective against radiolucent lines (p < 0.05). Patient-reported outcomes significantly improved following surgery (p < 0.01). Current smoking status and diagnosis of diabetes mellitus had no impact on radiographic or clinical outcomes. Risk factors were not identified for the 5 reoperations (4%). CONCLUSION: Three-level ACDF with rigid-plating and cortico-cancellous allograft is an effective procedure for degenerative diseases of the cervical spine without the application of additional adjuncts or combined anteriorposterior cervical surgeries. Significant improvements in cervical lordosis, segmental height, and segmental alignment can be achieved with little change in cervical SVA and a low rate of reoperations over short-term follow-up. Similarly, patient-reported outcomes show significant improvements.

Derivation of notochordal cells from human embryonic stem cells reveals unique regulatory networks by single cell-transcriptomics.

Intervertebral disc degeneration (IDD) is a public health dilemma as it is associated with low back and neck pain, a frequent reason for patients to visit the physician. During IDD, nucleus pulposus (NP), the central compartment of intervertebral disc (IVD) undergo degeneration. Stem cells have been adopted as a promising biological source to regenerate the IVD and restore its function. Here, we describe a simple, two-step differentiation strategy using a cocktail of four factors (LDN, AGN, FGF, and CHIR) for efficient derivation of notochordal cells from human embryonic stem cells (hESCs). We employed a CRISPR/Cas9 based genome-editing approach to knock-in the mCherry reporter vector upstream of the 3' untranslated region of the Noto gene in H9-hESCs and monitored notochordal cell differentiation. Our data show that treatment of H9-hESCs with the above-mentioned four factors for 6 days successfully resulted in notochordal cells. These cells were characterized by morphology, immunostaining, and gene and protein expression analyses for established notochordal cell markers including FoxA2, SHH, and Brachyury. Additionally, pan-genomic high-throughput single cell RNA-sequencing revealed an efficient and robust notochordal differentiation. We further identified a key regulatory network consisting of eight candidate genes encoding transcription factors including PAX6, GDF3, FOXD3, TDGF1, and SOX5, which are considered as potential drivers of notochordal differentiation. This is the first single cell transcriptomic analysis of notochordal cells derived from hESCs. The ability to efficiently obtain notochordal cells from pluripotent stem cells provides an additional tool to develop new cell-based therapies for the treatment of IDD.

FK506 Induces Ligand-Independent Activation of the Bone Morphogenetic Protein Pathway and Osteogenesis.

Osteoinductive bone morphogenetic proteins (BMPs), including BMP-2, have a unique capability of mediating bone formation both in orthotopic and ectopic locations. Immunosuppresive macrolides have been shown to potentiate BMP-2 activity through FKBP12, but these have yet to translate to effective osteoinductive therapies. Herein, we show the osteogenic activity of FK506 as a stand-alone agent in direct comparison to BMP-2 both in vitro and in vivo. FK506 was capable of producing stand-alone alkaline phosphatase induction in C2C12 cells comparable to that seen with rhBMP-2. FK506 treatment activated the BMP receptor, as shown by increased pSmad1/5 levels, and produced significantly higher mRNA levels of the early response genes in BMP and TGF-ß pathways. Additionally, the FK506 induction of alkaline phosphatase was shown to be resistant to Noggin treatment. In vivo osteogenic activity of FK506 was tested by local delivery on a collagen sponge in an ectopic subcutaneous implantation model in the rat. Dose responses of FK506 showed increasing levels of ectopic mineralization comparable to the mineral volume produced by BMP-2 delivery. These findings suggest that the use of FK506 can enhance osteoblastic differentiation in vitro and can induce mineralization when delivered locally in vivo.

Sagittal spinopelvic malalignment in degenerative scoliosis patients: isolated correction of symptomatic levels and clinical decision-making.

BACKGROUND: This study aims to determine if (1) loss of lumbar lordosis (LL), often associated with degenerative scoliosis (DS), is structural or rather largely due to positional factors secondary to spinal stenosis; (2) only addressing the symptomatic levels with a decompression and posterolateral fusion in carefully selected patients will result in improvement of sagittal malalignment; and (3) degree of sagittal plane correction achieved with such a local fusion could be predicted by routine pre-operative imaging. METHODS: A retrospective study design with prospectively collected imaging data of a consecutive series of surgically treated DS patients who underwent decompression and instrumented fusion at only symptomatic levels was performed. Pre- and post-operative plain radiographs and pre-operative magnetic resonance imaging (MRIs) of the spinopelvic region were analyzed. LL, pelvic incidence (PI), pelvic tilt (PT), and sacral slope (SS) were assessed in all patients. As a requirement for the surgical strategy, all patients presented with a pre-operative PI-LL mismatch greater than 10°. Post-operative complications were assessed. RESULTS: Pre-operative MRIs and lumbar extension radiographs revealed a mean LL of 42° (range 10-66°) and 48° (range 20-74°), respectively, in 68 patients (mean follow-up 29 months). LL post-operatively was corrected to a mean PI-LL of 10°. Of patients who achieved PI-LL mismatch within 10o on their pre-operative extension lateral lumbar radiographs, 62.5% were able to maintain a PI-LL mismatch within 10° on their initial post-operative films. Only 37.5% were not able to achieve that mismatch on extension radiographs (p = 0.001, OR = 9.58). Similarly, 54.2% were able to achieve a PI-LL < 10° on initial post-operative radiographs, when pre-operative MRI revealed a PI-LL mismatch within 10°. In contrast, only 20.5% achieved that goal post-operatively if their mismatch was greater than 10o on their MRI (p = 0.003, OR = 4.25). CONCLUSION: With a decompression and instrumented fusion of only the symptomatic levels in symptomatic DS patients, we were able to achieve a PI-LL mismatch to within 10°. The loss of LL observed pre-operatively may be largely positional rather than structural. The amount of LL correction observed immediately after surgery can be predicted from pre-operative lumbar extension radiographs and supine sagittal MRI.

There is no increased risk of adjacent segment disease at the cervicothoracic junction following an anterior cervical discectomy and fusion to C7.

BACKGROUND CONTEXT: Anterior cervical discectomy and fusion (ACDF) is a very common operative intervention for the treatment of cervical spine degenerative disease in those who have failed non-operative measures. However, studies examining long-term follow-up on patients who underwent ACDF reveal evidence of radiographic and clinical degenerative disc disease at the levels adjacent to the fusion construct. Consistent with other junctional regions of the spine, the cervicothoracic junction (CTJ) has significant morphologic variations. As a result, the CTJ undergoes significant static and dynamic stress. Given these findings, there has been some thought that ACDF down to C7 may experience additional risks for adjacent segment degeneration/disease (ASD) when compared with ASDFs that are cephalad to C7. PURPOSE: The goal of this study is to evaluate the rate of radiographic and clinical ASD in patients who have undergone single- or multilevel ACDF, down to C7. STUDY DESIGN: This is a retrospective cohort study. PATIENT SAMPLE: The sample included consecutive patients from a single orthopedic surgeon at one quaternary referral medical center who underwent an ACDF between January 2008 and November 2014. Indications for surgery included radiculopathy, myelopathy, or myeloradiculopathy in the setting of failed conservative treatments. Patients were excluded if they had an ACDF of which the caudal level was cephalad to C7 or if they had undergone a previous cervical fusion. OUTCOME MEASURES: Radiographic diagnosis of ASD was determined by the presence of disc space narrowing >50%, new or enlarged osteophytes, end plate sclerosis, or increased calcification of the anterior longitudinal ligament (ALL). Postoperatively, data were collected on the presence of new radicular or myelopathic symptoms indicative of pathology at C7-T1, indicating a diagnosis of clinical ASD. METHODS: Demographic information was collected for all patients, which included age, sex, body mass index, smoking status, and Charleston Comorbidity Index (CCI). Several radiographic parameters were measured preoperatively, immediately postoperatively, and at the last follow-up: C2-C7 lordosis, sagittal vertical axis (SVA), thoracic inlet angle (TIA), and T1 slope C2-C7 lordosis were measured using the Cobb angle between the inferior end plate of C2 to the inferior end plate of C7. Radiographic and clinical factors associated with ASD were analyzed postoperatively. RESULTS: Four patients (4.8%) presented with clinical evidence of ASD, all of whom also showed signs of radiographic ASD and improved with conservative measures. No patients underwent reoperation for ASD at the C7-T1 junction. Thirty patients (36.1%) presented radiographic evidence of ASD. These were generally older (54.4 vs. 48.4 years; p=.014). There were neither significant differences in radiographic parameters nor between single- versus multilevel ACDFs and the development of ASD. CONCLUSIONS: The cervicothoracic junction may present with vulnerability to ASD given the junctional biomechanics. However, this study provides evidence that an ACDF with the caudal level of C7 does not incur additional risk of ASD, showing similar outcomes to ACDFs at other levels.

PDGF-BB inhibits intervertebral disc cell apoptosis in vitro.

Degeneration of the intervertebral disc (IVD) results in deterioration of the spinal motion segment and can lead to debilitating back pain. Given the established mitotic and anti-apoptotic effects of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) in a variety of cell types we postulated that rhPDGF-BB might delay disc cell degeneration through inhibition of apoptosis. To address this hypothesis, we treated human IVD cells isolated from five independent patients with rhPDGF-BB in monolayer and 3D pellet cultures. The anti-apoptotic potential, cell proliferative capacity, morphology/pellet differentiation, and gene expression of PDGF-treated IVD cells were evaluated via flow cytometry/immunohistochemistry, MTT assays, histology, and quantitative RT-PCR, respectively. We found that rhPDGF-BB treatment significantly inhibited cell apoptosis, increased cell proliferation and matrix production, and maintained mRNA expression of critical extracellular matrix genes. This study suggests two possible mechanisms for the anti-degenerative effects of rhPDGF-BB on human IVD cells. First, PDGF treatment strongly inhibited IVD cell apoptosis in 3D cultures. Second, rhPDGF-BB acts as an anabolic agent, promoting maintenance of IVD cell phenotype in 3D culture, based on the molecular and protein expression analysis. We speculate that rhPDGF-BB may be used as a biologic treatment to target early degenerative IVD disease in the future.

Management decisions for adolescent idiopathic scoliosis significantly affect patient radiation exposure.

BACKGROUND CONTEXT: Adolescent idiopathic scoliosis (AIS) patients treated before the 1990s have a 1% to 2% increased lifetime risk of developing breast and thyroid cancer as a result of ionizing radiation from plain radiographs. Although present plain radiographic techniques have been able to reduce some of the radiation exposure, modern treatment algorithms for scoliosis often include computed tomography (CT) and intraoperative fluoroscopy. The exact magnitude of exposure to ionizing radiation in adolescents during modern scoliosis treatment is therefore unclear. PURPOSE: To determine the difference in radiation exposures in patients undergoing various forms of treatment for AIS. STUDY DESIGN: Retrospective cohort. PATIENT SAMPLE: Patients aged 9 to 18 years with a diagnosis of AIS, followed and/or treated with nonoperative or operative management for a minimum of 2 years. OUTCOME MEASURES: Number of radiographs and total radiation exposure calculated. METHODS: The charts and radiographs of patients managed for AIS at a single institution between September 2007 and January 2012 were reviewed. Patients were divided into three groups: operative group, braced group, and observation group. Patient demographics, Cobb angles, and curve types were recorded. The number of radiographs per year that each patient received and the total radiation dose were recorded. The plain radiographic radiation exposure was then combined with the direct exposure recording from ancillary tests, such as fluoroscopy and CT, and a radiation exposure rate was calculated (mrad/y). A single-factor analysis of variance (α=0.01) with a Tukey honest significant difference post hoc analysis was used to test significance between groups. RESULTS: Two hundred sixty-seven patients were evaluated: 86 operative, 80 brace, and 101 observation. All groups had similar demographics and curve type distribution. The mean initial Cobb angle at presentation was significantly different between the groups: operative (57°±11°), brace (24°±7.9°), and observation (18°±9.4°) (p<.01). There was a significant difference among the groups in terms of the mean number of radiographs received per year; operative group, 12.2 (95% confidence interval [CI]: 10.8-13.5; p<.001); braced group, 5.7 (95% CI: 5.2-6.2; p<.001), and observed group, 3.5 (95% CI: 3.160-3.864; p<.001). The operative group received 1,400 mrad per year (95% CI: 1,350-1,844; p<.001), braced group received 700 mrad per year (95% CI: 598-716; p<.001), and observed group received 400 mrad per year (95% CI: 363-444; p<.001). Importantly, 78% of radiation in the operative group was attributable to the operative fluoroscopy exposure. CONCLUSIONS: Significant differences exist in the total radiation exposure in scoliosis patients with different treatment regimens, with operative patients receiving approximately 8 to 14 times more radiation than braced patients or those undergoing observation alone, respectively. Operative patients also receive more than twice the radiation per year than braced or observed patients. Almost 78% of the annual radiation exposure for operative patients occurs intraoperatively. Because children are notably more sensitive to the carcinogenic effects of ionizing radiation, judicious use of present imaging methods and a search for newer imaging methods with limited ionizing radiation should be undertaken.

Mean subaxial space available for the cord index as a novel method of measuring cervical spine geometry to predict the chronic stinger syndrome in American football players.

OBJECT: The chronic stinger syndrome is a distinct entity from acute stingers and has been shown to have its own pathophysiology that, unlike acute stingers, may reflect long-standing geometrical changes of the subaxial spinal canal and chronic irritation/degeneration of the exiting nerve root complex. There is no method available, however, to accurately predict these symptoms in athletes. The mean subaxial cervical space available for the cord (MSCSAC) is a novel alternative to the Torg ratio for predicting neurological symptoms caused by cervical spondylosis in elite athletes. It is the goal of this study to determine critical values for this measurement index and to retrospectively correlate those values to neurological symptoms. METHODS: Magnetic resonance images obtained in 103 male athletes participating in the 2005 and 2006 National Football League Scouting Combine and a control group of 42 age-matched male nonathletes were retrospectively reviewed. The Torg ratio and SAC values were calculated in triplicate at each cervical level from C3-6 by using lateral radiographs and midsagittal T2-weighted MR images of the cervical spine, respectively. These values were then averaged for each individual to produce mean subaxial cervical Torg ratio (MSCTR) and MSCSAC values. Receiver operating characteristic curves were constructed for each measurement technique and were compared based on their respective area under the curves (AUCs). RESULTS: The MSCSAC difference between athletes with and without chronic stingers was statistically significant (p < 0.01). The difference between athletes with and without chronic stingers compared with controls was also statistically significant (p < 0.001 and p < 0.001, respectively). The AUC for the MSCSAC was 0.813, which was significantly greater than the AUC for both the MSCTR (p = 0.0475) and the individual Torg ratio (p = 0.0277). The MSCTR had the second largest AUC (0.676) and the conventional method of measuring individual Torg ratio values produced the lowest AUC (0.661). It was found that using the MSCSAC with a critical value of 5.0 mm produced a sensitivity of 80% and a negative likelihood ratio of 0.23 for predicting chronic stingers. Lowering the cutoff value to 4.3 mm for the MSCSAC resulted in a possible confirmatory test with a specificity of 96% and a positive likelihood ratio of 13.25. CONCLUSIONS: A critical value of 5.0 mm for the MSCSAC provides the clinician with a screening test for chronic stingers and anything < 4.3 mm adds additional confidence as a confirmatory test. These results are approximately 20% more accurate than the classic Torg ratio based on our AUC analysis. It was found that measuring the spinal geometry throughout the length of the subaxial cervical spine produced a more reliable method by which to predict neurological symptoms than the traditional approach of measuring individual levels. This shows that the underlying pathogenesis of the chronic stinger syndrome is best characterized as a process that involves the entire subaxial region uniformly.