Edward J. Vresilovic

Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro: implications for cell-based transplantation therapy.

Objective. Because mesenchymal stem cells can differentiate into chondrocyte-like cells, we ask the question, can mesenchymal stem cells commit to the nucleus pulposus phenotype? Background. Back pain, a significant source of morbidity in our society, is linked to degenerative changes of the intervertebral disc. Absence of suitable graft tissue limits therapeutic approaches for repair of disc tissue. For this reason, there is considerable interest in developing “tissue engineering” strategies for the regeneration of the nucleus pulposus. Methods. Rat mesenchymal stem cells were immobilized in 3-dimensional alginate hydrogels and cultured in a medium containing transforming growth factor-&bgr;1 under hypoxia (2% O2) and normoxia (20% O2). Mesenchymal stem cells were examined by confocal microscopy to evaluate their viability and metabolic status after labeling with Celltracker green, a thiol sensitive dye, and Mitotracker red, a dye sensitive to the mitochondrial membrane potential. Flow cytometry, semiquantitative reverse transcription polymerase chain reaction and Western blot analysis were carried out to evaluate phenotypic and biosynthetic activities and the signaling pathways involved in the differentiation process. Results. Under hypoxic conditions, mesenchymal stem cells formed large aggregates and exhibited positive Celltracker and Mitotracker signals. Glucose transporter-3, matrix metalloproteinase-2, collagen type II and type XI, and aggrecan mRNA and protein expression was upregulated, whereas there was no change in the levels of decorin, biglycan, fibromodulin, and lumican. Hypoxia maintained the expression of CD44 (hyaluronan receptor), ALCAM (CD166), and endoglin (transforming growth factor-&bgr; receptor). Likewise, expression of &bgr;3 and &agr;2 integrin was upregulated. Transforming growth factor-&bgr; treatment increased MAPK activity and Sox-9, aggrecan, and collagen type II gene expression. Basal levels of the phosphorylated MAPK isoform ERK1/2, but not p38, were higher under hypoxic conditions than normoxia, and its activation was further augmented by treatment of cells with transforming growth factor-&bgr;. In hypoxia, transforming growth factor-&bgr; sustained phosphorylated p38 expression for an extended time period. Pharmacological inhibition of ERK1/2 and p38 enzymatic activity resulted in a decrease in Sox-9, aggrecan, and collagen type II mRNA levels. Conclusions. Our results indicate that hypoxia and transforming growth factor-&bgr; drive mesenchymal stem cell differentiation towards a phenotype consistent with that of the nucleus pulposus. Measurement of selected signaling molecules and response to specific inhibitors suggest involvement of MAPK signaling pathways. It is concluded that mesenchymal stem cells could be used to repopulate the damaged or degenerate intervertebral disc.

Sacroiliac joint pain referral zones

OBJECTIVE To determine the patterns of pain referral from the sacroiliac joint. STUDY DESIGN Retrospective. PARTICIPANTS/METHODS Fifty consecutive patients who satisfied clinical criteria and demonstrated a positive diagnostic response to a fluoroscopically guided sacroiliac joint injection were included. Each patients preinjection pain description was used to determine areas of pain referral, and 18 potential pain-referral zones were established. OUTCOME MEASURES Observed areas of pain referral. RESULTS Eighteen men (36.0%) and 32 women (64.0%) were included with a mean age of 42.5 years (range, 20 to 75 yrs) and a mean symptom duration of 18.2 months (range, 1 to 72 mo). Forty-seven patients (94.0%) described buttock pain, and 36 patients (72.0%) described lower lumbar pain. Groin pain was described in 7 patients (14.0%). Twenty-five patients (50.0%) described associated lower-extremity pain. Fourteen patients (28.0%) described leg pain distal to the knee, and 6 patients (14.0%) reported foot pain. Eighteen patterns of pain referral were observed. A statistically significant relationship was identified between pain location and age, with younger patients more likely to describe pain distal to the knee. CONCLUSIONS Pain referral from the sacroiliac joint does not appear to be limited to the lumbar region and buttock. The variable patterns of pain referral observed may arise for several reasons, including the joints complex innervation, sclerotomal pain referral, irritation of adjacent structures, and varying locations of injury with the sacroiliac joint.

The predictive value of provocative sacroiliac joint stress maneuvers in the diagnosis of sacroiliac joint syndrome

OBJECTIVE To determine the clinical validity of provocative sacroiliac joint (SIJ) maneuvers in making the diagnosis of sacroiliac joint syndrome (SIJS). DESIGN Prospective constructive cohort study using sacroiliac joint block (SIJB) as the diagnostic gold standard. SETTING Tertiary care center. PATIENTS Consecutive patients describing low back pain including the region of the sacral sulcus. Physical examination revealed a positive response to three provocative SIJ maneuvers, two of which had to be Patricks test and pain with palpation over the ipsilateral sacral sulcus. INTERVENTIONS All subjects underwent fluoroscopically guided SIJB. MAIN OUTCOME MEASURES Response to SIJB was assessed with visual analog scale (VAS) ratings before and after the block. A reduction of the VAS rating by at least 80% was considered a positive response to SIJB. RESULTS Fifty consecutive patients met our criteria and underwent SIJB. Thirty patients had positive response to SIJB, making up the positive SIJS group. Twenty patients had less than 80% pain reduction with SIJB and thus comprised the negative SIJS group. The positive predictive value of provocative SIJ maneuvers in determining the presence of SIJS is therefore 60%. CONCLUSIONS Our results do not support the use of provocative SIJ maneuvers to confirm a diagnosis of SIJS. Rather, these physical examination techniques can, at best, enter SIJS into the differential diagnosis.

Comparison of animals used in disc research to human lumbar disc geometry.

Study Design. Measurement and normalization of disc geometry parameters for several animal models used in disc research. Objectives. To compare normalized values of disc geometry to the human disc geometry to aid in the selection and interpretation of animal model studies. Summary of Background Data. Animal models are widely used to study intervertebral disc degeneration and to evaluate disc treatment methods because of the availability of the tissue, the decreased variability between subjects compared with humans, and the feasibility to perform in vivo experiments. There is a general lack of comparative data with respect to the human disc analog for animal models. Methods. The disc height, lateral width, AP width, area, and the nucleus pulposus lateral width, AP width, area, and centroid offset were all measured and normalized by 2 scaling factors, lateral width and disc area, for comparison to human. Results. The species studied were ranked according to the average percent deviation of the normalized disc height, AP width and nucleus pulposus area from human geometry as: mouse lumbar (12%), rat lumbar (15%), mouse tail (18%), baboon (19%), bovine tail (22%), rabbit (26%), sheep (31%), and rat tail (46%). Conclusions. This paper provides a reference to compare disc geometries of experimental animal models to the human lumbar disc, to aid both in interpretation of and in planning for experimental disc research, and to provide normalized disc geometry parameters for computational models.

Therapeutic selective nerve root block in the nonsurgical treatment of atraumatic cervical spondylotic radicular pain: A retrospective analysis with independent clinical review

OBJECTIVE To investigate the outcomes resulting from the use of fluoroscopically guided therapeutic selective nerve root block (SNRB) in the nonsurgical treatment of atraumatic cervical spondylotic radicular pain. STUDY DESIGN Retrospective study with independent clinical review. PARTICIPANTS Twenty subjects (10 men, 10 women) with mean age 56.6 years. METHODS Each patient met specific physical examination, radiographic, and electrodiagnostic criteria to confirm a level of cervical involvement. Those patients whose root level remained indeterminate were required to demonstrate a positive response to a fluoroscopically guided diagnostic SNRB prior to the initiation of treatment. Therapeutic injections were administered in conjunction with physical therapy. Data collection and analysis were performed by an independent clinical reviewer. MAIN OUTCOME MEASURES Pain score, work status, medication usage, and patient satisfaction. RESULTS Twenty patients with an average symptom duration of 5.8 months were included. An average of 2.2 therapeutic injections was administered. Follow-up data collection transpired at an average of 21.2 months following discharge from treatment. A significant reduction (p = .001) in pain score was observed at the time of follow-up. Medication usage was also significantly improved (p = .005) at the time of follow-up. An overall good or excellent result was observed in 60%. Thirty percent of patients required surgery. Younger patients were more likely (p = .0047) to report the highest patient satisfaction rating following treatment. CONCLUSIONS This study suggests that fluoroscopically guided therapeutic SNRB is a clinically effective intervention in the treatment of atraumatic cervical spondylotic radicular pain.

Comparison of animal discs used in disc research to human lumbar disc: axial compression mechanics and glycosaminoglycan content.

Study Design. Experimental measurement and normalization of in vitro disc axial compression mechanics and glycosaminoglycan and water content for several animal species used in intervertebral disc research. Objective. To compare normalized axial mechanical properties and glycosaminoglycan and water content from other species to those of the human disc to aid in selection and interpretation of results in animal disc studies. Summary of Background Data. There is a lack of mechanical and biochemical comparative data from animal intervertebral discs with respect to the human disc. Methods. Intervertebral disc axial mechanical properties, glycosaminoglycan, and water content were evaluated for 9 disc types in 7 mammalian species: the calf, pig, baboon, sheep, rabbit, rat and mouse lumbar, and the cow and rat tail. Disc area and height were used for calculation of the normalized mechanical parameters. Glycosaminoglycan content was normalized by dry weight. Results. Many directly measured mechanical parameters varied by orders of magnitude. However, these parameters became comparable and often did not show significant differences after geometric normalization. Both glycosaminoglycan and water content revealed similarity across species. Conclusion. Disc axial mechanics are very similar across animal species when normalizing by the geometric parameters of disc height and area. This suggests that the disc tissue material properties are largely conserved across animal species. These results provide a reference to compare disc axial mechanics and glycosaminoglycan and water composition of experimental animal models to the human lumbar disc, to aid in both selection and interpretation of experimental disc research.

The Effect of Relative Needle Diameter in Puncture and Sham Injection Animal Models of Degeneration

Study Design. Biomechanical study and literature review. Objectives. To quantify the acute effect of needle diameter on the in vitro mechanical properties of cadaver lumbar discs in the rat and sheep. To review published in vivo animal studies and evaluate disc changes with respect to the relative needle size. Summary of Background Data. There are many cases where a disc needle puncture or injection is applied to animal models: puncture injuries to induce degeneration, chemonucleolysis to induce degeneration, and delivery of disc therapies. It is not clear what role the size of the needle may have in the outcome. Methods. Mechanics were measured after sham phosphate buffered saline injection with a 27 G or 33 G needle in the rat and with a 27 G needle in the sheep. A literature review was performed to evaluate studies in which animal discs were treated with a needle puncture or a sham injection. For each study, the ratio of the needle diameter to disc height (needle:height) was calculated. Results. When the rat was injected with a 27 G needle (52% of disc height), the compression, tension, and neutral zone stiffnesses were 20% to 60% below preinjected values and the neutral zone length was 130% higher; when injected with a 33 G needle (26% of disc height), the only affected property was the neutral zone length, which was only 20% greater. When the sheep was injected with a 27 G needle (10% of disc height), none of the axial properties were different from intact, the torsion stiffness was not different, and the torque range was 15% smaller. Twenty-three in vivo studies in the rat, rabbit, dog, or sheep were reviewed. The disc changes depended on the ratio of needle diameter to disc height as follows: significant changes were not observed for needle:height less than 40%, although between 25% and 40% results were variable and some minor nonsignificant effects were observed, disc changes were universal for needle:height over 40%. Conclusion. A needle puncture may directly alter mechanical properties via nucleus pulposus depressurization and/or anulus fibrosus damage, depending on the relative needle size. As more basic science research is aimed at treating disc degeneration via injection of therapeutic factors, these findings provide guidance in design of animal studies. Such studies should consider the relative needle size and include sham control groups to account for the potential effects of the needle injection.

Phenotypic characteristics of the nucleus pulposus: expression of hypoxia inducing factor-1, glucose transporter-1 and MMP-2

Abstract. Attempts to study the biology of the nucleus pulposus have been limited in scope due to the low rates of cell proliferation, difficulties in maintaining viable disc cells in culture and the absence of a clearly defined phenotype. The major objective of this communication is to construct a phenotypic signature for cells of the nucleus pulposus that is based on the hypothesis that in response to restriction on oxygen and nutrient flux, there is expression of HIF-1, GLUT-1 and MMP-2. Nucleus pulposus, as well as annulus fibrosus and cartilage of the vertebral end plates, was collected from rat spinal units. Western blot analysis and immunohistochemistry clearly showed that there was a significant level of expression of the HIF-1β isoform in the nucleus pulposus; HIF-1β was present at lower levels in cells of the annulus and the end plate. In contrast to HIF-1β, HIF-1α was expressed only in the nucleus pulposus. This isoform was absent from both the cartilage end plate and annulus. We detected HIF-1α immunohistochemically in the nucleus pulposus; however, the staining was light and diffuse. Cells of the nucleus pulposus expressed GLUT-1; in contrast, when probed by Western blot analysis the annulus and cartilage were negative for this protein. Western blot analysis also showed that in the nucleus pulposus the level of MMP-2 was high when compared to the adjacent tissues. We suggest that the differential expression of the two HIF isoforms, and GLUT-1 and MMP-2, provides a phenotypic signature that permits cells of the nucleus pulposus to be distinguished from neighboring tissues. Moreover, the presence of these isoforms provides evidence that cells of the disc respond to hypoxia and nutrient stress by upregulating stress-responsive genes.

The natural history of cervical spondylotic myelopathy

OBJECT The objective of this systematic review was to use evidence-based medicine to delineate the natural history of cervical spondylotic myelopathy (CSM) and identify factors associated with clinical deterioration. METHODS The National Library of Medicine and Cochrane Database were queried using MeSH headings and keywords relevant to the natural history of CSM. Abstracts were reviewed and studies meeting the inclusion criteria were selected. The guidelines group assembled an evidentiary table summarizing the quality of evidence (Classes I-III). Disagreements regarding the level of evidence were resolved through an expert consensus conference. The group formulated recommendations that contained the degree of strength based on the Scottish Intercollegiate Guidelines network. Validation was done through peer review by the Joint Guidelines Committee of the American Association of Neurological Surgeons and the Congress of Neurological Surgeons. RESULTS The natural history of CSM is mixed: it may manifest as a slow, stepwise decline or there may be a long period of quiescence (Class III). Long periods of severe stenosis are associated with demyelination and may result in necrosis of both gray and white matter. With severe and/or long lasting CSM symptoms, the likelihood of improvement with nonoperative measures is low. Objectively measurable deterioration is rarely seen acutely in patients younger than 75 years of age with mild CSM (modified Japanese Orthopaedic Association scale score > 12; Class I). In patients with cervical stenosis without myelopathy, the presence of abnormal electromyography findings or the presence of clinical radiculopathy is associated with the development of symptomatic CSM in this patient population (Class I). CONCLUSIONS The natural history of CSM is variable, which may affect treatment decisions.

Fluoroscopically guided therapeutic sacroiliac joint injections for sacroiliac joint syndrome.

Slipman CW, Lipetz JS, Plastaras CT, Jackson HB, Vresilovic EJ, Lenrow DA, Debra L. Braverman DL: Fluoroscopically guided therapeutic sacroiliac joint injections for sacroiliac joint syndrome. Am J Phys Med Rehabil 2001;80:425–432. ObjectiveTo investigate the outcomes resulting from the use of fluoroscopically guided therapeutic sacroiliac joint injections in patients with sacroiliac joint syndrome. DesignA retrospective study design with independent clinical review was utilized. Thirty-one patients were included; each patient met specific physical examination criteria and failed to improve clinically after at least 4 wk of physical therapy. Each patient demonstrated a positive response to a fluoroscopically guided diagnostic sacroiliac joint injection. Therapeutic sacroiliac joint injections were administered in conjunction with physical therapy. Outcome measures included Oswestry scores, Visual Analog Scale pain scores, work status, and medication usage. ResultsPatients’ symptom duration before diagnostic injection averaged 20.6 mo. An average of 2.1 therapeutic injections was administered. Follow-up data collection was obtained at an average of 94.4 wk. A significant reduction (P = 0.0014) in Oswestry disability score was observed at the time of follow-up. Visual Analog Scale pain scores were reduced (P < 0.0001) at the time of discharge and at follow-up. Work status was also significantly improved at the time of discharge (P = 0.0313) and at follow-up (P = 0.0010). A trend (P = 0.0645) toward less drug usage was observed. ConclusionsThese initial findings suggest that fluoroscopically guided therapeutic sacroiliac joint injections are a clinically effective intervention in the treatment of patients with sacroiliac joint syndrome. Controlled, prospective studies are necessary to further clarify the role of therapeutic injections in this patient population.