Lorin Michael Benneker

2004 Young Investigator Award Winner: vertebral endplate marrow contact channel occlusions and intervertebral disc degeneration.

Study Design. Intervertebral disc degeneration was evaluated by morphologic appearance, magnetic resonance imaging, and by biochemical matrix composition. Caliber and distribution of openings of the adjacent vertebral osseous endplates were measured. Objectives. Correlation between occlusion of vertebral endplate openings and intervertebral disc degeneration was quantified. Summary of Background Data. Calcifications of vertebral endplates with disease and age have suggested insufficient nutrition as a mechanism for intervertebral disc degeneration. It has been proposed that occlusion of endplate openings, which contain vascular sources for the disc, may limit the transport of nutrients, leading to disc degeneration. Methods. Fresh magnetic resonance images from 39 human lumbar discs were scored. Sectioned discs with endplates were morphologically graded. Samples of nuclear and anular regions were evaluated for proteoglycan and collagen contents. Backlight microscopic images of 4 endplate regions were obtained, and caliber and distribution of endplate openings for each disc were measured. Analysis of variance regression models were used to assess correlation between endplate openings and disc degeneration. Results. The decrease in opening density significantly correlated to morphologic degeneration grade, best for openings with 20 to 50 ìm equivalent diameter and in the nuclear region. Although the density of 20 to 50 ìm openings also significantly indirectly correlated to age, it was not as strong as the correlation to degeneration grade. Opening density was also significantly correlated to proteoglycan content in all regions. However, all other biochemical parameters as well as the T2 intensity score showed only weak or no correlation to opening density. Conclusions. A high indirect correlation between the density of openings in the osseous endplate (particularly of the size of the capillary buds) and the morphologicdegeneration grade of the disc support the hypothesis that occlusion of these openings may deprive the cells of nutrients, leading to insufficient maintenance of the extracellular matrix and disc degeneration.

Classification of Intervertebral Disk Degeneration with Axial T2 Mapping

OBJECTIVE The aim of this study was to establish an MRI classification system for intervertebral disks using axial T2 mapping, with a special focus on evaluating early degenerative intervertebral disks. MATERIALS AND METHODS Twenty-nine healthy volunteers (19 men, 10 women; age range, 20-44 years; mean age, 31.8 years) were studied, and axial T2 mapping was performed for the L3-L4, L4-L5, and L5-S1 intervertebral disks. Grading was performed using three classification systems for degenerative disks: our system using axial T2 mapping and two other conventional classification systems that focused on the signal intensity of the nucleus pulposus or the structural morphology in sagittal T2-weighted MR images. We analyzed the relationship between T2, which is known to correlate with change in composition of intervertebral disks, and degenerative grade determined using the three classification systems. RESULTS With axial T2 mapping, differences in T2 between grades I and II were smaller and those between grades II and III, and between grades III and IV, were larger than those with the other grading systems. The ratio of intervertebral disks classified as grade I was higher with the conventional classification systems than that with axial T2 mapping. In contrast, the ratio of intervertebral disks classified as grade II or III was higher with axial T2 mapping than that with the conventional classification systems. CONCLUSION Axial T2 mapping provides a more T2-based classification. The new system may be able to detect early degenerative changes before the conventional classification systems can.

Thermoreversible hyaluronan-based hydrogel supports in vitro and ex vivo disc-like differentiation of human mesenchymal stem cells

BACKGROUND CONTEXT The fate of human mesenchymal stem cells (hMSCs) supplied to the degenerating intervertebral disc (IVD) is still not fully understood and can be negatively affected by low oxygen, pH, and glucose concentration of the IVD environment. The hMSC survival and yield upon injection of compromised IVD could be improved by the use of an appropriate carrier and/or by predifferentiation of hMSCs before injection. PURPOSE To optimize hMSC culture conditions in thermoreversible hyaluronan-based hydrogel, hyaluronan-poly(N-isopropylacrylamide) (HA-pNIPAM), to achieve differentiation toward the disc phenotype in vitro, and evaluate whether preconditioning contributes to a better hMSC response ex vivo. STUDY DESIGN In vitro and ex vivo whole-organ culture of hMSCs. METHODS In vitro cultures of hMSCs were conducted in HA-pNIPAM and alginate for 1 week under hypoxia in chondropermissive medium alone and with the supplementation of transforming growth factor β1 or growth and differentiation factor 5 (GDF-5). Ex vivo, hMSCs were either suspended in HA-pNIPAM and directly supplied to the IVDs or predifferentiated with GDF-5 for 1 week in HA-pNIPAM and then supplied to the IVDs. Cell viability was evaluated by Live-Dead assay, and DNA, glycosaminoglycan (GAG), and gene expression profiles were used to assess hMSC differentiation toward the disc phenotype. RESULTS The HA-pNIPAM induced hMSC differentiation toward the disc phenotype more effectively than alginate: in vitro, higher GAG/DNA ratio and higher collagen type II, SOX9, cytokeratin-19, cluster of differentiation 24, and forkhead box protein F1 expressions were found for hMSCs cultured in HA-pNIPAM compared with those cultured in alginate, regardless of the addition of growth factors. Ex vivo, direct combination of HA-pNIPAM with the disc environment induced a stronger disc-like differentiation of hMSCs than predifferentiation of hMSCs followed by their delivery to the discs. CONCLUSIONS Hyaluronan-based thermoreversible hydrogel supports hMSC differentiation toward the disc phenotype without the need for growth factor supplementation in vitro and ex vivo. Further in vivo studies are required to confirm the suitability of this hydrogel as an effective stem cell carrier for the treatment of IVD degeneration.

Homing of Mesenchymal Stem Cells in Induced Degenerative Intervertebral Discs in a Whole Organ Culture System

Study Design. Homing of human bone marrow–derived mesenchymal stem cells (BMSCs) was studied using ex vivo cultured bovine caudal intervertebral discs (IVDs). Objective. To investigate in a whole organ culture whether metabolic and mechanical challenges can induce BMSC recruitment into the IVD. Summary of Background Data. Cells from injured tissues release cytokines and mediators that enable the recruitment of progenitor cells. BMSCs have the ability to survive within the IVD. Methods. Bovine IVDs with or without endplates were cultured for 1 week under simulated physiological or degenerative conditions; disc cells were analyzed for cell viability and gene expression, whereas media was analyzed for nitric oxide production and chemotaxis. Homing of BMSCs was investigated by supplying PKH-labeled human BMSCs onto cultured IVDs (1 × 106 cells/disc on d 8, 10, and 12 of culture); on day 14, the number of homed BMSCs was microscopically assessed. Moreover, a comparative study was performed between transduced BMSCs (transduced with an adenovirus encoding for insulin-like growth factor 1 [IGF-1]) and nontransduced BMSCs. Disc proteoglycan synthesis rate was quantified via 35S incorporation. The secretion of IGF-1 was evaluated by enzyme-linked immunosorbent assay on both simulated physiological and degenerative discs. Results. Discs cultured under degenerative conditions showed reduced cell viability, upregulation of matrix degrading enzymes, and increased nitric oxide production compared with simulated physiological discs. Greater homing occurred under degenerative compared with physiological conditions with or without endplate. Media of degenerative discs demonstrated a chemoattractive activity toward BMSCs. Finally, discs homed with IGF-1–transduced BMSCs showed increased IGF-1 secretion and significantly higher proteoglycan synthesis rate than discs supplied with nontransduced BMSCs. Conclusion. We have demonstrated for the first time that degenerative conditions induce the release of factors promoting BMSC recruitment in an ex vivo organ culture. Moreover, IGF-1 transduction of BMSCs strongly increases the rate of proteoglycan synthesis within degenerative discs. This finding offers a new delivery system for BMSCs and treatment strategy for IVD regeneration.

Severe spinal injuries in alpine skiing and snowboarding: a 6-year review of a tertiary trauma centre for the Bernese Alps ski resorts, Switzerland

Objective: To analyse the epidemiological data, injury pattern, clinical features and mechanisms of severe spinal injuries related to alpine skiing and snowboarding. Study design: A six-year review of all adult patients with severe spinal injuries sustained from alpine skiing or snowboarding. Setting: Tertiary trauma centre in Bern, Switzerland. Patients and methods: All adult patients (over 16 years of age) admitted to a tertiary trauma centre from 1 July 2000, through 30 June 2006, were reviewed using a computerised database. From these records, a total of 728 patients injured from snow sports were identified. Severe spinal injuries (defined as spinal fractures, subluxations, dislocations or concomitant spinal cord injuries) were found in 73 patients (17 female, 56 male). The clinical features of these patients were reviewed with respect to epidemiological factors, mechanism of injury, fracture pattern, and neurological status. Results: The majority of severe spinal injuries (n = 63) were related to skiing. Fatal central-nervous injuries and transient or persistent neurological symptoms occurred in 28 patients (23 skiers, 5 snowboarders). None of the snowboarders suffered from persistent neurological sequelae. Snowboarders with severe spinal injuries (n = 10) were all male (p<0.05), and were significantly younger than skiers (p<0.001). The most commonly affected site was the lumbar spine. However, 39 patients (53.4%) suffered from injury pattern at two or more levels. Conclusions: With advances in technology and slope maintenance, skiers and snowboarders progress to higher skill levels and faster speeds more rapidly than ever before. Great efforts have been focused on reducing extremity injuries in snow sports, but until recently very little attention has been given to spinal injury prevention on the slopes. Suggestions for injury prevention include the use of spine protectors, participation on appropriate runs for ability level, proper fit and adjustment of equipment, and taking lessons with the goal of increasing ability and learning hill etiquette.

Epidemiology and predictors of cervical spine injury in adult major trauma patients: a multicenter cohort study

BACKGROUND: Patients with cervical spine injuries are a high-risk group, with the highest reported early mortality rate in spinal trauma. METHODS: This cohort study investigated predictors for cervical spine injury in adult (≥ 16 years) major trauma patients using prospectively collected data of the Trauma Audit and Research Network from 1988 to 2009. Univariate and multivariate logistic regression analyses were used to determine predictors for cervical fractures/dislocations or cord injury. RESULTS: A total of 250,584 patients were analyzed. Median age was 47.2 years (interquartile range, 29.8–66.0) and Injury Severity Score 9 (interquartile range, 4–11); 60.2% were male. Six thousand eight hundred two patients (2.3%) sustained cervical fractures/dislocations alone. Two thousand sixty-nine (0.8%) sustained cervical cord injury with/without fractures/dislocations; 39.9% of fracture/dislocation and 25.8% of cord injury patients suffered injuries to other body regions. Age ≥65 years (odds ratio [OR], 1.45–1.92), males (females OR, 0.91; 95% CI, 0.86–0.96), Glasgow Coma Scale (GCS) score <15 (OR, 1.26–1.30), LeFort facial fractures (OR, 1.29; 95% confidence interval [CI], 1.05–1.59), sports injuries (OR, 3.51; 95% CI, 2.87–4.31), road traffic collisions (OR, 3.24; 95% CI, 3.01–3.49), and falls >2 m (OR, 2.74; 95% CI, 2.53–2.97) were predictive for fractures/dislocations. Age <35 years (OR, 1.25–1.72), males (females OR, 0.59; 95% CI, 0.53–0.65), GCS score <15 (OR, 1.35–1.85), systolic blood pressure <110 mm Hg (OR, 1.16; 95% CI, 1.02–1.31), sports injuries (OR, 4.42; 95% CI, 3.28–5.95), road traffic collisions (OR, 2.58; 95% CI, 2.26–2.94), and falls >2 m (OR, 2.24; 95% CI, 1.94–2.58) were predictors for cord injury. CONCLUSIONS: 3.5% of patients suffered cervical spine injury. Patients with a lowered GCS or systolic blood pressure, severe facial fractures, dangerous injury mechanism, male gender, and/or age ≥35 years are at increased risk. Contrary to common belief, head injury was not predictive for cervical spine involvement. LEVEL OF EVIDENCE: II.

Pelvic radiography in ATLS algorithms: A diminishing role?

BackgroundPelvic x-ray is a routine part of the primary survey of polytraumatized patients according to Advanced Trauma Life Support (ATLS) guidelines. However, pelvic CT is the gold standard imaging technique in the diagnosis of pelvic fractures. This study was conducted to confirm the safety of a modified ATLS algorithm omitting pelvic x-ray in hemodynamically stable polytraumatized patients with clinically stable pelvis in favour of later pelvic examination by CT scan.MethodsWe conducted a retrospective analysis of all polytraumatized patients in our emergency room between 01.07.2004 and 31.01.2006. Inclusion criteria were blunt abdominal trauma, initial hemodynamic stability and a stable pelvis on clinical examination. We excluded patients requiring immediate intervention because of hemodynamic instability.ResultsWe reviewed the records of n = 452 polytraumatized patients, of which n = 91 fulfilled inclusion criteria (56% male, mean age = 45 years). The mechanism of trauma included 43% road traffic accidents, 47% falls. In 68/91 (75%) patients, both a pelvic x-ray and a CT examination were performed; the remainder had only pelvic CT. In 6/68 (9%) patients, pelvic fracture was diagnosed by pelvic x-ray. None of these 6 patients was found having a false positive pelvic x-ray, i.e. there was no fracture on pelvic CT scan. In 3/68 (4%) cases a fracture was missed in the pelvic x-ray, but confirmed on CT (false negative on x-ray). None of the diagnosed fractures needed an immediate therapeutic intervention. 5 (56%) were classified type A fractures, and another 4 (44%) B 2.1 in computed tomography (AO classification). One A 2.1 fracture was found in a clinically stable patient who only received CT scan (1/23).ConclusionWhile pelvic x-ray is an integral part of ATLS assessment, this retrospective study suggests that in hemodynamically stable patients with clinically stable pevis, its sensitivity is only 67% and it may safely be omitted in favor of a pelvic CT examination if such is planned in adjunct assessment and available. The results support the safety and utility of our modified ATLS algorithm. A randomized controlled trial using the algorithm can safely be conducted to confirm the results.

Tenocytes of chronic rotator cuff tendon tears can be stimulated by platelet-released growth factors.

BACKGROUND Bone-to-tendon healing after rotator cuff repairs is mainly impaired by poor tissue quality. The tenocytes of chronic rotator cuff tendon tears are not able to synthesize normal fibrocartilaginous extracellular matrix (ECM). We hypothesized that in the presence of platelet-released growth factors (PRGF), tenocytes from chronically retracted rotator cuff tendons proliferate and synthesize the appropriate ECM proteins. MATERIALS AND METHODS Tenocytes from 8 patients with chronic rotator cuff tears were cultured for 4 weeks in 2 different media: standard medium (Iscoves Modified Dulbeccos Media + 10% fetal calf serum + 1% nonessential amino acids + 0.5 μg/mL ascorbic acid) and media with an additional 10% PRGF. Cell proliferation was assessed at 7, 14, 21, and 28 days. Messenger (m)RNA levels of collagens I, II, and X, decorin, biglycan, and aggrecan were analyzed using real time reverse-transcription polymerase chain reaction. Immunocytochemistry was also performed. RESULTS The proliferation rate of tenocytes was significantly higher at all time points when cultured with PRGF. At 21 days, the mRNA levels for collagens I, II, and X, decorin, aggrecan, and biglycan were significantly higher in the PRGF group. The mRNA data were confirmed at protein level by immunocytochemistry. CONCLUSIONS PRGFs enhance tenocyte proliferation in vitro and promote synthesis of ECM to levels similar to those found with insertion of the normal human rotator cuffs. CLINICAL RELEVANCE Biologic augmentation of repaired rotator cuffs with PRGF may enhance the properties of the repair tissue. However, further studies are needed to determine if application of PRGF remains safe and effective in long-term clinical studies. LEVEL OF EVIDENCE Basic Science Study, Cell Biology.

Papain-induced in vitro disc degeneration model for the study of injectable nucleus pulposus therapy

BACKGROUND CONTEXT Proteolytic enzyme digestion of the intervertebral disc (IVD) offers a method to simulate a condition of disc degeneration for the study of cell-scaffold constructs in the degenerated disc. PURPOSE To characterize an in vitro disc degeneration model (DDM) of different severities of glycosaminoglycans (GAG) and water loss by using papain, and to determine the initial response of the human mesenchymal stem cells (MSCs) introduced into this DDM. STUDY DESIGN Disc degeneration model of a bovine disc explant with an end plate was induced by the injection of papain at various concentrations. Labeled MSCs were later introduced in this model. METHODS Phosphate-buffered saline (PBS control) or papain in various concentrations (3, 15, 30, 60, and 150 U/mL) were injected into the bovine caudal IVD explants. Ten days after the injection, GAG content of the discs was evaluated by dimethylmethylene blue assay and cell viability was determined by live/dead staining together with confocal microscopy. Overall matrix composition was evaluated by histology, and water content was visualized by magnetic resonance imaging. Compressive and torsional stiffness of the DDM were also recorded. In the second part, MSCs were labeled with a fluorescence cell membrane tracker and injected into the nucleus of the DDM or a PBS control. Mesenchymal stem cell viability and distribution were evaluated by confocal microscopy. RESULTS A large drop of GAG and water content of the bovine disc were obtained by injecting >30 U/mL papain. Magnetic resonance imaging showed Grade II, III, and IV disc degeneration by injecting 30, 60, and 150 U/mL papain. A cavity in the center of the disc could facilitate later injection of the nucleus pulposus tissue engineering construct while retaining an intact annulus fibrosus. The remaining disc cell viability was not affected. Mesenchymal stem cells injected into the protease-treated DDM disc showed significantly higher cell viability than when injected into the PBS-injected control disc. CONCLUSIONS By varying the concentration of papain for injection, an increasing amount of GAG and water loss could be induced to simulate the different severities of disc degeneration. MSC suspension introduced into the disc has a very low short-term survival. However, it should be clear that this bovine IVD DDM does not reflect a clinical situation but offers exciting possibilities to test novel tissue engineering protocols.

Total-body digital X-ray in trauma An experience report on the first operational full body scanner in Europe and its possible role in ATLS

When patients enter our emergency room with suspected multiple injuries, Statscan provides a full body anterior and lateral image for initial diagnosis, and then zooms in on specific smaller areas for a more detailed evaluation. In order to examine the possible role of Statscan in the management of multiply injured patients we implemented a modified ATLS((R)) algorithm, where X-ray of C-spine, chest and pelvis have been replaced by single-total a.p./lat. body radiograph. Between 15 October 2006 and 1 February 2007 143 trauma patients (mean ISS 15+/-14 (3-75)) were included. We compared the time in resuscitation room to 650 patients (mean ISS 14+/-14 (3-75)) which were treated between 1 January 2002 and 1 January 2004 according to conventional ATLS protocol. The total-body scanning time was 3.5 min (3-6 min) compared to 25.7 (8-48 min) for conventional X-rays, The total ER time was unchanged 28.7 min (13-58 min) compared to 29.1 min (15-65 min) using conventional plain radiography. In 116/143 patients additional CT scans were necessary. In 98/116 full body trauma CT scans were performed. In 18/116 patients selective CT scans were ordered based on Statscan findings. In 43/143 additional conventional X-rays had to be performed, mainly due to inadequate a.p. views of fractured bones. All radiographs were transmitted over the hospital network (Picture Archiving and Communication System, PACS) for immediate simultaneous viewing at different places. The rapid availability of images for interpretation because of their digital nature and the reduced need for repeat exposures because of faulty radiography are also felt to be strengths.