Daniel Haschtmann

Establishment of a novel intervertebral disc/endplate culture model: analysis of an ex vivo in vitro whole-organ rabbit culture system.

Study Design. Ex vivo in vitro study evaluating a novel intervertebral disc/endplate culture system. Objectives. To establish a whole-organ intervertebral disc culture model for the study of disc degeneration in vitro, including the characterization of basic cell and organ function. Summary of Background Data. With current in vivo models for the study of disc and endplate degeneration, it remains difficult to investigate the complex disc metabolism and signaling cascades. In contrast, more controlled but simplified in vitro systems using isolated cells or disc fragments are difficult to culture due to the unconstrained conditions, with often-observed cell death or cell dedifferentiation. Therefore, there is a demand for a controlled culture model with preserved cell function that offers the possibility to investigate disc and endplate pathologies in a structurally intact organ. Methods. Naturally constrained intervertebral disc/endplate units from rabbits were cultured in multi-well plates. Cell viability, metabolic activity, matrix composition, and matrix gene expression profile were monitored using the Live/Dead® cell viability test (Invitrogen, Basel, Switzerland), tetrazolium salt reduction (WST-8), proteoglycan and deoxyribonucleic acid quantification assays, and quantitative polymerase chain reaction. Results. Viability and organ integrity were preserved for at least 4 weeks, while proteoglycan and deoxyribonucleic acid content decreased slightly, and matrix genes exhibited a degenerative profile with up-regulation of type I collagen and suppression of collagen type II and aggrecan genes. Additionally, cell metabolic activity was reduced to one third of the initial value. Conclusions. Naturally constrained intervertebral rabbit discs could be cultured for several weeks without losing cell viability. Structural integrity and matrix composition were retained. However, the organ responded to the artificial environment with a degenerative gene expression pattern and decreased metabolic rate. Therefore, the described system serves as a promising in vitro model to study disc degeneration in a whole organ.

Fracture of the vertebral endplates, but not equienergetic impact load, promotes disc degeneration in vitro

Vertebral endplate damage is associated with intervertebral disc (IVD) degeneration (DD) in vivo as confirmed by in‐vitro investigations. Our aims were to further characterize the process of DD using an in vitro full‐organ culture model and to elucidate whether significant endplate damage or impact loading alone is pivotal for the initiation of DD. Rabbit spinal segments (n = 80) were harvested, subjected to pure axial impact loading (n = 40) using a custom‐made device, and cultured for 28 days. The applied threshold energy (0.76 J) induced endplate fractures in 21 specimens (group A); 19 remained intact (group B). Markers for DD (cell viability, apoptosis, necrosis, matrix remodeling, and inflammation) were monitored for 28 days post‐trauma in the annulus fibrosus (AF) and nucleus pulposus and compared to non‐impacted control discs. Cell viability in both groups stayed at a control level. Group A compared to group B showed enhanced lactate dehydrogenase (LDH) and caspase‐3/7 activity, reduced glycosaminoglycan content, reduced aggrecan mRNA, but elevated mRNA for collagen‐2, catabolic enzymes (MMP‐1/‐3/‐13), and pro‐inflammatory (TNFα, IL‐6, IL‐8, MCP‐1) and pro‐apoptotic (fas ligand, caspase‐3) proteins. Group B compared to control only showed small changes in mRNA levels. Our findings demonstrate that burst endplates, but not equienergetic loading, promotes DD.

A Robust and Accurate Two-Stage Approach for Automatic Recovery of Distal Locking Holes in Computer-Assisted Intramedullary Nailing of Femoral Shaft Fractures

It has been recognized that one of the most difficult steps in intramedullary nailing of femoral shaft fractures is the distal locking- the insertion of distal transverse interlocking screws, for which it is necessary to know the positions and orientations of the distal locking holes (DLHs) of the intramedullary nail (IMN). This paper presents a robust and accurate approach for solving this problem based on two calibrated and registered fluoroscopic images. The problem is formulated as a two-stage model-based optimal fitting process. The first stage, nail detection, automatically estimates the axis of the distal part of the IMN (DP-IMN) by iteratively fitting a cylindrical model to the images. The second stage, pose recovery, resolves the translations and the rotations of the DLHs around the estimated axis by iteratively fitting the geometrical models of the DLHs to the images. An iterative best matched projection point (IBMPP) algorithm is combined with random sample strategies to effectively and robustly solve the fitting problems in both stages. We designed and conducted comprehensive experiments to validate the robustness and the accuracy of the present approach. Our in vitro experiments show on average less than 14 s execution time on a Linux machine, a mean angular error of 0.48deg (std = 0.25deg), and a mean translational error of 0.09 mm (std = 0.04 mm). We conclude that the present approach is fast, robust, and accurate for distal locking applications.

Accurate and reliable pose recovery of distal locking holes in computer-assisted intra-medullary nailing of femoral shaft fractures: A preliminary study

Objective: One of the difficult steps in intra-medullary nailing of femoral shaft fractures is distal locking – the insertion of distal interlocking screws. Conventionally, this is performed using repeated image acquisitions, which leads to considerable irradiation of the patient and surgical team. Virtual fluoroscopy has been used to reduce radiation exposure, but can only provide multi-planar two-dimensional projection views. In this study, two calibrated fluoroscopic images were used to automatically recover the positions and orientations of the distal locking holes (DLHs). The ultimate goal is to provide precise three-dimensional guidance during distal locking. Methods: A model-based optimal fitting process was used to reconstruct the positions and orientations of the DLHs from two calibrated fluoroscopic images. No human intervention is required. A preliminary in vitro validation study was conducted to analyze the accuracy and reliability of the technique using images acquired from different viewpoints. The ground truths of the DLH were obtained by inserting a custom-made steel rod through the hole and then digitizing both the top and bottom centers of the rod using a sharp pointer. The recovery errors were computed by comparing the computed results to the ground truths. Results: In all experiments, the poses of the DLHs could be recovered fully automatically. When the recovered positions and orientations of the DLHs were compared to their associated ground truths, a mean angular error of 0.5° (STD = 0.2°), and a mean translational error of 0.1 mm (STD = 0.0 mm) were found. Conclusions: Accurate and reliable pose recovery of distal locking holes from two calibrated fluoroscopic images is achievable. Our preliminary in vitro experimental results demonstrate that the recovered poses of the distal locking holes are sufficiently accurate for intra-operative use.

Apoptosis and gene expression of collagenases but not gelatinases in rabbit disc fragment cultures

OBJECT The object of this study was to characterize the biological response of isolated intervertebral disc fragments to in vitro culture conditions with respect to cell death and inflammatory and catabolic changes. The acquired data could help to gain a better understanding of the biological reaction of disc tissue when exposed to environmental changes along with altered nutritional and osmotic conditions, as are encountered in different in vitro disc models or disc diseases in vivo. METHODS Intervertebral disc anulus fragments were isolated from Burgundy rabbits and cultured in standard media for 3 days. The disc fragments were analyzed for their swelling properties, proteoglycan loss on histological studies, lactate dehydrogenase activity, apoptosis, gene expression of collagenases and gelatinases, and for proinflammatory (MCP-1, IL-8, and IL-6) and apoptosis-associated (TNF-alpha, Fas-L, and caspase 3) genes. RESULTS The results demonstrate that disc specimens were swelling, and a loss of proteoglycans with disarrangement of anulus architecture was observed. The disc cells underwent rapid apoptosis with upregulation of various proinflammatory genes. Both collagenases, matrix metalloproteinase (MMP)-1 and MMP-13, were increasingly transcribed, whereas the gelatinases MMP-2 and MMP-9 did not respond or were downregulated. CONCLUSIONS Cultured disc fragments swell and undergo necrotic and apoptotic cell death combined with a catabolic gene response and gene expression of proinflammatory and chemoattractant proteins. Some of these findings have been demonstrated before in various spinal disorders. In addition, disc fragments are not suitable for long-term culture if a stable disc metabolism is desired, and the described changes have to be considered when using isolated disc material for in vitro cultures.

Patient-reported outcome of surgical treatment for lumbar spinal epidural lipomatosis.

BACKGROUND CONTEXT Spinal epidural lipomatosis (SEL) is a rare condition characterized by an excessive accumulation of fat tissue in the spinal canal that can have a compressive effect, leading to clinical symptoms. This condition has a distinct pathology from spinal stenosis associated with degeneration of the intervertebral discs, ligaments, and facet joints. Several different conservative and surgical treatment strategies have been proposed for SEL, but its treatment remains controversial. There is a lack of evidence documenting the success of surgical decompression in SEL, and no previous studies have reported the postoperative outcome from the patients perspective. PURPOSE The aim of the present study was to evaluate patient-rated outcome after surgical decompression in SEL. STUDY DESIGN A retrospective analysis of prospectively collected data was carried out. PATIENT SAMPLE A total of 22 patients (19 males; age: 68.2±9.9 years) who had undergone spine surgery for SEL were identified from our local Spine Surgery Outcomes Database, which includes a total of 10,028 spine surgeries recorded between 2005 and 2012. Inclusion criteria were epidural lipomatosis confirmed by preoperative magnetic resonance imaging (MRI) scans and subsequent decompression surgery without spinal fusion. OUTCOME MEASURES The Core Outcome Measures Index (COMI) was used to assess patient-rated outcome. The COMI includes the domains pain (separate 0-10 scales for back and leg pain), back-specific function, symptom-specific well-being, general quality of life (QOL), work disability, and social disability. METHODS The questionnaires were completed preoperatively and at 3, 12, and 24 months postoperatively. Surgical data were retrieved from the patient charts and from our local Spine Surgery Outcomes Database, which we operate in connection with the International Spine Tango Registry. Differences between pre- and postoperative scores were analyzed using paired t tests and repeated measures analysis of variance. RESULTS At 3-months follow-up, the COMI score and scores for leg pain and back pain had improved significantly compared with their preoperative values (p<.005). The mean decrease in COMI score after 3 months was 2.6±2.4 (range: -1.3 to 6.5) points: from 7.5±1.7 (range: 3.5-10) to 4.9±2.5 (range: 0.5-9.6). A total of 11 patients (50%) had an improvement of the COMI of more than the minimal clinically important change (MCIC) score of 2.2 points. The mean decrease in leg pain after 3 months was 2.4±3.5 (-5 to 10) points. Overall, 17 patients (77.3%) reported a reduced leg pain, 12 (54.6%) of whom by at least the MCIC score of 2 points. The significant reductions from baseline in COMI and leg and back pain scores were retained up to 2 years postoperatively (p<.02). The general QOL item of the COMI improved significantly after surgery (p<.0001). Over 80% of the cohort rated their preoperative QOL as bad (n=13) or very bad (n=5), whereas 3 months after surgery, only 7 patients rated their QOL as bad, and one as very bad (36%). CONCLUSIONS The present study is the first to demonstrate that surgical decompression is associated with a statistically significant improvement in patient-rated outcome scores in patients with symptomatic SEL, with a clinically relevant change occurring in approximately half of them. Surgical decompression hence represents a reasonable treatment option for SEL, although the reason behind the less good response in some patients needs further investigation.

Could Less Be More When Assessing Patient-rated Outcome in Spinal Stenosis?

Study Design. Longitudinal study of the measurement properties of a brief outcome instrument. Objective. In patients undergoing surgery for lumbar spinal stenosis, we compared the responsiveness of the Core Outcome Measures Index (COMI) with that of the condition-specific Swiss Spinal Stenosis Measure (SSM), an instrument developed to assess patients with neurogenic claudication. Summary of Background Data. The COMI is a validated multidimensional questionnaire for assessing the key outcomes of importance to patients with back problems. Being brief, it is associated with minimal respondent burden and high completion rates. However, for a given pathology, intuitively it may be expected to be less responsive than a condition-specific instrument. Methods. A total of 91 patients (73 ± 8 yr; 53% males) completed the following questionnaires before surgery: COMI, SSM, Roland Morris Disability Questionnaire, back trouble “Feeling Thermometer,” pain numeric rating scale, EuroQoL-visual analogue scale. Twelve months postoperatively, 78/91 (86%) completed all the questionnaires again; they also rated the “global treatment outcome” (GTO; rated 1–5) and SSM “satisfaction with treatment result” (SSM-sat; rated 1–4), which were used as external criteria of treatment success. Results. Scores for the external criteria of success (GTO/SSM-sat) correlated with the change scores (baseline to 12 mo) in COMI (r = 0.57) and SSM (r = 0.54) to a similar extent. Using receiver operating characteristics, with GTO or SSM-sat dichotomized as external criterion, the area under the curve was similar for the COMI change score (0.86–0.90) and the SSM (sub)scales (0.80–0.90). Conclusion. With either SSM-sat or GTO serving as the external criterion, COMI was as responsive as the SSM. The COMI is well able to detect important change in lumbar spinal stenosis and has the added benefit of reducing the response burden for the patient and facilitating outcome comparisons with other spinal pathologies. Level of Evidence: 2

Patient-Rated Outcomes of Lumbar Fusion in Patients With Degenerative Disease of the Lumbar Spine: Does Age Matter?

Study Design. Single-center retrospective study of prospectively collected data, nested within the Eurospine Spine Tango data acquisition system. Objective. The aim of this study was to assess the patient-rated outcome and complication rates associated with lumbar fusion procedures in three different age groups. Summary of Background Data. There is a general reluctance to consider spinal fusion procedures in elderly patients due to the increased likelihood of complications. Methods. Before and at 3, 12, and 24 months after surgery, patients completed the multidimensional Core Outcome Measures Index. At the 3-, 12-, and 24-month follow-ups, they also rated the Global Treatment Outcome and their satisfaction with care. Patients were divided into three age groups: younger (≥50 years <65 years; n = 317), older (≥65 years <80 years; n = 350), and geriatric (≥80 years; n = 40). Results. A total of 707 consecutive patients were included. The preoperative comorbidity status differed significantly (P < 0.0001) between the age groups, with the highest scores in the geriatric group. Medical complications during surgery were lower in the younger age group (7%) than in the older (13.4%; P = 0.006) and geriatric groups (17.5%; P = 0.007); surgical complications tended to be higher in the elderly group (younger, 6.3%; older, 6.0%; geriatric, 15.0%; P = 0.09). There were no significant group differences (P > 0.05) for the scores on any of the Core Outcome Measures Index domains, Global Treatment Outcome, or patient-rated satisfaction at either 3-, 12-, and 24-months of follow-up. Conclusion. Despite greater comorbidity and complication rates in geriatric patients, the patient-rated outcome was as good in the elderly as it was in younger age groups up to 2 years after surgery. These data indicate that geriatric age needs careful consideration of associated risks but is not per se a contraindication for fusion for lumbar degenerative disease. Level of Evidence: 4

Leukocytes Enhance Inflammatory and Catabolic Degenerative Changes in the Intervertebral Disc After Endplate Fracture In Vitro Without Infiltrating the Disc.

Study Design. An established rabbit intervertebral disc (IVD)/endplate explant fracture model was extended with physiologic post-traumatic dynamic loading (PTDL) and coculturing of peripheral blood mononuclear cells (PBMCs). Objective. The aim of this study was to quantify the effects of PTDL and of cocultured PBMCs on post-traumatic disc degeneration (DD) and to determine whether PTDL facilitates homing of PBMC to fractured IVD/endplates. Summary of Background Data. DD is associated with endplate fracture. In vivo studies suggest a key role of immune cells in the pathogenesis of DD. However, the complexity of in vivo systems impedes the investigation of single factors governing the pathogenesis. Methods. Seventy-two IVD/endplate specimens were divided into 4 groups. In group A, endplate fractures were induced with a high-velocity axial load and exposed to PTDL in coculture with PBMCs for 14 days. Group A was compared with 3 control groups, with single-factor removal, in order to assess the relative contribution of PTDL (group B), PBMCs (group C), and endplate fracture (group D) to the biological response of the IVD. Disc gene transcription and serum nitric oxide (NO) serum concentration were measured to investigate differences in anabolism, catabolism, and inflammatory response between the groups. Changes in matrix composition and disc structure were assessed histologically. Results. PBMCs did not home to fractured IVDs, with or without PTDL. Group A compared with group D showed an enhanced transcription of anabolic, catabolic, and pro-inflammatory genes during the entire experiment, and an increased NO concentration for the first 3 days. Changes typical for DD were also found in histological sections. Group A compared with group C showed significant increases in catabolic and pro-inflammatory gene transcription after at least 7 days. No differences were found between groups A and B. Conclusion. Trauma induces degenerative changes; PTDL neither aggravates nor ameliorates this response. Although PBMCs do not infiltrate the disc, they aggravate the degenerative changes. Level of Evidence: N/A

Prior storage conditions and loading rate affect the in vitro fracture response of spinal segments under impact loading

Traumatic injuries of the spine are mostly the consequence of rapid overload e.g. impact loading. In vitro investigations on this topic usually encompass biomechanical testing using frozen/thawed specimens and employ quasi-static loading conditions. It is generally accepted that a freezing/thawing cycle does not alter mechanical properties for slow loading rates. However, this has never been investigated for high impact velocities. In order to assess the effects of freezing/thawing and the influence of different impact velocities, we loaded 27 fresh and 15 frozen/thawed cadaveric rabbit spinal segments (intervertebral disc with one third of the adjacent vertebrae) with different impact energies and velocities using a custom-made, dropped-weight loading device. Endplate fractures were assessed by micro-CT scans. Specimen dimensions (disk, bone, and total height) and vertebrae bone density (BV/TV) were compared pre- and post-trauma. Energy absorption by spinal segments was quantified by measuring the initial ball rebound. We found that freezing/thawing increased endplate fracture frequency and decreased the energy absorption of the segments. Higher impact velocities increased the energy absorption, while higher impact energy increased both energy absorption and fracture frequency. Two conclusions are drawn: first, under impact loading, freezing alters permanently the biomechanical response, and second, for different impact velocities, different fracture initiation mechanisms apply. Therefore, quasi-static loading of frozen/thawed spinal segments is not a valid model for traumatic endplate injuries. However, caution should be exercised in extrapolating these findings to human vertebrae until tests on larger vertebrae are performed.