Adverse effects of stromal vascular fraction during regenerative treatment of the intervertebral disc: observations in a goat model.

Stromal vascular fraction (SVF), an adipose tissue-derived heterogeneous cell mixture containing, among others, multipotent adipose stromal cells (ASCs) and erythrocytes, has proved beneficial for a wide range of applications in regenerative medicine. We sought to establish intervertebral disc (IVD) regeneration by injecting SVF intradiscally during a one-step surgical procedure in an enzymatically (Chondroitinase ABC; cABC) induced goat model of disc degeneration. Unexpectedly, we observed a severe inflammatory response that has not been described before, including massive lymphocyte infiltration, neovascularisation and endplate destruction. A second study investigated two main suspects for these adverse effects: cABC and erythrocytes within SVF. The same destructive response was observed in healthy goat discs injected with SVF, thereby eliminating cABC as a cause. Density gradient removal of erythrocytes and ASCs purified by culturing did not lead to adverse effects. Following these observations, we incorporated an extra washing step in the SVF harvesting protocol. In a third study, we applied this protocol in a one-step procedure to a goat herniation model, in which no adverse responses were observed either. However, upon intradiscal injection of an identically processed SVF mixture into our goat IVD degeneration model during a fourth study, the adverse effects surprisingly occurred again. Despite our quest for the responsible agent, we eventually could not identify the mechanism through which the observed destructive responses occurred. Although we cannot exclude that the adverse effects are species-dependent or model-specific, we advertise caution with the clinical application of autologous SVF injections into the IVD until the responsible agent(s) are identified.

MRI T2* mapping correlates with biochemistry and histology in intervertebral disc degeneration in a large animal model.

PURPOSE: To evaluate intervertebral disc (IVD) degeneration and treatments, an objective diagnostic tool is needed. Recently, T2* relaxation time mapping was proposed as a technique to assess early IVD degeneration, yet the correlation with biochemical content and histological features has not been investigated previously. Our objective was to validate T2* mapping for disc degeneration by correlating this technique with accepted parameters of IVD degeneration. METHODS: Mildly and severely degenerated lumbar discs were obtained from an in vivo large animal study; two healthy goat spines were acquired as control. In total, 48 IVDs were analysed using T2-weighted MRI, T2* relaxation time mapping, biochemical assays, macroscopic and histological scoring. Correlations between variables were expressed with Spearman's rho (ρ) coefficients. RESULTS: A complete range of degenerative grades were obtained (mean histological grade 2.2, range 0-6). A linear positive correlation was observed between T2* relaxation time and glycosaminoglycan content (ρ = 0.64, p < 0.001). T2* relaxation time decreased linearly with increasing degeneration as assessed with Pfirrmann scoring system (ρ = -0.67, p < 0.001), macroscopic (ρ = -0.33, p < 0.05) and histological (ρ = -0.45, p < 0.05) grading. CONCLUSIONS: T2* mapping is an MRI technique for IVD evaluation which allows for measurements on a continuous scale thus minimising observer bias compared to grading systems. Although limited by a small sample size, this study showed a relatively good and linear correlation between T2* relaxation time and accepted parameters of disc degeneration. This suggests that T2* mapping is a promising tool to assess disc degeneration in clinical practice.

Poor Radiological and Good Functional Long-term Outcome of Surgically Treated Scheuermann Patients.

STUDY DESIGN: Cohort study. OBJECTIVE: To analyze long-term clinical and radiological outcomes of surgically treated Scheuermann patients. SUMMARY OF BACKGROUND DATA: Long-term clinical and radiological outcomes of surgery for Scheuermann kyphosis are unknown. A single-center cohort of 33 consecutive, surgically treated (between 1991 and 1998) Scheuermann patients was studied. METHODS: Clinical and radiological data of 29 surgically treated Scheuermann patients were collected (posterior approach n = 13; combined anterior-posterior procedure n = 16), after a mean follow-up of 18 years. Oswestry Disability Index (ODI) scores were measured preoperatively (PRE) and twice postoperatively: 2 to 8 years postoperative (FU 1) and 14 to 21 years postoperative (FU 2). Visual Analog Score pain, Short Form-36 (SF-36), and EQ-5d scores were recorded at FU 2 only. Radiographs were analyzed for correction, distal and proximal junctional kyphosis, and implant failures. RESULTS: Mean preoperative kyphosis of the corrected levels was 76° (range 60°-105°) and decreased to a Cobb of 58°(range 30°-105°) at FU 2. Median Visual Analog Score was 2.5 points (range 0-8) and median ODI score was 12 (range 0-62) at FU 2. The ODI score at FU 1 was significantly better as compared to PRE (P < 0.001) and FU 2 (P < 0.001). Also, anterior-posterior treated group had a significantly better ODI score as compared to the posterior-only group (P = 0.023). EQ-5d scores on mobility, usual activities, and pain/discomfort were worse compared to an age-matched population control group; however, SF-36 outcome scores were comparable.Proximal junctional kyphosis was present in 53% of patients, distal junctional kyphosis did not occur, and implant failure/removal had occurred in 69% of patients. Radiological complications do not relate with the ODI, EQ-5d, and SF-36 and 72% of the patients were satisfied. CONCLUSION: Radiological results of this cohort were disappointing but did not relate to clinical outcome scores. Even lumbar pain could not prevent a high patient satisfaction and quality of life. Patients treated with a combined anterior-posterior approach tended to perform better. LEVEL OF EVIDENCE: 3.

Biomechanical and rheological characterization of mild intervertebral disc degeneration in a large animal model.

Biomechanical properties of healthy and degenerated nucleus pulposus (NP) are thought to be important for future regenerative strategies for intervertebral disc (IVD) repair. However, which properties are pivotal as design criteria when developing NP replacement materials is ill understood. Therefore, we determined and compared segmental biomechanics and NP viscoelastic properties in normal and mildly degenerated discs. In eight goats, three lumbar IVDs were chemically degenerated using chondroitinase ABC (CABC), confirmed with radiography and MRI after euthanasia 12 weeks post-operative. Neutral zone (NZ) stiffness and range of motion (ROM) were determined sagitally, laterally, and rotationally for each spinal motion segment (SMS) using a mechanical testing device. NPs were isolated for oscillatory shear experiments; elastic and viscous shear moduli followed from the ratio between shear stress and strain. Water content was quantified by weighing before and after freeze-drying. Disc height on radiographs and signal intensity on MRI decreased (6% and 22%, respectively, p < 0.01) after CABC treatment, confirming that chemical degeneration provides a good model of disc degeneration. Furthermore, CABC-injected IVDs had significantly lower NZ stiffness and larger ROM in lateral bending (LB) and axial rotation (AR) than controls. Rheometry consistently revealed significantly lower (10-12%) viscoelastic moduli after mild degeneration within goats, though the inter-animal differences were relatively large (complex modulus ∼12 to 41 kPa). Relative water content in the NP was unaffected by CABC, remaining at ∼75%. These observations suggest that viscoelastic properties have a marginal influence on mechanical behavior of the whole SMS. Therefore, when developing replacement materials the focus should be on other design criteria, such as biochemical cues and swelling pressure.

Reproducible long-term disc degeneration in a large animal model.

STUDY DESIGN: Twelve goats were chemically degenerated and the development of the degenerative signs was followed for 26 weeks to evaluate the progression of the induced degeneration. The results were also compared with a previous study to determine the reproducibility. OBJECTIVES: The purpose of this study was determine whether this Chondroitinase ABC (CABC) induced goat model is reproducible and to study the development of the degeneration in time up to 26 weeks. SUMMARY OF BACKGROUND DATA: Injecting CABC into goat intervertebral discs results in mild disc degeneration after 12 weeks. Spontaneous recovery or leveling off of the degeneration has been reported before and is relevant when the goat model is used in regeneration studies. Reproducibility of the induced degeneration is relevant as well. METHODS: Twelve goats were used in this study. The development of degeneration was studied after the injection of 0.25 U/mL CABC intradiscally. The development of degenerative signs was studied after 18 (n = 6) and 26 (n = 6) weeks by means of radiograph, magnetic resonance imaging, macroscopic analysis, and histology and biochemical evaluation. The induced degeneration was compared with the results from a previous study, in which degeneration was induced similarly and analysis was performed after 12 weeks. RESULTS: The severity of the degenerative signs was mild and was consequently present in all parameters analyzed. When compared with the results after 12 weeks, the degeneration was similar in the present study. Spontaneous recovery was not observed up to 26 weeks. CONCLUSION: The injection with CABC in the intervertebral disc reproducibly results in mild disc degeneration in the goat. These findings corroborate the goat model as a suitable large animal model to evaluate mild disc degeneration and potential new therapies.

Adjacent segment degeneration: observations in a goat spinal fusion study.

STUDY DESIGN: The adjacent discs of 13 goats, originally used in a lumbar spinal fusion model study, were analyzed for symptoms of intervertebral disc degeneration by means of magnetic resonance imaging (MRI), macroscopy, and histology. These goats were followed for 6 months and the results were compared with 6 control goats. OBJECTIVE: To evaluate the development of adjacent segment disc degeneration in vivo in a goat lumbar spinal fusion model. SUMMARY OF BACKGROUND DATA: There is ongoing debate on whether adjacent segment degeneration (ASD) develops through increased biomechanical load on discs adjacent to fusion sites, or by the natural process of pre-existing degenerative disease. Animal models offer an opportunity to separate these factors by evaluating the development of ASD in nondegenerated animal spines. METHODS: In a spinal fusion model study 2 segments (L3-L4 and L1-L2) were fixated and followed for 3 months (n = 6) and 6 months (n = 7) in 13 skeletally mature goats. Two adjacent discs (T13-L1 and L4-L5), 1 interjacent disc (L2-L3) and a control disc (L5-L6) were analyzed by means of magnetic resonance imaging, macroscopy, and histology. These results were compared with the discs of 6, nonoperated "normal" goats. RESULTS: No differences were observed in the adjacent and interjacent intervertebral discs after both follow-up periods. However, severe degenerative changes were observed in the L5-L6 level, originally included as controls. CONCLUSION: Large animal fusion models offer an excellent opportunity to study ASD in vivo, as pre-existing degenerative disc disease is not present and biomechanical effects of the fusion can be studied more isolated. Our results suggest that adjacent disc degeneration does not develop in our spinal goat fusion model. There is, however, an increased risk of disc degeneration in the L5-L6 level through an unclear mechanism.

Sterilization and strength of 70/30 polylactide cages: e-beam versus ethylene oxide.

STUDY DESIGN: In vitro and in vivo studies on the degradation of 70/30 poly(L,DL-lactide) (PLDLLA) cages. OBJECTIVE: To evaluate the effect of e-beam and ethylene oxide sterilization on degradation and strength. SUMMARY OF BACKGROUND DATA: e-beam-sterilized PLDLLA cages were shown to maintain mechanical strength for at least 6 months during degradation studies in vitro. Yet failure of the cages was observed after only 3 months in vivo. We hypothesized that degradation characteristics and mechanical strength could be improved by sterilizing the cages through ethylene oxide (EtO) instead of e-beam. METHODS: PLDLLA cages were sterilized either by e-beam or EtO, and degraded in phosphate-buffered saline. Each month, cages were compressed until failure. Inherent viscosity was determined as a measure of degradation. For the in vivo evaluation, e-beam- or EtO-sterilized cages were implanted at L3-L4 in a standardized goat model. After 3 or 6 months, retrieved segments were scanned by high-resolution magnetic resonance imaging. Also, inherent viscosity of the polymer was measured. RESULTS: e-beam sterilization strongly decreased inherent viscosity of PLDLLA compared with EtO sterilization, but initial strength was only affected marginally. After 6 months, the strength of the e-beam-sterilized cages dropped, while that of EtO-sterilized cages was maintained. Degradation in vivo was slightly faster than in vitro. In both groups, however, mechanical failure occurred at 3 months after implantation. CONCLUSIONS: Inherent viscosity decreases with degradation time, but strength only decreases when inherent viscosity is below a certain threshold. Above this threshold, mechanical strength is a property of the polymer and independent of inherent viscosity. e-beam sterilization strongly decreases inherent viscosity and thus advances mechanical degradation. EtO sterilization delays degradation but does not increase initial strength. Early failure of PLDLLA cages in the goat model thus is unrelated to sterilization method and requires further study.

In vivo comparison of Dhvar-5 and gentamicin in an MRSA osteomyelitis prevention model.

OBJECTIVES: The continued rise in drug-resistant pathogens has led to global research efforts into new antimicrobial agents. A promising class of new agents are the antimicrobial peptides. The aim of the study was to investigate the efficacy of the antimicrobial peptide Dhvar-5 in a prophylactic, methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis model. METHODS: Dhvar-5 (12 mg or 24 mg/rabbit) was incorporated into polymethyl methacrylate (PMMA) beads as a local drug delivery system. For comparison, plain beads (control) and beads containing gentamicin as a sulphate (10 mg or 24 mg per rabbit) were also prepared. The beads were inserted into the inoculated femoral cavity of 36 rabbits, and 1 week later they were killed. The presence and severity of MRSA osteomyelitis was assessed by culture and histology. RESULTS: Both the 24 mg Dhvar-5 beads and the 24 mg gentamicin sulphate beads significantly reduced the bacterial load of the inoculated femora compared with the control chain. Although a 24 mg Dhvar-5 dose inhibited MRSA growth, it did not completely sterilize the femora. Sterilization occurred only in some of the gentamicin-treated specimens. CONCLUSION: We conclude that both the gentamicin beads and the Dhvar-5 beads were only partially effective at preventing MRSA infection in this model.