Controversies in spine research: organ culture versus in vivo models for studies of the intervertebral disc

Intervertebral disc degeneration is a common cause of low back pain, the leading cause of disability worldwide. Appropriate preclinical models for intervertebral disc research are essential to achieving a better understanding of underlying pathophysiology and for the development, evaluation, and translation of more effective treatments. To this end, in vivo animal and ex vivo organ culture models are both widely used by spine researchers; however, the relative strengths and weaknesses of these two approaches are a source of ongoing controversy. In this article, members from the Spine and Preclinical Models Sections of the Orthopedic Research Society, including experts in both basic and translational spine research, present contrasting arguments in support of in vivo animal models versus ex vivo organ culture models for studies of the disc, supported by a comprehensive review of the relevant literature. The objective is to provide a deeper understanding of the respective advantages and limitations of these approaches, and advance the field toward a consensus with respect to appropriate model selection and implementation. We conclude that complementary use of several model types and leveraging the unique advantages of each is likely to result in the highest impact research in most instances

Muscle atrophy and fatty infiltration after an acute rotator cuff repair in a sheep model.

Introductionrotator cuff tears (RCTs) are the most common tendon injury seen in orthopedic patients. Muscle atrophy and fatty infiltration of the muscle are crucial factors that dictate the outcome following rotator cuff surgery. Though less studied in humans, rotator cuff muscle fibrosis has been seen in animal models as well and may influence outcomes as well. The purpose of this study was to determine if the rotator cuff would develop muscle changes even in the setting of an acute repair in a sheep model. We hypothesized that fatty infiltration and fibrosis would be present even after an acute repair six months after initial surgery.Methodstwelve female adult sheep underwent an acute rotator cuff tear and immediate repair on the right shoulder. The left shoulder served as a control and did not undergo a tear or a repair. Six months following acute rotator cuff repairs, sheep muscles were harvested to study atrophy, fatty infiltration, and fibrosis by histological analysis, western blotting, and reverse transcription polymerase chain reaction (RT-PCR).Resultsthe repair group demonstrated an increase expression of muscle atrophy, fatty infiltration, and fibrosis related genes. Significantly increased adipocytes, muscle fatty infiltration, and collagen deposition was observed in rotator cuff muscles in the tendon repair group compared to the control group.Conclusionsrotator cuff muscle undergoes degradation changes including fatty infiltration and fibrosis even after the tendons are repair immediately after rupture.Level of evidenceBasic Science Study

Computed Tomography Provides Improved Quantification of Trabecular Lumbar Spine Bone Loss Compared to Dual‐Energy X‐Ray Absorptiometry in Ovariectomized Sheep

ABSTRACT Early detection of osteoporosis using advanced imaging is imperative to the successful treatment and prevention of high morbidity fractures in aging patients. In this preclinical study, we aimed to compare dual‐energy X‐ray absorptiometry (DXA) and quantitative computed tomography (QCT) to quantify bone mineral density (BMD) changes in the sheep lumbar spine. We also aimed to determine the relationship of BMD to microarchitecture in the same animals as an estimate of imaging modality precision. Osteoporosis was induced in 10 ewes via laparoscopic ovariectomy and administration of high‐dose corticosteroids. We performed DXA and QCT imaging to measure areal BMD (aBMD) and trabecular volumetric BMD (Tb.vBMD)/cortical vBMD (Ct.vBMD), respectively, at baseline (before ovariectomy) and at 3, 6, 9, and 12 months after ovariectomy. Iliac crest bone biopsies were collected at each time point for micro‐computed tomography (microCT) analysis; bone volume fraction (BV/TV), trabecular number (Tb.N), thickness (Tb.Th), and spacing (Tb.Sp) were reported. aBMD and Tb.vBMD both decreased significantly by 3 and 6 months (p < 0.05) compared with baseline, whereas no changes to Ct.vBMD were observed. Combined (Tb. and Ct.) vBMD was significantly correlated with aBMD at all time points (all p < 0.05). Additionally, greater significant correlations were found between BV/TV and Tb.vBMD at all five time points (R2 = 0.54, 0.57, 0.66, 0.46, and 0.56, respectively) than with aBMD values (R2 = 0.23, 0.55, 0.41, 0.20, and 0.19, respectively). The higher correlation of microCT values with QCT than with DXA indicates that QCT provides additional detailed information regarding bone mineral density changes in preclinical settings. Because trabecular bone is susceptible to rapid density loss and structural changes during osteoporosis, QCT can capture these subtle changes more precisely than DXA in a large animal preclinical model. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research

Electronics module design and function.

<p>(a) Diagram of adherence IVR electronics module showing major functional components (boxes) and dataflow (arrows). (b) Plot showing relationships between reference temperature, comparator reference voltage (V_ref), and the 4-bit V_ref generator parameter. Each point represents a reference temperature that is set in software by the generator 4-bit value (0–15). Here a generator value of 11 is set to select a reference temperature of 34°C. Dotted lines indicate V_ref values selected for IN/OUT determination and “Logging On” “Logging Off” functionality in the <i>in vitro</i> and <i>in vivo</i> testing of adherence IVR devices.</p

<i>In vitro</i> evaluation of adherence IVR.

<p>Response of adherence IVR over 100 h using a simulated insertion/removal cycle of 2.5 h in and 0.5 h out (lower plot) using a programmed temperature test apparatus. Data represent the IN/OUT status (0 or 1) logged at a 5 min interval. The upper plot shows expansion of data at 6, 48, and, 96 h for three adherence IVRs measuring simultaneously. Vertical dotted lines indicate transition to “IN” temperature for each cycle shown. The temporal alignment of “IN” measurements does not vary between the three devices over 96 h of measurement. The time required for the device to respond to a simulated insertion or removal event was determined using the temperature-time data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174729#pone.0174729.g005" target="_blank">Fig 5</a>. The device was exposed to three repeated step changes in temperature between 28°C and 37°C, and response was calculated as the time required for the device to measure the range of 10% to 90% of the total 9°C temperature change (28.9°C and 36.1°C). For the step from 28°C to 37°C (simulated insertion), the 10%-90% time was 1.84 ± 0.006 min. For the step from 37°C to 28°C (simulated removal), the 90% to 10% time was 1.96 ± 0.096 min.</p

Effects of Fisetin Treatment on Cellular Senescence of Various Tissues and Organs of Old Sheep

Fisetin has been shown to be beneficial for brain injury and age-related brain disease via different mechanisms. The purpose of this study was to determine the presence of senescent cells and the effects of fisetin on cellular senescence in the brain and other vital organs in old sheep, a more translational model. Female sheep 6–7 years old (N = 6) were treated with 100 mg/kg fisetin or vehicle alone on two consecutive days a week for 8 weeks. All vital organs were harvested at the time of sacrifice. Histology, immunofluorescence staining, and RT-Q-PCR were performed on different regions of brain tissues and other organs. Our results indicated that fisetin treatment at the current regimen did not affect the general morphology of the brain. The presence of senescent cells in both the cerebral brain cortex and cerebellum and non-Cornu Ammonis (CA) area of the hippocampus was detected by senescent-associated β-galactosidase (SA-β-Gal) staining and GL13 (lipofuscin) staining. The senescent cells detected were mainly neurons in both gray and white matter of either the cerebral brain cortex, cerebellum, or non-CA area of the hippocampus. Very few senescent cells were detected in the neurons of the CA1-4 area of the hippocampus, as revealed by GL13 staining and GLB1 colocalization with NEUN. Fisetin treatment significantly decreased the number of SA-β-Gal+ cells in brain cortex white matter and GL13+ cells in the non-CA area of the hippocampus, and showed a decreasing trend of SA-β-Gal+ cells in the gray matter of both the cerebral brain cortex and cerebellum. Furthermore, fisetin treatment significantly decreased P16+ and GLB1+ cells in neuronal nuclear protein (NEUN)+ neurons, glial fibrillary acidic protein (GFAP)+ astrocytes, and ionized calcium binding adaptor molecule 1 (IBA1)+ microglia cells in both gray and white matter of cerebral brain cortex. Fisetin treatment significantly decreased GLB1+ cells in microglia cells, astrocytes, and NEUN+ neurons in the non-CA area of the hippocampus. Fisetin treatment significantly decreased plasma S100B. At the mRNA level, fisetin significantly downregulated GLB1 in the liver, showed a decreasing trend in GLB1 in the lung, heart, and spleen tissues, and significantly decreased P21 expression in the liver and lung. Fisetin treatment significantly decreased TREM2 in the lung tissues and showed a trend of downregulation in the liver, spleen, and heart. A significant decrease in NRLP3 in the liver was observed after fisetin treatment. Finally, fisetin treatment significantly downregulated SOD1 in the liver and spleen while upregulating CAT in the spleen. In conclusion, we found that senescent cells were widely present in the cerebral brain cortex and cerebellum and non-CA area of the hippocampus of old sheep. Fisetin treatment significantly decreased senescent neurons, astrocytes, and microglia in both gray and white matter of the cerebral brain cortex and non-CA area of the hippocampus. In addition, fisetin treatment decreased senescent gene expressions and inflammasomes in other organs, such as the lung and the liver. Fisetin treatment represents a promising therapeutic strategy for age-related diseases