Annulus fibrosus (AF) defects from annular tears, herniation, and discectomy procedures are associated with painful conditions and accelerated intervertebral disc (IVD) degeneration. Currently, no effective treatments exist to repair AF damage, restore IVD biomechanics and promote tissue regeneration. An injectable fibrin-genipin adhesive hydrogel (Fib-Gen) was evaluated for its performance repairing large AF defects in a bovine caudal IVD model using ex vivo organ culture and biomechanical testing of motion segments, and for its in vivo longevity and biocompatibility in a rat model by subcutaneous implantation. Fib-Gen sealed AF defects, prevented IVD height loss, and remained well-integrated with native AF tissue following approximately 14,000 cycles of compression in 6-day organ culture experiments. Fib-Gen repair also retained high viability of native AF cells near the repair site, reduced nitric oxide released to the media, and showed evidence of AF cell migration into the gel. Biomechanically, Fib-Gen fully restored compressive stiffness to intact levels validating organ culture findings. However, only partial restoration of tensile and torsional stiffness was obtained, suggesting opportunities to enhance this formulation. Subcutaneous implantation results, when compared with the literature, suggested Fib-Gen exhibited similar biocompatibility behaviour to fibrin alone but degraded much more slowly. We conclude that injectable Fib-Gen successfully sealed large AF defects, promoted functional restoration with improved motion segment biomechanics, and served as a biocompatible adhesive biomaterial that had greatly enhanced in vivo longevity compared to fibrin. Fib-Gen offers promise for AF repairs that may prevent painful conditions and accelerated degeneration of the IVD, and warrants further material development and evaluation.

IntroductionIncreased expression of the proinflammatory cytokine TNF-α in intervertebral discs (IVDs) leads to inflammation, which results in progressive IVD degeneration. We have previously reported that activation of Wnt-β-catenin (hereafter called Wnt) signaling suppresses the proliferation of nucleus pulposus cells and induces cell senescence, suggesting that Wnt signaling triggers the process of degeneration of the IVD. However, it is not known whether cross talk between TNF-α and Wnt signaling plays a role in the regulation of nucleus pulposus cells. The goal of the present study was to examine the effect of the interaction between Wnt signaling and the proinflammatory cytokine TNF-α in nucleus pulposus cells.MethodsCells isolated from rat nucleus pulposus regions of IVDs were cultured in monolayers, and the expression and promoter activity of Wnt signaling and TNF-α were evaluated. We also examined whether the inhibition of Wnt signaling using cotransfection with Dickkopf (DKK) isoforms and Sclerostin (SOST) could block the effects of pathological TNF-α expression in nucleus pulposus cells.ResultsTNF-α stimulated the expression and promoter activity of Wnt signaling in nucleus pulposus cells. In addition, the activation of Wnt signaling by 6-bromoindirubin-3′-oxime (BIO), which is a selective inhibitor of glycogen synthase kinase 3 (GSK-3) activity that activates Wnt signaling, increased TNF-α expression and promoter activity. Conversely, the suppression of TNF-α promoter activity using a β-catenin small interfering RNA was evident. Moreover, transfection with DKK-3, DKK-4, or SOST, which are inhibitors of Wnt signaling, blocked Wnt signaling-mediated TNF-α activation; these effects were not observed for DKK-1 or DKK-2.ConclusionsHere, we have demonstrated that Wnt signaling regulates TNF-α and that Wnt signaling and TNF-α form a positive-feedback loop in nucleus pulposus cells. The results of the present study provide in vitro evidence that activation of Wnt signaling upregulates the TNF-α expression and might cause the degeneration of nucleus pulposus cells. We speculate that blocking this pathway might protect nucleus pulposus cells against degeneration.

Characterization of cells is important for facilitating cell-based therapies for degenerative diseases of intervertebral discs. For this purpose, we analyzed mouse annulus fibrosus cells by flowcytometory to detect phenotypic change in their primary cultures. After examination of sixteen cell surface proteins, we focused on CD146 that solely increased during culture expansion. CD146 is known to be a marker for mesenchymal stem cells and for their vascular smooth muscle commitment with expression of contractile phenotype enhanced by SM22a. We sorted CD146þ cells to elucidate their characteristics and the key factors that play a role in this change. Whole cell cultures showed the ability for tripotent differentiation toward mesenchymal lineages, whereas sorted CD146þ cells did not. Expression of CD146 was elevated by addition of transforming growth factor b1, and sorted CD146þ cells expressed higher levels of mRNA for SM22a and Elastin than did CD146À cells. Morphologically, CD146þ cells more broadly deposited extracellular type I collagen than CD146À cells and showed filamentous actin bundles traversing their cytoplasm and cell-cell junctions. Moreover, CD146þ cells demonstrated significantly higher gel contraction properties than CD146À cells when they were embedded in collagen gels. Human annulus fibrosus CD146þ cells also showed higher contractility. Immunohistochemistry determined CD146þ cells localized to the outermost annulus layers of mouse intervertebral disc tissue with co-expression of SM22a. These results suggest that increment of CD146 expression indicates gradual change of cultured annulus fibrosus cells to express a contractile phenotype and that transforming growth factor b1 enhances this cellular commitment. ß

A multitude of studies has indicated the potential of cell therapy as a method for intervertebral disc (IVD) regeneration. Transplantation of a variety of cells has been assessed and shown capable of deterring the rate of degeneration in animal models and in human clinical trials. In this study, a novel approach using human discogenic nucleus pulposus cells directly from their cryopreserved state was assessed. In an established canine disc degeneration model, the degeneration process was evaluated in IVDs receiving precultured discogenic cells, thawedonly discogenic cells, and a saline sham injection after induction of degeneration. Degeneration progression was followed over time by the evaluation of the disc height index (DHI). Finally, after 12 weeks, the manipulated and control discs were explanted, histologically stained, and of the estimated 632 million low back pain patients globally will advance to a chronic condition. 1,2 Together, these compelling numbers engender a critical social-economic burden on society. For example, within the USA, 100 billion USD is spent annually on low back pain associated costs. 3,4 Currently, treatment options are limited and fail to restore or halt further advancement of the underlying pathology. 5 Degeneration of the intervertebral disc (IVD) is widely considered to be a predominant cause of low back pain. IVD degeneration is hallmarked by a dysregulation in extracellular matrix (ECM) homeostasis. 6,7 The exact origin of IVD degeneration remains to be elucidated; however, the nucleus pulposus (NP) is believed to be the place of onset. 8,9 Progression of IVD degeneration is characterized by a decline in proteoglycan production, an increase in matrix degenerative proteins, and a switch from type II collagen to type I collagen production. 6,7 Moreover, NP cells undergo senescence and dedifferentiation toward a more fibrotic phenotype. 10 The NP disorganization and height loss causes incorrect loading of adhering tendons promoting reorganization of tendon ECM to thicker and stiffer structures, further disrupting the mechanical features along the spine.

We investigated how skeletal muscle mass (SMM) affects spinal sagittal balance (radiographic parameters) in symptomatic spinal patients. The first purpose of this study was to evaluate the body composition and the spinal sagittal alignment in symptomatic spinal patients. The second purpose of this study was to compare whether the body composition and the spinal sagittal alignment is different in patients with cervical spine disease and lumbar spine disease. We retrospectively evaluated 313 patients who were hospitalized for surgery to treat spinal degenerative disease, who were divided into cervical and lumbar spine disease groups. All patients underwent full-length standing whole-spine radiography and bioimpedance analysis (BIA) before surgery. We used standard measurements to assess the sagittal vertical axis (SVA), cervical lordosis (CL; C2–C7), lumbar lordosis (LL; T12–S1), thoracic kyphosis (TK; T5–12), pelvic incidence (PI), pelvic tilt (PT), and sacral slope (SS). We also analyzed radiological and body composition parameters, patient characteristics, and the correlation between SMM and each sagittal parameters. In the overall cohort, the mean age at the time of operation was 66.5 ± 15.3 years and 59.2% of the patients were men. The correlation coefficients (r) between SMM and PT were negative weak correlation (r = −0.343, P < 0.001). The correlation with SMM for other LL, PI, SS, and SVA was statistically significant, but the correlation was none. In addition, our results also suggested strong correlations (r > 0.5) between LL and SS (r = 0.744), between LL and SVA (r = −0.589), between PT and SS (r = −0.580), and LL and PT (r = −0.506). Fifty-seven patients (18.2%, cervical group) had cervical spine disease and 256 patients (81.8%, lumbar group) had lumbar spine disease. No significant differences in age, height, body weight, and body mass index were observed between the two groups. The SMM of patients with cervical and lumbar spine disease also did not differ significantly. In the lumbar group, correlations were found between SMM and PT (r = −0.288, P < 0.001), between SMM and LL (r = 0.179, P < 0.01), and between SMM and SS (r = 0.170, P < 0.01), while only PT (r = −0.480, P < 0.001) was negatively correlated with SMM in the cervical group. This analysis indicated that PT is the sagittal parameter most closely related to SMM in patients with the spinal degenerative disease. The SMM might be one of the important factors that influenced the posterior inclination of the pelvis in symptomatic spinal patients, especially in cervical spine disease.