Histological analysis of nucleus pulposus tissue from patients with lumbar disc herniation after condoliase administration.

Background: Condoliase is an enzyme used as a treatment for lumbar disc herniation (LDH). This enzyme degrades chondroitin sulfate (CS) in the nucleus pulposus of the intervertebral disc (IVD). However, there are cases in which symptoms do not improve, despite condoliase administration. This study reports histological analysis of lumbar disc tissue of LDH patients who underwent surgery because condoliase had no therapeutic effect. Methods: Between March 2019 and August 2019, 12 LDH patients who underwent full endoscopic spine surgery (FESS) discectomy at the Dezawa Akira PED Clinic were the subjects of the study. There are two study groups: six cases underwent FESS after condoliase administration, while six underwent FESS without condoliase administration. The average duration from drug administration to surgery was 152 days. Herniated disc removed at surgery was evaluated by histological staining including immunohistochemistry by anti-CS antibodies. Results: Multiple large clusters (40-120 µm in diameter) were observed in the nucleus pulposus of those who received condoliase, but no clusters were observed in those who did not. The lumbar disc tissues, including the nucleus pulposus of recipients, were stained with anti-CS antibodies that recognize the CS unsaturated disaccharide, but non-administration tissue was not stained. These findings suggest that the enzyme acted on the nucleus pulposus, even in cases where symptoms were not improved by condoliase administration. Furthermore, there was no histological difference between stained images of the extracellular matrix in those who did or did not receive condoliase, suggesting that condoliase acted specifically on CS in the nucleus pulposus. Conclusions: We demonstrated that CS in the nucleus pulposus was degraded in patients in whom condoliase did not have a therapeutic effect. Moreover, condoliase acts in human IVD without causing necrosis of chondrocytes and surrounding tissues.

Selective Chemonucleolysis With Condoliase in Cynomolgus Monkeys.

Selective chemonucleolytic effects of condoliase, a glycosaminoglycan degrading enzyme, was investigated histopathologically in cynomolgus monkeys. Condoliase was administered once into the lumber intervertebral disc (IVD), and as a comparative control, chymopapain, a proteolytic enzyme, was administered in a similar manner. Histopathological changes of the IVD and the adjacent vertebral body (VB) were examined at 1 to 26 weeks after administration. Major changes induced by condoliase in the IVD were degenerative and necrotic changes in the nucleus pulposus, annulus fibrosus, cartilaginous endplate (CEP), and epiphyseal growth plate (EGP); focal disappearance of the EGP; and neovascularization and ossification of the CEP. Decreased/necrosis of bone marrow cells with new bone formation was observed in the VB. Cellular regeneration in the IVD was observed as a recovery changes on and after week 4. The changes in the IVD and VB subsided at week 26. Chymopapain induced qualitatively similar but more widely extended changes. The degrees of the changes in the IVD and VB were more severe than those of condoliase, and the changes were exacerbated even at week 26. These results indicated that histopathological changes caused by condoliase were less severe and more selective than those by chymopapain.

Isolation and characterization of monoclonal antibodies specific for chondroitin sulfate E.

Chondroitin sulfate E (CSE) is a polysaccharide containing mainly disaccharide units of D-glucuronic acid (GlcA) and 4,6-O-disulfated N-acetyl-D-galactosamine (GalNAc) residues (E-unit) in the amount of ∼ 60%. CSE is involved in many biological and pathological processes. In this study, we established new monoclonal antibodies, termed E-12C and E-18H, by using CSE that contained more than 70% of E-units as an immunogen. These antibodies recognized CSE but not other CSs isomers or dermatan sulfate (DS). We evaluated the reactivities of the antibodies to 6-O-sulfated CSA (6S-CSA) and DS (6S-DS) that possessed ∼ 60% of GalNAc (4S, 6S) moieties in their structures. Neither of the antibodies reacted with 6S-DS. The antibodies strictly distinguished the structural difference of GlcA and L-iduronic acid in the polysaccharide. Binding affinities of the antibodies were determined by a surface plasmon resonance assay using CSE and 6S-CSA. The binding affinities were strongly associated with the molecular weight of CSE and the E-unit content of 6S-CSA. Moreover, we demonstrated that the antibodies are applicable to histochemical analysis. In conclusion, the new anti-CSE monoclonal antibodies specifically recognize the E-unit of CSE. The antibodies will become useful tools for the investigation of the biological and pathological significance of CSE.

Effects of Repeated Intravenous Administration of Dextrans,Water-soluble Macromolecules, in Rats.

We investigated the influence of repeated intravenous administration of dextrans (DEXs) to rats. Seven-week-old Sprague Dawley rats (6 males/group) were given intravenously 10% saline solutions of dextrans (DEXs), 40 kDa or 200-300 kDa, at a dose level of 5 mL/kg/day for 28 days and they were examined histopathologically. Another group (3 males/group) was administered saline in a similar manner and served as the control. Histopathological changes indicating accumulation of DEXs in the mononuclear phagocyte system (MPS) and the liver were noted in the treated groups. The incidence and severity of the findings were molecular weight-dependent, except for the lungs. These results are considered useful in interpreting data from preclinical studies, in which DEXs or their derivatives are administered as test or control substances.

Evaluation of in vivo biocompatibility and biodegradation of photocrosslinked hyaluronate hydrogels (HADgels).

HADgels are newly developed photocrosslinked hyaluronate hydrogels. They are produced from an aqueous solution of a hyaluronan derivative (HAD) in which cinnamic acid is introduced into the carboxyl moiety of hyaluronan using 3-aminopropanol as a spacer. High-energy ultraviolet irradiation of the HAD solution induces photodimerization of cinnamic acid, resulting in the development of a macromolecular network of each hyaluronan to water-insoluble hydrogels. The biocompatibility and biodegradation of HADgels were evaluated by guinea pig intracutaneous injection testing for up to 28 days. By macroscopic and histological observations, HADgels showed good tissue compatibility and did not induce excess inflammation at the injection sites. Biodegradation of the HADgels clearly depended on the degree of crosslinking at the fixed weight concentrations of HAD (0.5% and 1.0%). In addition, serum analyses showed that the injected guinea pigs did not produce specific antibodies against HADgels. These results indicate that HADgels have preferable biocompatibility and can be used as a new class of injectable, absorbable biomaterial, especially for preventing postsurgical adhesion formations.