Kosaku Mizuno

Immunohistologic study of the ruptured intervertebral disc of the lumbar spine

Study Design This study analyzed immunohistological features of the extruded or sequestrated intervertebral disc of the lumbar spine. To clarify the pathogenesis of neovascularization, cells isolated from herniated disc were cultured and examined biologically. Objectives The objectives of this study were to characterize the histologic features of extruded or sequestrated discs and inflammatory cells that infiltrate along the margins of the disc tissue and to clarify the pathogenesis of neovascularization observed at the edge of the disc tissue. Summary of Background Data When some of the contents of the disc extrudes into the epidural space and is considered “foreign,” an autoimmune response develops, which can lead to a chronic inflammatory response. However, the pathogenesis of inflammatory cell infiltrations and neovascularization are not clearly defined. Methods The herniated discs were obtained during surgery and were stained with anti-interleukin-1, intercellular adhesion molecule-1, lymphocyte function-associated antigen, and basic fibroblast growth factor antibodies by using an indirect immunoperoxidase method. Cells isolated from herniated disc were cocultured with human endothelial cells and basic fibroblast growth factor contained by cultured disc cells were measured by radioimmunoassay. Results The ingrowth of granulation tissue with vascularization, occurring at the edge of fibrocartilage fragment, was present at 11 of 16 of extruded and 3 of 5 of sequestrated discs. Anti-interleukin-1, intracellular adhesion molecule-1, lymphocyte function-associated antigen, and basic fibroblast growth factor were expressed on most of mononuclear cells infiltrating into the extruded or sequestrated disc. Cells from the extruded or sequestrated disc demonstrated significantly greater levels of basic fibroblast growth factor than those from the protruded disc, and they enhanced the proliferation of endothelial cells. Conclusions This study showed that mononuclear cells infiltrating along the margins of extruded discs expressed inflammatory mediators and might induce neovascularization and persistence of inflammation.

Low-intensity pulsed ultrasound initiates bone healing in rat nonunion fracture model

Low‐intensity pulsed ultrasound exposure has been shown clinically to shorten the fracture repair process and to induce healing of nonunions in humans, but its mechanism of action remains unclear. In this study we investigated the effect and mechanism of low‐intensity pulsed ultrasound on nonunion fracture healing in rat tibias. A consistently reproducible nonunion was produced in rat tibias by muscle interposition without osteotomy. This model was produced by creating a closed tibial fracture with only the distal end of the tibialis anterior muscle interposed into the fracture site. One limb was noninvasively exposed to low‐intensity pulsed ultrasound (a 200‐millisecond burst of sine waves of 1.5 MHz, repeating at 1.0 kHz) for 20 minutes daily. The incident intensity was approximately 30 mW/cm2. Rats were killed at intervals between 2 and 6 weeks. The events were assessed by radiographs, microfocus X‐ray computed tomograms, and histologic examination. After 6 weeks of exposure, 7 of 14 nonunion fractures showed healing on radiologic assessment. The results of three‐dimensional microfocus X‐ray computed tomographic reconstruction and histologic examination also supported this finding. On the other hand, all control tibias remained in a state of nonunion during the same period. These results indicate that low‐intensity pulsed ultrasound promotes healing in the rat nonunion fracture model.

Effect of basic fibroblast growth factor on the healing of defects in the canine anterior cruciate ligament.

Abstract The effect of basic fibroblast growth factor (bFGF) on the healing of partial anterior cruciate ligament (ACL) lacerations was investigated in 17 adult mongrel canines. The defect was created in the midsubstance of both ACLs using a biopsy punch. In the bFGF group, a bFGF-impregnated pellet was sutured to the infrapatellar fat pad close to the defect. In the control group, the same pellet without bFGF was used. The healing process was evaluated macroscopically and histologically at 1, 3, 6, and 24 weeks postoperatively. In the bFGF group, a pannus-like tissue which contained abundant blood vessels extended into the defect from the adjacent synovial tissue in the early postoperative phase. Histological examination of the tissue which filled the defect revealed initial remodeling processes with a decreased number of cells and better orientation of the collagen fibers at 6–24 weeks. On the other hand, in the control group, poor healing processes were observed at each examination period. This study demonstrated that the application of a bFGF-impregnated pellet may enhance the healing potential of a partially injured ACL. Enhanced neovascularization and the formation of granulation tissue induced by bFGF early in the healing process accounted for the increased healing response.

Graft healing in the bone tunnel in anterior cruciate ligament reconstruction

The histologic sequence in the bone tunnel after anterior cruciate ligament reconstruction using the bone-patellar tendon-bone graft was investigated in this study. Eighteen adult mongrel dogs were used. After excision of the anterior cruciate ligament, the graft was routed through the bone tunnels and fixed with interference fit screws. After the dogs were sacrificed at intervals, the bone blocks containing the bone tunnels were isolated and processed for histologic examination. At the bone-bone interface, incorporation of the bone plug at each end of the graft was completed at 12 weeks. The structure of the tendon insertion of the grafted patellar tendon, consisting of four distinct zones, was observed without apparent necrotic and degenerative change for as long as 12 weeks. Between the tendon and the bone tunnel, a layer of hypercellular fibrous tissue gradually became mature with time. Thus, it appeared the morphologic characteristics and location of the reestablished attachment of the bone-patellar tendon-bone graft were more similar to those of the native anterior cruciate ligament compared with the graft to bone healing in the hamstring tendon graft.

Evaluation of functional deficits determined by four different hop tests in patients with anterior cruciate ligament deficiency

Abstract The purpose of this study was to analyze the validity of the newly designed functional ability test (FAT) for the normal population and patients with deficiency of the anterior cruciate ligament (ACL). The FAT consists of four tests: the figure-of-eight hop, the up-down hop, the side hop, and the single hop. Sixty control subjects and 50 patients with unilateral ACL deficiency were tested. In the control group, the values measured were significantly different between males and females in all of the tests. On the other hand, when left/right difference values were compared, no significant difference was found between males and females in any of the tests. More than 95% of control group exhibited symmetrical function in each part of the FAT, whereas in the ACL-deficient group, the percentage of patients who showed abnormal symmetry was 68% in the figure-of-eight hop, 58% in the up-down hop, 44% in the side hop, and 42% in the single hop. The percentage of ACL-deficient patients with functional asymmetry in at least one of the four tests was 82%. The FAT was found to be useful in evaluating lower limb function in ACL-deficient patients.

Expression of platelet-derived growth factor proteins and their receptor α and β mRNAs during fracture healing in the normal mouse

Abstract Platelet-derived growth factor (PDGF), abundant in bone tissue, has been reported to stimulate mesenchymal cell proliferation and migration. To elucidate the functional roles of PDGF during fracture healing, we investigated the expression of PDGF-A and -B chain proteins and receptor α and β mRNAs in fractured mouse tibiae. Twelve-week-old male BALB/c mice were operated on to make a closed fracture on the proximal tibia. On days 2, 4, 7, 10, 14, 21, and 28 after the operation, the fractured tibiae were excised, fixed with 4% paraformaldehyde, decalcified with 20% EDTA, and embedded in paraffin to prepare 7-µm sections. Immunohistochemistry using polyclonal antibodies against human PDGF-A and -B chains was carried out by the avidin-biotin-peroxidase method. For in situ hybridization, we used digoxigenin-labeled single-stranded DNA probes specific for mouse PDGF receptors α and β generated by unidirectional polymerase chain reaction. In the inflammatory phase on days 2–4 after the fracture, mesenchymal cells gathering at the fracture site expressed the PDGF-B chain and β receptor mRNA. At the stage of cartilaginous callus formation on day 7, the immunoreactivity for PDGF-A and -B chains on proliferating and hypertrophic chondrocytes and the signals of α and β receptor mRNAs on proliferating chondrocytes became manifest. At the stage of bony callus and bone remodeling on days 14–21, the predominant expression of the PDGF-B chain and β receptor was observed on both osteoclasts and osteoblasts. On day 28, signals for PDGF ligand proteins and receptor mRNAs diminished. The coincidental localization of PDGF ligands and their receptors implies a paracrine and autocrine mechanism. Our data suggested that PDGF contributed in part to the promotion of the chondrogenic and osteogenic changes of mesenchymal cells from the early to the midphase of fracture healing; the functions mediated by the β receptor, including cell migration, might be prerequisites to the recruitment of mesenchymal cells in the initial step and to the interaction between osteoclasts and osteoblasts in the bone remodeling phase.

Graft tension and knee stability after anterior cruciate ligament reconstruction

This study examined the effect of the initial graft tension on the clinical outcome of anterior cruciate ligament reconstruction in a prospective randomized design. Fifty patients were enrolled in the study. In the reconstructive procedure, a bone-patellar tendon-bone graft was used. A set force of 25 N (5.6 lb) was applied in 25 patients and 50 N (11.2 lb) tension was maintained during fixation for the remaining 25 patients with the knee in full extension. In the immediate postoperative period, the anterior cruciate ligament reconstructed knee was similarly overconstrained in both groups. At 3 months, laxity of the surgically treated knee in both groups had increased significantly and become close to that of the contralateral knee. Measured laxity value showed a slight increase between 3 and 6 months, and remained similar thereafter. No significant difference in the clinical outcome was observed between the groups throughout the followup.

Localization of growth factors in the reconstructed anterior cruciate ligament: immunohistological study in dogs

Abstract This study was designed to examine localization of the growth factors in the autogenous patellar tendon graft used to reconstruct the anterior cruciate ligament (ACL) in the canine model. Among the various growth factors, basic fibroblast growth factor, transforming growth factor-β1, and platelet-derived growth factor were selected for analysis as potential factors that regulate graft remodeling processes. In the control patellar tendon and the ACL only basic fibroblast growth factor was positively stained. In the reconstructed graft increased levels of staining for all the three factors were observed in the early postoperative period, reaching the greatest expression 3 weeks after implantation. Thereafter immunoreactivity of these growth factors decreased and returned to the preoperative levels, which were similar to that of the control ACL 12 weeks postoperatively. This rapid reduction in the level of their localization indicates that once the extrinsic cells are infiltrated to the graft and revascularization completed, these growth factors may have less significance for subsequent remodeling.

Use of hydroxyapatite to fill cavities after excision of benign bone tumours

We treated 75 patients with benign bone tumours by curettage and filling the defect with calcium hydroxyapatite (HA). There were 28 women and 47 men with a mean age of 27.7 years (3 to 80). The mean follow-up was for 41.3 months. Postoperative radiological assessment revealed that the implanted HA was well incorporated into the surrounding host bone in all patients. Two patients suffered fractures in the postoperative period. Two patients complained of pain associated with HA in the soft tissues, but this diminished within six months. No patient had local pain at the final follow-up. Recurrence of the tumour was seen in three cases. Histopathological study of the implanted area showed removal of the HA by histiocytes and multinucleated giant cells, and the formation of much appositional bone. We conclude that HA is an excellent bone-graft substitute in surgery for benign bone tumours.

The role of cyclooxygenase-2 and inflammatory cytokines in pain induction of herniated lumbar intervertebral disc

Lumbar disc herniation (LDH) is the disease which is the major cause of radiculopathy. In terms of the pathogenesis of disease, it is reported that prostaglandinE2 (PGE2) plays an important role to induce radiculopathy. Arachidonate cascade, which is the process of PGE2 synthesis, is mainly regulated by two kinds of enzymes, phospholipaseA2 (PLA2) and cyclooxy genase (COX). Previously, PLA2 was recognized as the rate-limiting enzyme of this cascade, and some authors reported the clinical significance of PLA2 at the site of LDH concerning the radicular pain. Recently, COX was elucidated to consist of 2 types of isoform, a constitutive form of COX-1 and an inducible form of COX-2. COX-2 has been focused as a key enzyme to regulate PGE2 synthesis and plays an important role in inflammation, because COX-2 was induced in many types of cells by the stimulation of inflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha). However, it is not fully discussed whether or not, COX-2 is induced in lumbar disc tissue and if it plays a significant role in the pathogenesis of LDH. To clarify the role of COX-2 in the pathomechanism of radiculopathy of LDH, we have investigated the expression of COX-2, IL-1 beta and TNF alpha in herniated lumbar disc tissue. Immunohistologically, they were detected in the cytosol of chondrocytes constituting the disc tissue. RT-PCR showed that herniated lumbar disc-derived cells expressed mRNA of COX-2, IL-1 beta and TNF alpha in the presence of inflammatory cytokines in vitro. The disc-derived cells also produced much PGE2 by stimulating of inflammatory cytokines at the same time and this PGE2 production was distinctly suppressed by a selective inhibitor of COX-2, 6-methoxy-2-naphtyl acetic acids (6MNA). These results suggest that COX-2 and inflammatory cytokines might play a causative role in the radiculopathy of LDH through upregulating PGE2 synthesis.