Masashi Miyazaki

An update on bone substitutes for spinal fusion

With the current advances in spinal surgery, an understanding of the precise biological mechanism of each bone substitute is necessary for inducing successful spinal fusion. In this review, the categories of bone substitutes include allografts, ceramics, demineralized bone matrix, osteoinductive factors, autogenous platelet concentrate, mesenchymal stem cells, and gene therapy. Further, clinical studies have been evaluated by their levels of evidence in order to elucidate the precise effect of the bone substitute employed and to establish clinical guidance. This article will review both clinical studies based on evidence and basic research in current advances in order to avoid as far as possible any chances of failure in the future and to understand cellular biology in novel technologies.

A porcine collagen-derived matrix as a carrier for recombinant human bone morphogenetic protein-2 enhances spinal fusion in rats

BACKGROUND CONTEXTnRecombinant bone morphogenetic proteins (rhBMPs) have been used successfully in clinical trials. However, large doses of rhBMPs were required to induce adequate bone repair. Collagen sponges (CSs) have failed to allow a more sustained release of rhBMPs. Ongoing research aims to design carriers that allow a more controlled and sustained release of the protein. E-Matrix is a injectable scaffold matrix that may enhance rhBMP activity and stimulate bone regeneration.nnnPURPOSEnThe purpose of this study was to test E-Matrix as a carrier for rhBMPs in a CS and examine its feasibility in clinical applications by using a rat spinal fusion model.nnnPATIENT SAMPLEnA total of 80 Lewis rats aged 8-16 weeks were divided into nine groups.nnnSTUDY DESIGN/SETTINGnRat spinal fusion model.nnnOUTCOME MEASURESnRadiographs were obtained at 4, 6, and 8 weeks. The rats were sacrificed and their spines were explanted and assessed by manual palpation, high-resolution microcomputed tomography (micro-CT), and histologic analysis.nnnMETHODSnGroup I animals were implanted with CS alone (negative control); Group II animals with CS containing 10microg rhBMP-2 (positive control); Group III animals with CS containing 3microg rhBMP-2; Group IV animals with CS containing 3microg rhBMP-2 and E-Matrix; Group V animals with CS containing 1microg rhBMP-2; Group VI animals with CS containing 1microg rhBMP-2 and E-Matrix; Group VII animals with CS containing 0.5microg rhBMP-2; Group VIII animals with CS containing 0.5microg rhBMP-2 and E-Matrix; and Group IX animals with CS and E-Matrix without rhBMP-2.nnnRESULTSnRadiographic evaluation, micro-CT, and manual palpation revealed spinal fusion in all rats in the BMP-2 and E-Matrix groups (IV, VI, and VIII) and high-dose BMP-2 groups (II and III). Four spines in the 3microg rhBMP-2 group (V) fused, and one spine in the 0.5microg rhBMP-2 group (VII) exhibited fusion. No spines were fused in Groups I (CS alone) and IX (E-Matrix alone). The volume of new bone in the area between the tip of the L4 transverse process and the base of the L5 transverse process in Group IV was equivalent to the volumes observed in Group II.nnnCONCLUSIONnE-matrix enhances spinal fusion as a carrier for rhBMP-2 in a rat spinal fusion model. The results of this study suggest that E-Matrix as a growth factor carrier may be applicable to spinal fusion and may improve rhBMP-2s activity at the fusion site.

Tacrolimus and cyclosporine A inhibit human osteoclast formation via targeting the calcineurin-dependent NFAT pathway and an activation pathway for c-jun or MITF in rheumatoid arthritis

In the present study, we aimed to determine whether tacrolimus (FK506) and cyclosporine A act directly on human osteoclast precursors obtained from patients with rheumatoid arthritis (RA) and influence monocyte–osteoclast differentiation induced by receptor activator of NF-κB ligand (RANKL) in vitro, the stage at which differentiation was affected and the manner in which tacrolimus or cyclosporine A affected the osteoclast signaling pathway. Peripheral blood mononuclear cells (PBMCs) were isolated from RA patients and cultured in the presence of RANKL and macrophage-colony stimulating factor (M-CSF). Tacrolimus or cyclosporine A was added to these cultures to determine the effect on the osteoclast differentiation. Osteoclast formation was determined by assessing the number of tartrate resistant acid phosphatase (TRAP) staining cells and measuring the extent of lacunar resorption. The expression of osteoclast transcription factors, such as TNF receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells c1 (NFATc1), c-Fos, c-Jun, microphthalmia transcription factor (MITF) and PU.1 in mononuclear cells (MNCs) was assayed by quantitative reverse transcription-polymerase chain reaction. Addition of tacrolimus or cyclosporine A resulted in a decrease in the number of TRAP-positive multinucleated cells (TRAP+ MNCs) and a decrease in the extent of lacunar resorption pit formation as compared to the control cultures; thus, human monocyte–osteoclast differentiation was more effectively inhibited at the late stage and addition of tacrolimus or cyclosporine A resulted in a decrease in the mRNA expression of NFATc1, c-Jun, and MITF at the late stage. Our results suggest that tacrolimus or cyclosporine A acts directly on human osteoclast precursors in RA patients and exerts their immunosuppressive effects on human monocyte–osteoclast formation via targeting both the calcineurin-dependent NFAT pathway and activation pathway for c-Jun or MITF.

Manipulation of the anabolic and catabolic responses with BMP-2 and zoledronic acid in a rat femoral fracture model

Bone repair involves a complex set of regulated signaling pathways that control the formation of new bone matrix and the resorption of damaged bone matrix at the fracture site. It has been reported that the optimal time point for single-dose zoledronic acid (ZA) administration systemically increased the strength of bone morphogenetic protein (BMP)-7-mediated callus. However, its repair mechanism during bone fracture healing remains unknown. We aimed to investigate the synergic effect of recombinant human (rh) BMP-2 and ZA in a rat femoral fracture model. Fifty-eight rats were divided into 4 groups. Group I (n=14) animals were implanted with a carrier alone. Group II (n=15) animals were implanted with a carrier containing 1-μg rhBMP-2. Group III (n=14) animals were implanted with a carrier and a subcutaneous systemic ZA injection 2 weeks after surgery. Group IV (n=15) animals were implanted with a carrier containing 1-μg rhBMP-2 and ZA subcutaneous injection 2 weeks after surgery. The rats were euthanized after 6 weeks and their fractured femurs were explanted and assessed by manual palpation, radiographs, and high-resolution micro-computerized tomography (micro-CT) and were subjected to biomechanical and histological analysis. The fusion rates in Group IV (93.3%) were considerably higher than those in Groups I (28.6%), II (53.3%), and III (57.1%). Additionally, the radiographic scores of Group IV were higher than those in Groups I, II, and III. In micro-CT analysis, the tissue volume (TV) of the callus was higher in Group IV than in Groups I and II (p<0.05). New bone volume (BV) and trabecular spacing (Tb.Sp) also showed essentially the same trend as that of TV. The ratio of BV to TV (BV/TV), the trabecular number (Tb.N), and the trabecular thickness (Tb.Th) was higher in Groups III and IV than in Groups I and II (p<0.05). In biomechanical analysis, the ultimate loads at failure and stiffness in Groups III and IV were on average higher than those in Groups I and II (p<0.05), while the energy absorption of Group IV was higher than those of Groups I and II (p<0.05). The synergic effect of rhBMP-2 and ZA given systemically as a single dose at the optimal time was efficacious for fracture repair and significantly enhanced bone fusion. Our results suggest that this combination facilitates bone healing and has potential clinical application.

Rabbit Model for in vivo Study of Intervertebral Disc Degeneration and Regeneration

OBJECTIVEnThe purpose of this study is to verify the usefulness of the rabbit model for disc degeneration study.nnnMATERIALSnThe L1-L2, L2-L3, L3-L4, or L4-L5 lumbar intervertebral disc (IVD) of 9 mature male New Zealand White rabbits were injured by inserting a 16-gauge needle to a depth of 5 mm in the left anterolateral annulus fibrosus while leaving L5-L6 IVD uninjured. Three other rabbits also received intradiscal injections of rabbit disc cells transfected with adenovirus and bone morphogenetic protein-2 (ad-BMP-2) at L4-L5 in addition to injury by 16-gauge needle at the L1-L2 level. Using digitized radiographs, measurements of IVD height were made and analyzed by using the disc height index (DHI). Magnetic resonance imaging (MRI) scans of the injured discs, injected discs, and uninjured L5-L6 discs were performed at 15 weeks post surgery and compared with preoperative MRI scans.nnnRESULTSnAll twelve rabbits showed consistent results of disc degeneration within 15 weeks following annular puncture. DHIs of injured discs were significantly lower than that of the uninjured L5-L6 discs (p<0.05). The mean value of disc degeneration grade of injured discs was significantly higher than that of uninjured discs (p<0.05). The injection of disc cell transfected with ad-BMP-2 did not induce disc regeneration at 15 weeks after injection.nnnCONCLUSIONnThis study showed that the injured disc had a significant change in DHI on simple lateral radiograph and disc degeneration grade on MRI scans within 15 weeks in all rabbits. Rabbit annular puncture model can be useful as a disc degeneration model in vivo.

Manipulation of anabolic and catabolic responses with bone morphogenetic protein and zoledronic acid in a rat spinal fusion model.

Bone fusion involves a complex set of regulated signaling pathways that control the formation of new bone matrix and the resorption of damaged bone matrix at the surgical site. It has been reported that systemically administering a single dose of zoledronic acid (ZA) at the optimal time increases the strength of the bone morphogenetic protein (BMP)-mediated callus. In the present study, we aimed to investigate the effect of BMP-2 and ZA in a rat spinal model. Sixty-seven rats were divided into 6 groups: group I (n=11) animals were implanted with a carrier alone, group II (n=12) animals were implanted with a carrier and a subcutaneous injection of ZA was administered 2weeks after surgery, group III (n=12) animals were implanted with a carrier containing 1μg of rhBMP-2, group IV (n=12) animals were implanted with a carrier containing 1μg of rhBMP-2 and a subcutaneous injection of ZA was administered 2weeks after surgery, group V (n=10) animals were implanted with a carrier containing 3μg of rhBMP-2, and group VI (n=10) animals were implanted with a carrier containing 3μg of rhBMP-2 and a subcutaneous injection of ZA was administered 2weeks after surgery. The rats were euthanized after 6weeks, and their spines were explanted and assessed by manual palpation, radiography, high-resolution micro-computerized tomography (micro-CT), and histologic analysis. The fusion rates in group VI (60%) were considerably higher than those in the groups I (0%), II (0%), III (12.5%), IV (20.8%), and V (35%), (P<0.05). Additionally, the radiographic scores of group VI were higher than those in the other groups, (P<0.05). In micro-CT analysis, the tissue and bone volumes of the callus were significantly higher in group VI than those in the other groups, (P<0.05). The trabecular number was significantly higher and the trabecular spacing was significantly lower in group VI than those in the other groups, (P<0.05). The combination of rhBMP-2 and ZA administered systemically as a single dose at the optimal time was efficacious in our rat spinal fusion model. Our results suggest that this combination facilitates spinal fusion and has potential clinical application.

Analysis of the Relationship between Ligamentum Flavum Thickening and Lumbar Segmental Instability, Disc Degeneration, and Facet Joint Osteoarthritis in Lumbar Spinal Stenosis

Study Design Cross-sectional study. Purpose To investigate the relationship between ligamentum flavum (LF) thickening and lumbar segmental instability and disc degeneration and facet joint osteoarthritis. Overview of Literature Posterior spinal structures, including LF thickness, play a major role in lumbar spinal canal stenosis pathogenesis. The cause of LF thickening is multifactorial and includes activity level, age, and mechanical stress. LF thickening pathogenesis is unknown. Methods We examined 419 patients who underwent computed tomography (CT) myelography and magnetic resonance imaging after complaints of clinical symptoms. To investigate LF hypertrophy, 57 patients whose lumbar vertebra had normal disc heights at L4–5 were selected to exclude LF buckling as a hypertrophy component. LF thickness, disc space widening angulation in flexion, segmental angulation, presence of a vacuum phenomenon, and lumbar lordosis at T12–S1 were investigated. Disc and facet degeneration were also evaluated. Facet joint orientation was measured via an axial CT scan. Results The mean LF thickness in all patients was 4.4±1.0 mm at L4–5. There was a significant correlation between LF thickness and disc degeneration; LF thickness significantly increased with severe disc degeneration and facet joint osteoarthritis. There was a tendency toward increased LF thickness in more sagittalized facet joints than in coronalized facet joints. Logistic regression analysis showed that LF thickening was influenced by segmental angulation and facet joint osteoarthritis. Patient age was associated with LF thickening. Conclusions LF hypertrophy development was associated with segmental instability and severe disc degeneration, severe facet joint osteoarthritis, and a sagittalized facet joint orientation.

Tumor suppressive microRNA-138 inhibits metastatic potential via the targeting of focal adhesion kinase in Ewing's sarcoma cells

Short non-coding RNAs, called microRNAs (miRNAs), regulate cell biology by affecting the expression of target genes. However, we know little about the miRNAs regulating the growth and progression of Ewings sarcoma (ES). To identify possible oncogenic factors in ES, we used a microarray-based approach to profile the changes in the expression of miRNAs and the downstream mRNAs in five ES cell lines. One miRNA, miR‑138, was significantly downregulated, whereas the expression of focal adhesion kinase (FAK) was significantly upregulated in all tested ES cells. When miR‑138 was transfected into ES cell lines, the expression of FAK in these cells was greatly suppressed and inhibited the proliferation and mobility of ES cells. Overexpression of miR‑138 in vitro resulted in further inhibition of the cell cycle at the G1 phase and in the induction of anoikis, in a dose- and time-dependent manner. Moreover, miR‑138 overexpression in ES cells significantly suppressed the number of distant metastases in vivo. The data in the present study demonstrates for the first time a novel mechanism that regulates the expression of FAK via miR‑138 in ES cells.

Analysis of the Relationship between Hypertrophy of the Ligamentum Flavum and Lumbar Segmental Motion with Aging Process

Study Design Retrospective cross-sectional study. Purpose To investigate the relationship between ligamentum flavum (LF) hypertrophy and lumbar segmental motion. Overview of Literature The pathogenesis of LF thickening is unclear and whether the thickening results from tissue hypertrophy or buckling remains controversial. Methods 296 consecutive patients underwent assessment of the lumbar spine by radiographic and magnetic resonance imaging (MRI). Of these patients, 39 with normal L4–L5 disc height were selected to exclude LF buckling as one component of LF hypertrophy. The study group included 27 men and 12 women, with an average age of 61.2 years (range, 23–81 years). Disc degeneration and LF thickness were quantified on MRI. Lumbar segmental spine instability and presence of a vacuum phenomenon were identified on radiographic images. Results The distribution of disc degeneration and LF thickness included grade II degeneration in 4 patients, with a mean LF thickness of 2.43±0.20 mm; grade III in 10 patients, 3.01±0.41 mm; and grade IV in 25 patients, 4.16±1.12 mm. LF thickness significantly increased with grade of disc degeneration and was significantly correlated with age (r=0.55, p<0.01). Logistic regression analysis identified predictive effects of segmental angulation (odds ratio [OR]=1.55, p=0.014) and age (OR=1.16, p=0.008). Conclusions Age-related increases in disc degeneration, combined with continuous lumbar segmental flexion-extension motion, leads to the development of LF hypertrophy.

Clinical presentation of geriatric polytrauma patients with severe pelvic fractures: comparison with younger adult patients

BackgroundElderly polytrauma patients with pelvic fractures are at higher risk than young adults for severe medical outcomes and/or death in the early post-trauma phase. The aim of our study was to identify predictive factors of medical severity among geriatric polytrauma patients.MethodsWe conducted a retrospective cross-sectional study of polytrauma patients treated at our center, who had a pelvic fracture and at least two other injuries with an abbreviated injury scorexa0≥3. Our study group included 15 geriatric (mean age, 65xa0years) and 13 young (mean age, 39xa0years) adults. Factors related to medical status on admission were compared between the groups to identify those predictive of a severe medical outcome, defined by massive transfusion (>10 units of red blood cells) within the first 24xa0h of admission and/or death.ResultsGroups were comparable in terms of injury severity score (mean, 29), systolic blood pressure, heart rate, shock index, hemoglobin level, the prothrombin time-to-international normalized ratio (PT-INR) and base deficit. Over two-third of geriatric patients required a massive transfusion, with two patients dying, compared with the death of one young patient. Among geriatric patients, predictors of a severe medical outcome were extravasation of contrast medium on computed tomography, a hemoglobin levelxa0<11xa0g/dl, a PT-INRxa0>1.1 and a base deficitxa0>2xa0mmol/l.ConclusionsEven with our aggressive treatment algorism of pelvic fractures, particularly for the elderly, most of the geriatric polytrauma patients with severe pelvic fractures were at a high risk of massive transfusion. Extravasation on enhanced computed tomography and abnormal levels of select blood serum markers could assist in the early identification of geriatric polytrauma patients at risk for a severe medical outcome.