Junichi Kushioka, MD, PhD

All Publications

• Bone regeneration in inflammation with aging and cell-based immunomodulatory therapy. Inflammation and regeneration Kushioka, J., Chow, S. K., Toya, M., Tsubosaka, M., Shen, H., Gao, Q., Li, X., Zhang, N., Goodman, S. B. 2023; 43 (1): 29

  Abstract

  Aging of the global population increases the incidence of osteoporosis and associated fragility fractures, significantly impacting patient quality of life and healthcare costs. The acute inflammatory reaction is essential to initiate healing after injury. However, aging is associated with "inflammaging", referring to the presence of systemic low-level chronic inflammation. Chronic inflammation impairs the initiation of bone regeneration in elderly patients. This review examines current knowledge of the bone regeneration process and potential immunomodulatory therapies to facilitate bone healing in inflammaging.Aged macrophages show increased sensitivity and responsiveness to inflammatory signals. While M1 macrophages are activated during the acute inflammatory response, proper resolution of the inflammatory phase involves repolarizing pro-inflammatory M1 macrophages to an anti-inflammatory M2 phenotype associated with tissue regeneration. In aging, persistent chronic inflammation resulting from the failure of M1 to M2 repolarization leads to increased osteoclast activation and decreased osteoblast formation, thus increasing bone resorption and decreasing bone formation during healing.Inflammaging can impair the ability of stem cells to support bone regeneration and contributes to the decline in bone mass and strength that occurs with aging. Therefore, modulating inflammaging is a promising approach for improving bone health in the aging population. Mesenchymal stem cells (MSCs) possess immunomodulatory properties that may benefit bone regeneration in inflammation. Preconditioning MSCs with pro-inflammatory cytokines affects MSCs' secretory profile and osteogenic ability. MSCs cultured under hypoxic conditions show increased proliferation rates and secretion of growth factors. Resolution of inflammation via local delivery of anti-inflammatory cytokines is also a potential therapy for bone regeneration in inflammaging. Scaffolds containing anti-inflammatory cytokines, unaltered MSCs, and genetically modified MSCs can also have therapeutic potential. MSC exosomes can increase the migration of MSCs to the fracture site and enhance osteogenic differentiation and angiogenesis.In conclusion, inflammaging can impair the proper initiation of bone regeneration in the elderly. Modulating inflammaging is a promising approach for improving compromised bone healing in the aging population.

  View details for DOI 10.1186/s41232-023-00279-1

  View details for PubMedID 37231450

  View details for PubMedCentralID 2880220

• Metabolic profile of mesenchymal stromal cells and macrophages in the presence of polyethylene particles in a 3D model. Stem cell research & therapy Teissier, V., Gao, Q., Shen, H., Li, J., Li, X., Huang, E. E., Kushioka, J., Toya, M., Tsubosaka, M., Hirata, H., Alizadeh, H. V., Maduka, C. V., Contag, C. H., Yang, Y. P., Zhang, N., Goodman, S. B. 2023; 14 (1): 99

  Abstract

  Continuous cross talk between MSCs and macrophages is integral to acute and chronic inflammation resulting from contaminated polyethylene particles (cPE); however, the effect of this inflammatory microenvironment on mitochondrial metabolism has not been fully elucidated. We hypothesized that (a) exposure to cPE leads to impaired mitochondrial metabolism and glycolytic reprogramming and (b) macrophages play a key role in this pathway.We cultured MSCs with/without uncommitted M0 macrophages, with/without cPE in 3-dimensional gelatin methacrylate (3D GelMA) constructs/scaffolds. We evaluated mitochondrial function (membrane potential and reactive oxygen species-ROS production), metabolic pathways for adenosine triphosphate (ATP) production (glycolysis or oxidative phosphorylation) and response to stress mechanisms. We also studied macrophage polarization toward the pro-inflammatory M1 or the anti-inflammatory M2 phenotype and the osteogenic differentiation of MSCs.Exposure to cPE impaired mitochondrial metabolism of MSCs; addition of M0 macrophages restored healthy mitochondrial function. Macrophages exposed to cPE-induced glycolytic reprogramming, but also initiated a response to this stress to restore mitochondrial biogenesis and homeostatic oxidative phosphorylation. Uncommitted M0 macrophages in coculture with MSC polarized to both M1 and M2 phenotypes. Osteogenesis was comparable among groups after 21 days.This work confirmed that cPE exposure triggers impaired mitochondrial metabolism and glycolytic reprogramming in a 3D coculture model of MSCs and macrophages and demonstrated that macrophages cocultured with MSCs undergo metabolic changes to maintain energy production and restore homeostatic metabolism.

  View details for DOI 10.1186/s13287-023-03260-4

  View details for PubMedID 37085909

  View details for PubMedCentralID PMC10122387

• The efficiency of genetically modified mesenchymal stromal cells combined with a functionally graded scaffold for bone regeneration in corticosteroid-induced osteonecrosis of the femoral head in rabbits. Journal of biomedical materials research. Part A Tsubosaka, M., Maruyama, M., Lui, E., Moeinzadeh, S., Huang, E. E., Kushioka, J., Hirata, H., Jain, C., Storaci, H. W., Chan, C., Toya, M., Gao, Q., Teissier, V., Shen, H., Li, X., Zhang, N., Matsumoto, T., Kuroda, R., Goodman, S. B., Yang, Y. P. 2023

  Abstract

  Core decompression (CD) with mesenchymal stromal cells (MSCs) is an effective therapy for early-stage osteonecrosis of the femoral head (ONFH). Preconditioning of MSCs, using inflammatory mediators, is widely used in immunology and various cell therapies. We developed a three-dimensional printed functionally graded scaffold (FGS), made of beta-TCP and PCL, for cell delivery at a specific location. The present study examined the efficacy of CD treatments with genetically modified (GM) MSCs over-expressing PDGF-BB (PDGF-MSCs) or GM MSCs co-over-expressing IL-4 and PDGF-BB and preconditioned for three days of exposure to lipopolysaccharide and tumor necrosis factor-alpha (IL-4-PDGF-pMSCs) using the FGS for treating steroid-induced ONFH in rabbits. We compared CD without cell-therapy, with IL-4-PDGF-pMSCs alone, and with FGS loaded with PDGF-MSCs or IL-4-PDGF-pMSCs. For the area inside the CD, the bone volume in the CD alone was higher than in both FGS groups. The IL-4-PDGF-pMSCs alone and FGS+PDGF-MSCs reduced the occurrence of empty lacunae and improved osteoclastogenesis. There was no significant difference in angiogenesis among the four groups. The combined effect of GM MSCs or pMSCs and the FGS was not superior to the effect of each alone. To establish an important adjunctive therapy for CD for early ONFH in the future, it is necessary and essential to develop an FGS that delivers biologics appropriately and provides structural and mechanical support.

  View details for DOI 10.1002/jbm.a.37495

  View details for PubMedID 36606330

• Gait Variability to Phenotype Common Orthopedic Gait Impairments Using Wearable Sensors. Sensors (Basel, Switzerland) Kushioka, J., Sun, R., Zhang, W., Muaremi, A., Leutheuser, H., Odonkor, C. A., Smuck, M. 2022; 22 (23)

  Abstract

  Mobility impairments are a common symptom of age-related degenerative diseases. Gait features can discriminate those with mobility disorders from healthy individuals, yet phenotyping specific pathologies remains challenging. This study aims to identify if gait parameters derived from two foot-mounted inertial measurement units (IMU) during the 6 min walk test (6MWT) can phenotype mobility impairment from different pathologies (Lumbar spinal stenosis (LSS)-neurogenic diseases, and knee osteoarthritis (KOA)-structural joint disease). Bilateral foot-mounted IMU data during the 6MWT were collected from patients with LSS and KOA and matched healthy controls (N = 30, 10 for each group). Eleven gait parameters representing four domains (pace, rhythm, asymmetry, variability) were derived for each minute of the 6MWT. In the entire 6MWT, gait parameters in all four domains distinguished between controls and both disease groups; however, the disease groups demonstrated no statistical differences, with a trend toward higher stride length variability in the LSS group (p = 0.057). Additional minute-by-minute comparisons identified stride length variability as a statistically significant marker between disease groups during the middle portion of 6WMT (3rd min: p ≤ 0.05; 4th min: p = 0.06). These findings demonstrate that gait variability measures are a potential biomarker to phenotype mobility impairment from different pathologies. Increased gait variability indicates loss of gait rhythmicity, a common feature in neurologic impairment of locomotor control, thus reflecting the underlying mechanism for the gait impairment in LSS. Findings from this work also identify the middle portion of the 6MWT as a potential window to detect subtle gait differences between individuals with different origins of gait impairment.

  View details for DOI 10.3390/s22239301

  View details for PubMedID 36502003

  View details for PubMedCentralID PMC9739785

• Therapeutic effects of MSCs, genetically modified MSCs, and NFkB-inhibitor on chronic inflammatory osteolysis in aged mice. Journal of orthopaedic research : official publication of the Orthopaedic Research Society Kushioka, J., Toya, M., Shen, H., Hirata, H., Zhang, N., Huang, E., Tsubosaka, M., Gao, Q., Teissier, V., Li, X., Utsunomiya, T., Goodman, S. B. 2022

  Abstract

  The number of total joint replacements is increasing, especially in elderly patients, and so too are implant-related complications such as prosthesis loosening. Wear particles from the prosthesis induce a chronic inflammatory reaction and subsequent osteolysis, leading to the need for revision surgery. This study investigated the therapeutic effect of NF-kB decoy oligodeoxynucleotides (ODN), mesenchymal stem cells (MSCs), and genetically-modified NF-kB sensing interleukin-4 over-secreting MSCs (IL4-MSCs) on chronic inflammation in aged mice. The model was generated by continuous infusion of contaminated polyethylene particles into the intramedullary space of the distal femur of aged mice (15-17-month-old) for six weeks. Local delivery of ODN showed increased bone mineral density (BMD), decreased osteoclast-like cells, increased alkaline phosphatase (ALP)-positive area, and increased M2/M1 macrophage ratio. Local injection of MSCs and IL4-MSCs significantly decreased osteoclast-like cells and increased the M2/M1 ratio, with a greater trend for IL4-MSCs than MSCs. MSCs significantly increased ALP-positive area and BMD values compared to the control. The IL4-MSCs demonstrated higher values for both ALP-positive area and BMD. These findings demonstrated the therapeutic effects of ODN, MSCs, and IL4-MSCs on chronic inflammatory osteolysis in aged mice. The two MSC-based therapies were more effective than ODN in increasing the M2/M1 macrophage ratio, reducing bone resorption, and increasing bone formation. Specifically, MSCs were more effective in increasing bone formation, and IL4-MSCs were more effective in mitigating inflammation. This study suggests potential therapeutic strategies for treating wear particle-associated inflammatory osteolysis after arthroplasty in the elderly. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/jor.25434

  View details for PubMedID 36031590

• Effects of rhBMP-2-loaded hydroxyapatite granules/beta-tricalcium phosphate hydrogel (HA/beta-TCP/hydrogel) composite on a rat model of caudal intervertebral fusion. Scientific reports Nakagawa, S., Okada, R., Kushioka, J., Kodama, J., Tsukazaki, H., Bal, Z., Tateiwa, D., Ukon, Y., Hirai, H., Makino, T., Takenaka, S., Okada, S., Kaito, T. 2022; 12 (1): 7906

  Abstract

  The effects and inflammation-related side effects of bone morphogenetic protein (BMP)-2 on posterior lumbar interbody fusion are controversial. One of the potential causes for the inconsistent results is the uncontrolled release of BMP-2 from the collagen carrier. Therefore, BMP delivery systems that support effective bone regeneration while attenuating the side effects are strongly sought for. We developed NOVOSIS putty (NP), a novel composite material of hydroxyapatite (HA), beta-tricalcium phosphate (beta-TCP)/hydrogel, and BMP-2, which can sustainably release BMP-2 over 2weeks. This study was aimed at comparing the effects and side effects of NP and collagen sponge (CS) containing BMP-2 using a rat caudal intervertebral fusion model. The fusion rates of NP with low and high doses of BMP-2 were significantly higher than those of an iliac bone (IB) graft, but those of CS with low and high doses of BMP-2 were not different from those of the IB graft. Furthermore, the incidences of ectopic bone formation and soft tissue swelling were significantly lower in the NP group than in the CS group. The HA/beta-TCP/hydrogel carrier enabled superior bone induction with low-dose BMP-2 and decreased the incidence of side effects caused by high-dose BMP-2 vis-a-vis the collagen carrier.

  View details for DOI 10.1038/s41598-022-12082-y

  View details for PubMedID 35550600

• Ageing attenuates bone healing by mesenchymal stem cellsin a microribbon hydrogelwith a murine long bone critical-size defect model. Immunity & ageing : I & A Hirata, H., Zhang, N., Ueno, M., Barati, D., Kushioka, J., Shen, H., Tsubosaka, M., Toya, M., Lin, T., Huang, E., Yao, Z., Wu, J. Y., Zwingenberger, S., Yang, F., Goodman, S. B. 2022; 19 (1): 14

  Abstract

  BACKGROUND: Despite the high incidence of fractures and pseudoarthrosis in the aged population, a potential role for the use of mesenchymal stem cells (MSCs) in the treatment of bone defects in elderly patients has not been elucidated. Inflammation and the innate immune system, including macrophages, play crucial roles in the differentiation and activation of MSCs. We have developed lentivirus-transduced interleukin 4 (IL4) over-expressing MSCs (IL4-MSCs) to polarize macrophages to an M2 phenotype to promote bone healing in an established young murine critical size bone defect model. In the current study, we explore the potential of IL4-MSCs in aged mice.METHODS: A 2mm femoral diaphyseal bone defect was created and fixed with an external fixation device in 15- to 17-month-old male and female BALB/c mice. Microribbon (RB) scaffolds (Sc) with or without encapsulation of MSCs were implanted in the defect sites. Accordingly, the mice were divided into three treatment groups: Sc-only, Sc+MSCs, and Sc+IL4-MSCs. Mice were euthanized six weeks after the surgery; subsequently, MicroCT (CT), histochemical and immunohistochemical analyses were performed.RESULTS: CT analysis revealed that bone formation was markedly enhanced in the IL4-MSC group. Compared with the Sc-only, the amount of new bone increased in the Sc+MSCs and Sc+IL4-MSC groups. However, no bridging of bone was observed in all groups. H&E staining showed fibrous tissue within the defect in all groups. Alkaline phosphatase (ALP) staining was increased in the Sc+IL4-MSC group. The Sc+IL4-MSCs group showed a decrease in the number of M1 macrophages and an increase in the number of M2 macrophages, with a significant increase in the M2/M1 ratio.DISCUSSION: IL4 promotes macrophage polarization to an M2 phenotype, facilitating osteogenesis and vasculogenesis. The addition of IL4-MSCs in the RB scaffold polarized macrophages to an M2 phenotype and increased bone formation; however, complete bone bridging was not observed in any specimens. These results suggest that IL4-MSCs are insufficient to heal a critical size bone defect in aged mice, as opposed to younger animals. Additional therapeutic strategies are needed in this challenging clinical scenario.

  View details for DOI 10.1186/s12979-022-00272-1

  View details for PubMedID 35279175

• Selective Retinoic Acid Receptor gamma Antagonist 7C is a Potent Enhancer of BMP-Induced Ectopic Endochondral Bone Formation. Frontiers in cell and developmental biology Tateiwa, D., Kaito, T., Hashimoto, K., Okada, R., Kodama, J., Kushioka, J., Bal, Z., Tsukazaki, H., Nakagawa, S., Ukon, Y., Hirai, H., Tian, H., Alferiev, I., Chorny, M., Otsuru, S., Okada, S., Iwamoto, M. 2022; 10: 802699

  Abstract

  Bone morphogenetic proteins (BMPs) have been clinically applied for induction of bone formation in musculoskeletal disorders such as critical-sized bone defects, nonunions, and spinal fusion surgeries. However, the use of supraphysiological doses of BMP caused adverse events, which were sometimes life-threatening. Therefore, safer treatment strategies for bone regeneration have been sought for decades. Systemic administration of a potent selective antagonist of retinoic acid nuclear receptor gamma (RARgamma) (7C) stimulated BMP-induced ectopic bone formation. In this study, we developed 7C-loaded poly lactic nanoparticles (7C-NPs) and examined whether local application of 7C enhances BMP-induced bone regeneration. The collagen sponge discs that absorbed recombinant human (rh) BMP-2 were implanted into the dorsal fascia of young adult mice to induce ectopic bone. The combination of rhBMP-2 and 7C-NP markedly increased the total bone volume and thickness of the bone shell of the ectopic bone in a dose-dependent manner compared to those with rhBMP-2 only. 7C stimulated sulfated proteoglycan production, expression of chondrogenic marker genes, and Sox9 reporter activity in both chondrogenic cells and MSCs. The findings suggest that selective RARgamma antagonist 7C or the related compounds potentiate the bone inductive ability of rhBMP-2, as well as support any future research to improve the BMP-2 based bone regeneration procedures in a safe and efficient manner.

  View details for DOI 10.3389/fcell.2022.802699

  View details for PubMedID 35359440

• Sex differences in the therapeutic effect of unaltered versus NFkappaB sensing IL-4 over-expressing mesenchymal stromal cells in a murine model of chronic inflammatory bone loss. Frontiers in bioengineering and biotechnology Shen, H., Kushioka, J., Toya, M., Utsunomiya, T., Hirata, H., Huang, E. E., Tsubosaka, M., Gao, Q., Li, X., Teissier, V., Zhang, N., Goodman, S. B. 2022; 10: 962114

  Abstract

  Wear particles from joint arthroplasties induce chronic inflammation associated with prolonged upregulation of nuclear factor kappa-B (NF-kappaB) signaling in macrophages and osteoclasts, which leads to osteolysis and implant loosening. Mesenchymal stromal cell (MSC)-based therapy showed great potential for immunomodulation and mitigation of osteolysis in vivo, especially in the chronic phase of inflammation. We previously generated genetically modified MSCs that secrete the anti-inflammatory cytokine interleukin 4 (IL-4) in response to NF-kappaB activation (NFkappaB-IL-4 MSCs). However, whether the impact of sexual difference in the internal environment can alter the therapeutic effects of IL-4 over-secreting MSCs that simultaneously mitigate prolonged inflammation and enhance bone formation remains unknown. This study investigated the therapeutic effects of unaltered MSCs versus NFkappaB-IL-4 MSCs in mitigating chronic inflammation and enhancing bone formation in male and female mice. The murine model was established by continuous infusion of polyethylene particles contaminated with lipopolysaccharide (cPE) into the medullary cavity of the distal femur for 6 weeks to induce chronic inflammation. Unaltered MSCs or NFkappaB-IL-4 MSCs were infused into the femoral intramedullary cavity in sex-matched groups beginning 3 weeks after primary surgery. Femurs were harvested at 6 weeks, and bone marrow density was measured with micro-computational tomography. Numbers of osteoclast-like cells, osteoblasts, and macrophages were evaluated with histochemical and immunofluorescence staining. cPE infusion resulted in severe bone loss at the surgery site, increased tartrate-resistant acid phosphatase positive osteoclasts and M1 pro-inflammatory macrophages, and decreased alkaline phosphatase expression. MSC-based therapy effectively decreased local bone loss and polarized M1 macrophages into an M2 anti-inflammatory phenotype. In females, unaltered MSCs demonstrated a larger impact in enhancing the osteogenesis, but they demonstrated similar anti-inflammatory effects compared to NFkappaB-IL-4 MSCs. These results demonstrated that local inflammatory bone loss can be effectively modulated via MSC-based treatments in a sexually dimorphic manner, which could be an efficacious therapeutic strategy for treatment of periprosthetic osteolysis in both genders.

  View details for DOI 10.3389/fbioe.2022.962114

  View details for PubMedID 36046680

• Effect on Osteogenic Differentiation of Genetically Modified IL4 or PDGF-BB Over-Expressing and IL4-PDGF-BB Co-Over-Expressing Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro. Bioengineering (Basel, Switzerland) Tsubosaka, M., Maruyama, M., Huang, E. E., Zhang, N., Utsunomiya, T., Gao, Q., Shen, H., Li, X., Kushioka, J., Hirata, H., Yao, Z., Yang, Y. P., Goodman, S. B. 2021; 8 (11)

  Abstract

  The use of genetically modified (GM) mesenchymal stromal cells (MSCs) and preconditioned MSCs (pMSCs) may provide further opportunities to improve the outcome of core decompression (CD) for the treatment of early-stage osteonecrosis of the femoral head (ONFH). GM interleukin-4 (IL4) over-expressing MSCs (IL4-MSCs), platelet-derived growth factor (PDGF)-BB over-expressing MSCs (PDGF-BB-MSCs), and IL4-PDGF-BB co-over-expressing MSCs (IL4-PDGF-BB-MSCs) and their respective pMSCs were used in this in vitro study and compared with respect to cell proliferation and osteogenic differentiation. IL4-MSCs, PDGF-BB-MSCs, IL4-PDGF-BB-MSCs, and each pMSC treatment significantly increased cell proliferation compared to the MSC group alone. The percentage of Alizarin red-stained area in the IL4-MSC and IL4-pMSC groups was significantly lower than in the MSC group. However, the percentage of Alizarin red-stained area in the PDGF-BB-MSC group was significantly higher than in the MSC and PDGF-BB-pMSC groups. The percentage of Alizarin red-stained area in the IL4-PDGF-BB-pMSC was significantly higher than in the IL4-PDGF-BB-MSC group. There were no significant differences in the percentage of Alizarin red-stained area between the MSC and IL4-PDGF-BB-pMSC groups. The use of PDGF-BB-MSCs or IL4-PDGF-BB-pMSCs increased cell proliferation. Furthermore, PDGF-BB-MSCs promoted osteogenic differentiation. The addition of GM MSCs may provide a useful supplementary cell-based therapy to CD for treatment of ONFH.

  View details for DOI 10.3390/bioengineering8110165

  View details for PubMedID 34821731

• Amine modification of calcium phosphate by low-pressure plasma for bone regeneration. Scientific reports Kodama, J., Harumningtyas, A. A., Ito, T., Michlicek, M., Sugimoto, S., Kita, H., Chijimatsu, R., Ukon, Y., Kushioka, J., Okada, R., Kamatani, T., Hashimoto, K., Tateiwa, D., Tsukazaki, H., Nakagawa, S., Takenaka, S., Makino, T., Sakai, Y., Necas, D., Zajickova, L., Hamaguchi, S., Kaito, T. 2021; 11 (1): 17870

  Abstract

  Regeneration of large bone defects caused by trauma or tumor resection remains one of the biggest challenges in orthopedic surgery. Because of the limited availability of autograft material, the use of artificial bone is prevalent; however, the primary role of currently available artificial bone is restricted to acting as a bone graft extender owing to the lack of osteogenic ability. To explore whether surface modification might enhance artificial bone functionality, in this study we applied low-pressure plasma technology as next-generation surface treatment and processing strategy to chemically (amine) modify the surface of beta-tricalcium phosphate (beta-TCP) artificial bone using a CH4/N2/He gas mixture. Plasma-treated beta-TCP exhibited significantly enhanced hydrophilicity, facilitating the deep infiltration of cells into interconnected porous beta-TCP. Additionally, cell adhesion and osteogenic differentiation on the plasma-treated artificial bone surfaces were also enhanced. Furthermore, in a rat calvarial defect model, the plasma treatment afforded high bone regeneration capacity. Together, these results suggest that amine modification of artificial bone by plasma technology can provide a high osteogenic ability and represents a promising strategy for resolving current clinical limitations regarding the use of artificial bone.

  View details for DOI 10.1038/s41598-021-97460-8

  View details for PubMedID 34504247

• A novel BMP-2-loaded hydroxyapatite/beta-tricalcium phosphate microsphere/hydrogel composite for bone regeneration. Scientific reports Tateiwa, D., Nakagawa, S., Tsukazaki, H., Okada, R., Kodama, J., Kushioka, J., Bal, Z., Ukon, Y., Hirai, H., Kaito, T. 2021; 11 (1): 16924

  Abstract

  Although bone morphogenetic protein (BMP) has potent osteoinductivity, the potential adverse events attributed to its burst release prevent its widespread clinical application. Therefore, there is a strong need for BMP delivery systems that maximize osteoinductivity while preventing adverse effects. We evaluated the bone-regenerating potential of NOVOSIS putty (NP), a novel composite combining hydroxyapatite, beta-tricalcium phosphate microsphere/poloxamer 407-based hydrogel, and recombinant human (rh) BMP-2. In vitro assessment of release kinetics by enzyme-linked immunosorbent assay demonstrated sustained release of rhBMP-2 from NP and burst release from collagen sponge (CS), and in vivo assessment of release kinetics by longitudinal tracking of fluorescently labeled rhBMP-2 showed a longer biological half-life of rhBMP-2 with NP than with CS. Furthermore, osteogenic gene expression in MC3T3-E1 cells was significantly higher after co-culture with NP than after co-culture with CS, suggesting that the sustained release of rhBMP-2 from NP effectively contributed to the differentiation of osteoblasts. In a rat spinal fusion model, the volume and quality of newly formed bone was higher in the NP group than in the CS group. Use of NP results in efficient bone regeneration through sustained release of rhBMP-2 and improves the quality of BMP-induced bone.

  View details for DOI 10.1038/s41598-021-96484-4

  View details for PubMedID 34413442

• Low magnetic field promotes recombinant human BMP-2-induced bone formation and influences orientation of trabeculae and bone marrow-derived stromal cells. Bone reports Okada, R., Yamato, K., Kawakami, M., Kodama, J., Kushioka, J., Tateiwa, D., Ukon, Y., Zeynep, B., Ishimoto, T., Nakano, T., Yoshikawa, H., Kaito, T. 2021; 14: 100757

  Abstract

  Effects of high magnetic fields [MFs,≥1T (T)] on osteoblastic differentiation and the orientation of cells or matrix proteins have been reported. However, the effect of low MFs (< 1T) on the orientation of bone formation is not well known. This study was performed to verify the effects of low MFs on osteoblastic differentiation, bone formation, and orientation of both cells and newly formed bone. An apparatus was prepared with two magnets (190 mT) aligned in parallel to generate a parallel MF. In vitro, bone marrow-derived stromal cells of rats were used to assess the effects of low MFs on cell orientation, osteoblastic differentiation, and mineralization. A bone morphogenetic protein (BMP)-2-induced ectopic bone model was used to elucidate the effect of low MFs on microstructural indices, trabecula orientation, and the apatite c-axis orientation of newly formed bone. Low MFs resulted in an increased ratio of cells oriented perpendicular to the direction of the MF and promoted osteoblastic differentiation in vitro. Moreover, in vivo analysis demonstrated that low MFs promoted bone formation and changed the orientation of trabeculae and apatite crystal in a direction perpendicular to the MF. These changes led to an increase in the mechanical strength of rhBMP-2-induced bone. These results suggest that the application of low MFs has potential to facilitate the regeneration of bone with sufficient mechanical strength by controlling the orientation of newly formed bone.

  View details for DOI 10.1016/j.bonr.2021.100757

  View details for PubMedID 33681430

• Antibacterial efficacy of quaternized chitosan coating on 3D printed titanium cage in rat intervertebral disc space. The spine journal : official journal of the North American Spine Society Kodama, J., Chen, H., Zhou, T., Kushioka, J., Okada, R., Tsukazaki, H., Tateiwa, D., Nakagawa, S., Ukon, Y., Bal, Z., Tian, H., Zhao, J., Kaito, T. 2021

  Abstract

  BACKGROUND CONTEXT: Infection around intervertebral fusion cages can be intractable because of the avascular nature of the intervertebral disc space. Intervertebral cages with antibacterial effects may be a method by which this complication can be prevented.PURPOSE: To investigate the bacterial load on the antibacterial coating cages for spinal interbody fusion STUDY DESIGN: An experimental in vitro and in vivo study.METHODS: Based on the micro-computed tomography (CT) data of rat caudal discs, mesh-like titanium (Ti) cages that anatomically fit into the discs were fabricated by three-dimensional (3D) printing. Additionally, an antibacterial coating was applied with quaternized chitosan (hydroxypropyltrimethyl ammonium chloride chitosan, HACC). In vitro release kinetics of the HACC was performed, and the antibacterial performance of the HACC-coated (Ti-HACC) cages (via inhibition zone assay, bacterial adhesion assay, and biofilm formation assay) was evaluated. Then, Ti-HACC- or noncoated (Ti) cages were implanted in the caudal discs of rats with bioluminescent Staphylococcus aureus. Bacterial survival was investigated using an in vivo imaging system (IVIS) on postoperative days 1, 3, and 5. On day 5, the infection-related changes (bone destruction and migration of cages) were assessed using micro-CT, and the healing status of the surgical wounds was also assessed. After the removal of the cages, the quantification of bacteria attached to the cages was obtained by IVIS. Histological evaluation was performed by hematoxylin and eosin staining and TRAP (tartrate-resistant acid phosphatase) staining.RESULTS: Release kinetic analysis showed the sustained release of HACC over 3 days from Ti-HACC cages. Antibacterial effects of Ti-HACC cages were demonstrated in all in vitro assays. IVIS evaluation indicated that the in vivo implantation of Ti-HACC cages with S. aureus exhibited better wound healing, less infection-related changes on micro-CT, and reduced bacterial quantity in the extracted cages compared to Ti cages. Histological evaluation demonstrated an increased number of TRAP-positive osteoclasts and severe bone destruction in the rats treated with Ti cages.CONCLUSIONS: We developed a novel antibacterial HACC-coated intervertebral cage that exhibited prominent antibacterial efficacy and prevented the structural damage caused by the infection in rat caudal discs.CLINICAL SIGNIFICANCE: HACC-coated titanium intervertebral cages may be a promising option for preventing intractable postoperative infection in spinal interbody fusion surgery.

  View details for DOI 10.1016/j.spinee.2021.02.016

  View details for PubMedID 33621666

• A novel nano-hydroxyapatite/synthetic polymer/bone morphogenetic protein-2 composite for efficient bone regeneration. The spine journal : official journal of the North American Spine Society Bal, Z., Korkusuz, F., Ishiguro, H., Okada, R., Kushioka, J., Chijimatsu, R., Kodama, J., Tateiwa, D., Ukon, Y., Nakagawa, S., Dede, E. C., Gizer, M., Korkusuz, P., Yoshikawa, H., Kaito, T. 2021

  Abstract

  BACKGROUND: Efficient bone regeneration using recombinant human bone morphogenetic protein-2 (BMP-2) is needed to reduce side effects caused by high-dose BMP-2 use. The composite material of polylactic acid-polyethene glycol (PLA-PEG) for sustained release and an osteogenic nano-hydroxyapatite (nHAp) can contribute to efficient bone regeneration by BMP-2.STUDY DESIGN: An experimental in vitro and in vivo study.PURPOSE: The objective of this study is to investigate the effectiveness of a novel composite material of PLA-PEG and nHAp as a carrier for BMP-2.METHODS: The release kinetics of BMP-2 from the composites was investigated by ELISA. Thirty-six male Sprague-Dawley rats underwent posterolateral spinal fusion on L4-L5 with three different doses of BMP-2 (0 g [control], 3 g [low dose], and 10 g [high dose]). Weekly CT results and histology and a manual palpation test at 8 weeks postoperatively were used for assessment of the spinal fusion.RESULTS: ELISA demonstrated the sustained release of BMP-2 until day 21. CT and manual palpation test demonstrated a solid fusion in 91.6% (11/12) of specimens in both the low- and high-dose groups. N mice in the control group attained bony fusion (0%, 0/9). nHAp was resorbed between 2 and 4 weeks postoperatively, and regenerated fusion mass at 8 weeks postoperatively consisted of only newly formed bone.CONCLUSIONS: The nHAp/PLA-PEG composite enabled efficient bone regeneration with low-dose BMP-2. The sustained release of BMP-2 by PLA-PEG and the osteogenic and biodegradable scaffold of nHAp might contribute to efficient bone regeneration.CLINICAL SIGNIFICANCE: This novel composite material has potential in clinical applications (spinal fusion, large bone defect and non-union) by enabling efficient bone formation by BMP-2.

  View details for DOI 10.1016/j.spinee.2021.01.019

  View details for PubMedID 33493682

• A novel negative regulatory mechanism of Smurf2 in BMP/Smad signaling in bone BONE RESEARCH Kushioka, J., Kaito, T., Okada, R., Ishiguro, H., Bal, Z., Kodama, J., Chijimatsu, R., Pye, M., Narimatsu, M., Wrana, J. L., Inoue, Y., Ninomiya, H., Yamamoto, S., Saitou, T., Yoshikawa, H., Imamura, T. 2020; 8 (1): 41

  Abstract

  Transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) play important roles in bone metabolism. Smad ubiquitination regulatory factors (Smurfs) regulate TGF-β/BMP signaling via ubiquitination, resulting in degradation of signaling molecules to prevent excessive activation of TGF-β/BMP signaling. Though Smurf2 has been shown to negatively regulate TGF-β/Smad signaling, its involvement in BMP/Smad signaling in bone metabolism has not been thoroughly investigated. In the present study, we sought to evaluate the role of Smurf2 in BMP/Smad signaling in bone metabolism. Absorbable collagen sponges containing 3 μg of recombinant human BMP2 (rhBMP2) were implanted in the dorsal muscle pouches of wild type (WT) and Smurf2-/- mice. The rhBMP2-induced ectopic bone in Smurf2-/- mice showed greater bone mass, higher mineral apposition and bone formation rates, and greater osteoblast numbers than the ectopic bone in WT mice. In WT mice, the ectopic bone consisted of a thin discontinuous outer cortical shell and scant inner trabecular bone. In contrast, in Smurf2-/- mice, the induced bone consisted of a thick, continuous outer cortical shell and abundant inner trabecular bone. Additionally, rhBMP2-stimulated bone marrow stromal cells (BMSCs) from Smurf2-/- mice showed increased osteogenic differentiation. Smurf2 induced the ubiquitination of Smad1/5. BMP/Smad signaling was enhanced in Smurf2-/- BMSCs stimulated with rhBMP2, and the inhibition of BMP/Smad signaling suppressed osteogenic differentiation of these BMSCs. These findings demonstrate that Smurf2 negatively regulates BMP/Smad signaling, thereby identifying a new regulatory mechanism in bone metabolism.

  View details for DOI 10.1038/s41413-020-00115-z

  View details for Web of Science ID 000591559700001

  View details for PubMedID 33298874

  View details for PubMedCentralID PMC7680794

• The small compound, TD-198946, protects against intervertebral degeneration by enhancing glycosaminoglycan synthesis in nucleus pulposus cells SCIENTIFIC REPORTS Kushioka, J., Kaito, T., Chijimatsu, R., Okada, R., Ishiguro, H., Bal, Z., Kodama, J., Yano, F., Saito, T., Chung, U., Tanaka, S., Yoshikawa, H. 2020; 10 (1): 14190

  Abstract

  Degeneration of the nucleus pulposus (NP) might serve as a trigger for intervertebral disc degeneration (IDD). A recent drug screening study revealed that the thienoindazole derivative, TD-198946, is a novel drug for the treatment of osteoarthritis. Because of the environmental and functional similarities between articular cartilage and intervertebral disc, TD-198946 is expected to prevent IDD. Herein, we sought to evaluate the effects of TD-198946 on IDD. TD-198946 enhanced glycosaminoglycan (GAG) production and the related genes in mouse NP cells and human NP cells (hNPCs). Further, Kyoto Encyclopedia of Genes and Genomes pathway analysis using the mRNA sequence of hNPCs suggested that the mechanism of action of TD-198946 primarily occurred via the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. The Akt inhibitor suppressed the enhancement of GAG production induced by TD-198946. The effects of TD-198946 on IDD at two different time points (immediate treatment model, immediately after the puncture; latent treatment model, 2 weeks after the puncture) were investigated using a mouse tail-disc puncture model. At both time points, TD-198946 prevented a loss in disc height. Histological analysis also demonstrated the preservation of the NP structures. TD-198946 exhibited therapeutic effects on IDD by enhancing GAG production via PI3K/Akt signaling.

  View details for DOI 10.1038/s41598-020-71193-6

  View details for Web of Science ID 000567061600031

  View details for PubMedID 32843678

  View details for PubMedCentralID PMC7447806

• Resting-state Amplitude of Low-frequency Fluctuation is a Potentially Useful Prognostic Functional Biomarker in Cervical Myelopathy CLINICAL ORTHOPAEDICS AND RELATED RESEARCH Takenaka, S., Kan, S., Seymour, B., Makino, T., Sakai, Y., Kushioka, J., Tanaka, H., Watanabe, Y., Shibata, M., Yoshikawa, H., Kaito, T. 2020; 478 (7): 1667–80

  Abstract

  Cervical MRI is the standard diagnostic imaging technique for patients with cervical myelopathy. However, the utility of conventional cervical MRI as a predictive biomarker for surgical recovery remains unclear, partly because of the limited information obtained from this anatomically small area. Brain resting-state functional MRI (rs-fMRI) may help identify candidate predictive biomarkers. Two analytical methods that assess local spontaneous brain activity are widely used for rs-fMRI: functional connectivity between two brain regions and amplitude of low-frequency fluctuation (ALFF). In our previous analysis of functional connectivity, we discovered that brain functional connectivity may be a predictive biomarker for neurologic recovery in patients with cervical myelopathy; however, the functional connectivity analysis identified a correlation with only one clinical outcome (the 10-second test). To establish a comprehensive prediction measure, we need to explore other brain biomarkers that can predict recovery of other clinical outcomes in patients with cervical myelopathy.We aimed to (1) elucidate preoperative ALFF alterations in patients with cervical myelopathy and how ALFF changes after surgery, with a focus on postoperative normalization and (2) establish a predictive model using preoperative ALFF by investigating the correlation between preoperative ALFF and postoperative clinical recovery in patients with cervical myelopathy.Between August 2015 and June 2017, we treated 40 patients with cervical myelopathy. Thirty patients met our prespecified inclusion criteria, all were invited to participate, and 28 patients opted to do so (93%; 14 men and 14 women; mean age: 67 years). The 28 patients and 28 age- and sex-matched controls underwent rs-fMRI (twice for patients with cervical myelopathy: before and 6 months after cervical decompression surgery). We analyzed the same study population that was used in our earlier study investigating functional connectivity. Controls had none of the following abnormalities: neck or arm pain, visual or auditory disorders, cognitive disorder, structural brain disorder, a history of brain surgery, mental and neurologic disorders, and medications for the central nervous system. We performed ALFF comparisons between preoperative patients with cervical myelopathy and controls, analyzed postoperative ALFF changes in patients with cervical myelopathy, and performed a correlation analysis between preoperative ALFF and clinical recovery in these patients. Clinical outcomes in the cervical myelopathy group were assessed using the 10-second test, the Japanese Orthopaedic Association upper-extremity motor (JOA-UEM) score, JOA upper-extremity sensory score (JOA-UES), and Japanese Orthopaedic Association Cervical Myelopathy Evaluation Questionnaire for upper-extremity function (JOACMEQ-UEF) score before and 6 months after surgery, which is when we believe these scores generally reach a plateau. A total of 93% of those enrolled (26 of 28 patients) were analyzed both preoperatively and postoperatively; the other two were lost to follow-up.The cervical myelopathy group had an increase in ALFF in the bilateral primary sensorimotor cortices (right, cluster size = 850 voxels, t-value = 6.10; left, cluster size = 370 voxels, t-value = 4.84) and left visual cortex (cluster size = 556 voxels, t-value = 4.21) compared with the control group. The cervical myelopathy group had a decrease in ALFF in the bilateral posterior supramarginal gyrus (right, cluster size = 222 voxels, t-value = 5.09; left, cluster size = 436 voxels, t-value = 5.28). After surgery, the bilateral sensorimotor cortices (right, cluster size = 468 voxels, t-value = 6.74; left, cluster size = 167 voxels, t-value = 5.40) and left visual cortex (cluster size = 3748 voxels, t-value = 6.66) showed decreased ALFF compared with preoperative ALFF, indicating postoperative normalization of spontaneous brain activities in these regions. However, the bilateral posterior supramarginal gyrus did not show an increase in ALFF postoperatively, although ALFF in this region decreased preoperatively. Greater levels of ALFF at the left and right frontal pole and left pars opercularis of the inferior frontal gyrus before surgery in the cervical myelopathy group were correlated with larger improvements in the JOACMEQ-UEF score 6 months after surgery (r = 0.784; p < 0.001, r = 0.734; p < 0.001 and r = 0.770, respectively; p < 0.001). The prediction formula, based on preoperative ALFF values in the left frontal pole, was as follows: the predicted postoperative improvement in the JOACMEQ-UEF score = 34.6 × preoperative ALFF value - 7.0 (r = 0.614; p < 0.001).Our findings suggest that preoperative ALFF may be a biomarker for postoperative recovery in that it predicted postoperative JOACMEQ-UEF scores. To establish a comprehensive prediction measure for neurologic recovery in patients with cervical myelopathy, a multicenter study is underway.Level II, diagnostic study.

  View details for DOI 10.1097/CORR.0000000000001157

  View details for Web of Science ID 000549947700047

  View details for PubMedID 32011371

  View details for PubMedCentralID PMC7310516

• Assessment of effects of rhBMP-2 on interbody fusion with a novel rat model SPINE JOURNAL Okada, R., Kaito, T., Ishiguro, H., Kushioka, J., Otsuru, S., Kanayama, S., Bal, Z., Kitaguchi, K., Hashimoto, K., Makino, T., Takenaka, S., Sakai, Y., Yoshikawa, H. 2020; 20 (5): 821–29

  Abstract

  The effects of using off-label recombinant human bone morphogenetic protein (rhBMP)-2 for interbody fusion are controversial. Although animal models of posterolateral fusion are well-established, establishing animal models to validate the safety and efficacy of interbody fusion is difficult, which may contribute to the inconsistent clinical results.To develop a novel animal model of interbody fusion in rat coccygeal vertebrae without destroying bony endplates.An experimental animal study.Forty-five male Sprague-Dawley rats underwent coccygeal interbody fusion without violating vertebral endplates. The animals were divided into three different groups based on the materials that were implanted into the interbody space (1) allogeneic iliac bone (IB) alone (IB group), (2) IB and 3 µg of rhBMP-2 (BMP low-dose group), or (3) IB and 10 µg of rhBMP-2 (BMP high-dose group). Fusion rates were investigated using microcomputed tomography 6 weeks after the operation. The incidence of adverse events, including soft-tissue swelling, delayed wound healing, osteolysis, and ectopic bone formation were evaluated. The total number of adverse events (using the adverse event score) in each group and the swelling ratio (calculated using the surgical site tissue volume [TV; TV on postoperative day 1/preoperative TV]) were also evaluated.The fusion rates in the BMP low- and high-dose groups (33.3% and 46.7%) were not significantly different, but both were significantly higher than that in the IB group (0%) (p=.042 and .006, respectively). Significant differences in the incidence of osteolysis, adverse event scores, and swelling ratios were observed only between the BMP high-dose and IB groups (p=.043, .006 and .014, respectively).We developed a novel rat model of interbody fusion in which the vertebral endplates were not violated, reflecting the normal clinical setting. rhBMP-2 use increased the fusion rate, but a higher dose of rhBMP-2 did not lead to a higher fusion rate than that for low-dose rhBMP-2; conversely, it led to an increase in the occurrence of adverse events.This novel rat model of coccygeal interbody fusion that preserved bony endplates has clinical significance for validating the effectiveness of biologics or bone graft substitutes before clinical trial.

  View details for DOI 10.1016/j.spinee.2019.12.014

  View details for Web of Science ID 000534521900017

  View details for PubMedID 31901554

• Risk factors for in-hospital mortality after spine surgery: a matched case-control study using a multicenter database SPINE JOURNAL Kushioka, J., Takenaka, S., Makino, T., Sakai, Y., Kashii, M., Iwasaki, M., Yoshikawa, H., Kaito, T. 2020; 20 (3): 321–28

  Abstract

  It is yet unclear what preoperative and intraoperative factors affect mortality after spine surgery.To identify the preoperative and intraoperative risk factors for in-hospital mortality after spine surgery using a matched case-control study based on a multicenter database.A retrospective matched case-control study based on a registry of prospectively collected multicenter data.We identified 25 patients who died in the hospital (the mortality group) from the 26,604 patients in the database who underwent spine surgery at our 27 affiliated institutions between 2012 and 2018. An age-, sex-, spinal disease-, and surgical procedure-matched control group of patients (n=100, 4:1 ratio with the mortality group) was selected from the same database.Data relating to comorbidities, preoperative blood tests, operative factors, and perioperative complications.We retrospectively reviewed all the medical records of each patient in the two groups to nullify the effects of overt risk factors such as age, sex, diseases, and surgical procedures. Risk factors for in-hospital mortality were initially evaluated by univariate analysis. Then, multivariate logistic regression models were generated to analyze independent risk factors for in-hospital mortality.The overall in-hospital mortality rate was 0.09% (25/26,604). Mortality was lowest in patients with degenerative cervical (0.04%, 2/5,027) or lumbar disease (0.03%, 5/15,630). In contrast, mortality was highest in patients with dialysis-related spondyloarthropathy (3.0%, 3/99), patients with infectious spondylodiscitis (1.5%, 6/401), and patients with metastatic spinal tumors (0.9%, 3/334). Multivariate logistic regression analysis revealed that massive intraoperative hemorrhage (>2 L) (odds ratio [OR], 28.2; 95% confidence interval [CI], 2.27-349), preoperative renal comorbidity (OR, 4.33; 95% CI, 1.38-13.6), and elevated preoperative aspartate aminotransferase levels (OR, 1.51 per 10 units; 95% CI, 1.04-2.20) were risk factors.Spine surgery for patients with dialysis-dependency, infectious diseases or metastatic tumors had much more potential of in-hospital mortality compared with those for patients with degenerative diseases. Massive intraoperative hemorrhage and preoperative renal and liver comorbidities were identified as risk factors for in-hospital mortality in patients who underwent spine surgery.

  View details for DOI 10.1016/j.spinee.2019.10.008

  View details for Web of Science ID 000518474900002

  View details for PubMedID 31669616

• BMP and TGF beta use and release in bone regeneration TURKISH JOURNAL OF MEDICAL SCIENCES Bal, Z., Kushioka, J., Kodama, J., Kaito, T., Yoshikawa, H., Korkusuz, P., Korkusuz, F. 2020; 50: 1707–22

  Abstract

  A fracture that does not unite in nine months is defined as nonunion. Nonunion is common in fragmented fractures and large bone defects where vascularization is impaired. The distal third of the tibia, the scaphoid bone or the talus fractures are furthermore prone to nonunion. Open fractures and spinal fusion cases also need special monitoring for healing. Bone tissue regeneration can be attained by autografts, allografts, xenografts and synthetic materials, however their limited availability and the increased surgical time as well as the donor site morbidity of autograft use, and lower probability of success, increased costs and disease transmission and immunological reaction probability of allografts oblige us to find better solutions and new grafts to overcome the cons. A proper biomaterial for regeneration should be osteoinductive, osteoconductive, biocompatible and mechanically suitable. Cytokine therapy, where growth factors are introduced either exogenously or triggered endogenously, is one of the commonly used method in bone tissue engineering. Transforming growth factor β (TGFβ) superfamily, which can be divided structurally into two groups as bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs) and TGFβ, activin, Nodal branch, Mullerian hormone, are known to be produced by osteoblasts and other bone cells and present already in bone matrix abundantly, to take roles in bone homeostasis. BMP family, as the biggest subfamily of TGFβ superfamily, is also reported to be the most effective growth factors in bone and development, which makes them one of the most popular cytokines used in bone regeneration. Complications depending on the excess use of growth factors, and pleiotropic functions of BMPs are however the main reasons of why they should be approached with care. In this review, the Smad dependent signaling pathways of TGFβ and BMP families and their relations and the applications in preclinical and clinical studies will be briefly summarized.

  View details for DOI 10.3906/sag-2003-127

  View details for Web of Science ID 000602339800015

  View details for PubMedID 32336073

• A novel and efficient method for culturing mouse nucleus pulposus cells SPINE JOURNAL Kushioka, J., Kaito, T., Chijimatsu, R., Okada, R., Ishiguro, H., Bal, Z., Kodama, J., Takenaka, S., Makino, T., Sakai, Y., Yoshikawa, H. 2019; 19 (9): 1573–83

  Abstract

  As degeneration of the nucleus pulposus (NP) is a major cause of intervertebral disc degeneration, research directed toward nucleus pulposus cells (NPCs) is drawing increased attention. However, caused by the difficulties associated with their harvest and culture, there are few reports describing cultivation methods for mouse NP cells (mNPCs).To establish efficient culture methods for mNPCs.In vitro animal study.After primary 3-dimensional (3D) gel culture of mNPCs and analysis of gene expression, cells digested from the gel were cultured in various bio-coated dishes with and without basic fibroblast growth factor (bFGF), and their growth kinetics and changes in gene expression profiles were evaluated. Next, the mNPCs obtained after sequential 3D gel and 2D culture were subjected to micromass culture and the effects of adding transforming growth factor-β3 (TGF-β3) on their gene expression profile and extracellular matrix (ECM) synthesis were evaluated.The cell morphology and gene expression pattern of mNPCs proliferated in primary 3D collagen gel culture resembled those of mNP. In contrast, mNPCs could not proliferate in conventional monolayer culture. Cell adhesion (colony number) and proliferation (colony size) were greater in fibronectin-coated dishes than in dishes with other bio-coatings. The addition of bFGF enhanced mNPCs proliferation, but the gene expression characteristics of mNPCs were lost as passage number increased. 2D culture with bFGF followed by micromass culture allowed for the recovery of the mNPC gene expression profile in primary 3D-gel culture, and TGF-β3 supplementation during micromass culture enhanced ECM synthesis.We established novel culture methods for mNPCs. These methods will benefit basic cell-based and molecular research involving these cells.

  View details for DOI 10.1016/j.spinee.2019.04.005

  View details for Web of Science ID 000482120700014

  View details for PubMedID 30986578

• Administration of ONO-2506 suppresses neuropathic pain after spinal cord injury by inhibition of astrocytic activation SPINE JOURNAL Ishiguro, H., Kaito, T., Hashimoto, K., Kushioka, J., Okada, R., Tsukazaki, H., Kodama, J., Bal, Z., Ukon, Y., Takenaka, S., Makino, T., Sakai, Y., Yoshikawa, H. 2019; 19 (8): 1434–42

  Abstract

  Spinal cord injury (SCI) results in not only motor dysfunction but also chronic neuropathic pain. Allodynia, an abnormal sensation that evokes pain against non-noxious stimuli, is a major symptom of post-SCI neuropathic pain. Astrocytic activation is a cause of post-SCI neuropathic pain and is considered a key treatment target. However, no effective treatment for these problems is available to date. ONO-2506 is a novel agent that suppresses astrocytic activation by inhibition of S100B production from astrocytes. Recently, it has been demonstrated that ONO-2506 inhibits secondary injury and improves motor function after SCI.This study aimed to investigate the effect of ONO-2506 on post-SCI neuropathic pain.Animal study of a rat model of spinal cord contusion.A total of 22 male Sprague-Dawley rats aged 6 weeks were used. Incomplete SCI was created at T10 level. Animals were divided into two groups: Saline group and ONO-2506 group. Nine animals in each group were finally included for this study. Intraperitoneal administration of ONO-2506 (20 mg/kg) or saline was continued daily for 1 week following SCI. Recovery of hind limb motor function was assessed using the Basso, Beattie, and Bresnahan (BBB) score. Mechanical and thermal allodynia of hind paws were evaluated by the withdrawal threshold using a von Frey filament and the withdrawal latency using the plantar test device. At 6 weeks after SCI, sagittal sections at the injured site and axial sections at L 4/5 were evaluated by fluorescent immunohistochemistry staining using S100B and glial fibrillary acidic protein (GFAP) antibodies.The improvement course of BBB scores was similar between the two groups. However, the withdrawal thresholds for mechanical stimuli and the withdrawal latency for thermal stimuli were significantly higher in the ONO-2506 group than in the Saline group over 6 weeks after SCI. The histologic assessments at the injured site demonstrated a significant reduction in the cross-sectional area of the cysts and a high fluorescence intensity area of S100B and GFAP in the ONO-2506 group. By correlation analysis, a high absolute value of the correlation coefficient was confirmed between the intensity of S100B expression at the injured site and the allodynia severity.Administration of ONO-2506 attenuated post-SCI neuropathic pain in a rat model of incomplete SCI. Histologic results support that the inhibition of S100B production and subsequent suppression of astrocytic activation contributed to the reduction in neuropathic pain.

  View details for DOI 10.1016/j.spinee.2019.04.006

  View details for Web of Science ID 000476725600015

  View details for PubMedID 30974239

• Towards prognostic functional brain biomarkers for cervical myelopathy: A resting-state fMRI study SCIENTIFIC REPORTS Takenaka, S., Kan, S., Seymour, B., Makino, T., Sakai, Y., Kushioka, J., Tanaka, H., Watanabe, Y., Shibata, M., Yoshikawa, H., Kaito, T. 2019; 9: 10456

  Abstract

  Recently, there has been increasing interest in strategies to predict neurological recovery in cervical myelopathy (CM) based on clinical images of the cervical spine. In this study, we aimed to explore potential preoperative brain biomarkers that can predict postoperative neurological recovery in CM patients by using resting-state functional magnetic resonance imaging (rs-fMRI) and functional connectivity (FC) analysis. Twenty-eight patients with CM and 28 age- and sex-matched healthy controls (HCs) underwent rs-fMRI (twice for CM patients, before and six months after surgery). A seed-to-voxel analysis was performed, and the following three statistical analyses were conducted: (i) FC comparisons between preoperative CM and HC; (ii) correlation analysis between preoperative FCs and clinical scores; and (iii) postoperative FC changes in CM. Our analyses identified three FCs between the visual cortex and the right superior frontal gyrus based on the conjunction of the first two analyses [(i) and (ii)]. These FCs may act as potential biomarkers for postoperative gain in the 10-second test and might be sufficient to provide a prediction formula for potential recovery. Our findings provide preliminary evidence supporting the possibility of novel predictive measures for neurological recovery in CM using rs-fMRI.

  View details for DOI 10.1038/s41598-019-46859-5

  View details for Web of Science ID 000475845400060

  View details for PubMedID 31320690

  View details for PubMedCentralID PMC6639260

• Intervertebral disc regeneration with an adipose mesenchymal stem cell-derived tissue-engineered construct in a rat nucleotomy model ACTA BIOMATERIALIA Ishiguro, H., Kaito, T., Yarimitsu, S., Hashimoto, K., Okada, R., Kushioka, J., Chijimatsu, R., Takenaka, S., Makino, T., Sakai, Y., Moriguchi, Y., Otsuru, S., Hart, D. A., Fujie, H., Nakamura, N., Yoshikawa, H. 2019; 87: 118–29

  Abstract

  Low back pain results in more global disabilities than any other condition, and intervertebral disc (IVD) degeneration is commonly involved in the etiology. Supplementation of IVDs with reparative cells is a rational strategy to address such clinical problems. We have previously developed a scaffold-free tissue-engineered construct (TEC) as a novel cell therapy system for repair of articular cartilage and meniscus. We now show the regenerative potential of adipose mesenchymal stem cells derived TEC (ADSC-TEC) for IVD degeneration using a rat tail model of total nucleotomy. The regenerative efficacy of ASDC-TEC was investigated structurally and biomechanically up to 6 months after implantation. ADSC-TEC implantation into IVDs preserved the disc height, endplate, and annulus fibrosus structure, and showed similar biomechanical characteristics to the sham group at postoperative 6 weeks. The structure of regenerated IVD was maintained until 6 months. Furthermore, ADSC-TEC implantation attenuated the impact of age-related biomechanical deterioration when assessed at 6 months post-implantation. These results demonstrate that use of ADSC-TECs can be an effective treatment for IVD degeneration. STATEMENT OF SIGNIFICANCE: We developed adipose mesenchymal stem cell-derived scaffold-free tissue engineered construct (ADSC-TEC) as a novel cell therapy system. The ADSC-TEC implantation into a rat total-nucleotomized disc space regenerated intervertebral discs (IVDs) histologically and biomechanically. The regenerative capacity of the ADSC-TEC was exerted by its trophic effects on annulus fibrosus cells and the load-sharing effect at intervertebral space. Interestingly, the regenerated IVDs by the ADSC-TEC was less susceptible to the age-related deterioration than the IVDs of normal rats. Thus, the application of ADSC-TEC into the degenerated disc can be an alternative therapy for various disease associated with structural and functional failure of IVDs.

  View details for DOI 10.1016/j.actbio.2019.01.050

  View details for Web of Science ID 000460713600009

  View details for PubMedID 30690206

• Effect of the small compound TD-198946 on glycosaminoglycan synthesis and transforming growth factor beta 3-associated chondrogenesis of human synovium-derived stem cells in vitro JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE Chijimatsu, R., Yano, F., Saito, T., Kobayashi, M., Hamamoto, S., Kaito, T., Kushioka, J., Hart, D. A., Chung, U., Tanaka, S., Yoshikawa, H., Nakamura, N. 2019; 13 (3): 446–58

  Abstract

  As an alternative to chondrocytes-based cartilage repair, stem cell-based therapies have been investigated. Specifically, human synovium-derived stem cells (hSSCs) are a promising cell source based on their highly capacities for chondrogenesis, but some methodological improvements are still required towards optimal cartilage regeneration. Recently, a small compound, TD-198946, was reported to promote chondrogenesis of several stem cells, but the effect on hSSCs is still unknown. This study aimed to examine the effects of TD-198946 on chondrocyte differentiation and cartilaginous tissue formation with hSSCs. A range of concentrations of TD-198946 were examined in chondrogenic cultures of hSSC-derived cell pellets. The effect of TD-198946 on glycosaminoglycan (GAG) production, chondrocyte marker expression, and cartilaginous tissue formation was assessed. At concentrations >1 nM, TD-198946 dose-dependently enhanced GAG production, particularly hyaluronan, whereas chondrocyte differentiation was not impacted. When combined with transforming growth factor β3 (TGFβ3), TD-198946 promoted chondrocyte differentiation and production of cartilaginous matrices at doses <1 nM as judged by SOX9, S100, and type 2 collagen upregulation. Conversely, doses >1 nM TD-198946 attenuated TGFβ3-associated chondrocyte differentiation, but aggrecan was efficiently produced at 1 to 10 nM TD-198946 as judged by safranin O staining. Thus, TD-198946 exhibited different dose ranges for either GAG synthesis or chondrocyte differentiation. Regarding use of TD-198946 for in vitro engineering of cartilage, cartilaginous particles rich in type 2 collagen and GAG were predominately created with TGFβ3 + 0.25 nM TD-198946. These studies have demonstrated that TD-198946 synergistically enhances chondrogenesis of hSSCs in a unique dose range, and such findings may provide a novel strategy for stem cell-based cartilage therapy.

  View details for DOI 10.1002/term.2795

  View details for Web of Science ID 000462352100009

  View details for PubMedID 30650248

• Tranexamic acid for reducing intra- and postoperative blood loss in posterior lumbar interbody fusion: Is it safe enough? Response JOURNAL OF NEUROSURGERY-SPINE Kushioka, J., Yamashita, T., Okuda, S., Maeno, T., Matsumoto, T., Iwasaki, M., Yamasaki, R. 2018; 29 (2): 227

  View details for DOI 10.3171/2017.12.SPINE171255

  View details for Web of Science ID 000440474700017

• Difference in the fusion rate and bone formation between artificial bone and iliac autograft inside an inter-body fusion cage - A comparison between porous hydroxyapatite/type 1 collagen composite and autologous iliac bone JOURNAL OF ORTHOPAEDIC SCIENCE Kushioka, J., Kaito, T., Makino, T., Fujiwara, H., Tsukazaki, H., Takenaka, S., Sakai, Y., Yoshikawa, H. 2018; 23 (4): 622–26

  Abstract

  Lateral inter-body fusion (LIF) using cages with a large bone grafting space can lead to a shortage of autologous grafting materials. The use of artificial bone is an option to increase the volume of grafting materials. However, the rate of bony fusion for these materials compared to that of autologous bone is unclear.The bone fusion rate for artificial bone (HAp/Col) and autologous iliac bone (IBG) graft among 23 patients who had undergone LIF (total 66 disc levels) combined with multilevel posterior corrective fusion for the treatment of adult spinal deformity was retrospectively evaluated. To allow comparison, one of the two separate bone grafting holes in each LIF cage was filled with HAp/Col and the other, with IBG. The change in Hounsfield units (HU) inside the implanted holes at 1-year post surgery (PO1Y) from baseline and immediately after surgery and bony fusion between adjacent vertebrae, defined by the extent of trabecular continuity at PO1Y, were evaluated using computed tomography. Differences between the convex and concave sides as well as effects of the side of approach were investigated.HU values increased significantly for IBG, from 228.9 at baseline to 286.1 at PO1Y (p < 0.001), with no change for HAp/Col. The fusion rate was higher for IBG (71.2%) than for HAp/Col (19.7%; p < 0.001). A significant effect of the location of the holes on fusion rate was identified for HAp/Col but not IBG. No effects of the side of approach were identified.A higher rate of fusion in LIF cages was obtained with IBG than with HAp/Col, with no effect of location of implantation (convex or concave) for IBG. Therefore, exclusive use of artificial bone, particularly on the convex side, should be avoided during LIF.

  View details for DOI 10.1016/j.jos.2018.03.006

  View details for Web of Science ID 000438317000003

  View details for PubMedID 29627140

• Modified Scarf Osteotomy with Medial Capsule Interposition for Hallux Valgus in Rheumatoid Arthritis A Study of Cases Including Severe First Metatarsophalangeal Joint Destruction JOURNAL OF BONE AND JOINT SURGERY-AMERICAN VOLUME Kushioka, J., Hirao, M., Tsuboi, H., Ebina, K., Noguchi, T., Nampei, A., Tsuji, S., Akita, S., Hashimoto, J., Yoshikawa, H. 2018; 100 (9): 765–76

  Abstract

  Arthrodesis of the first metatarsophalangeal (MTP) joint has been recommended for severe hallux valgus deformity in patients with rheumatoid arthritis (RA). However, with the progress of medical treatment of RA, joint preservation surgery has recently been performed. The aim of this study was to investigate the clinical and radiographic outcomes of modified Scarf osteotomy with medial capsule interposition for RA cases including severe destruction of the first MTP joint and to evaluate risk factors for recurrence.A retrospective observational study of 76 cases (60 patients) followed for a mean of 35.3 months (range, 24 to 56 months) after a modified Scarf osteotomy was performed. Scores on the Japanese Society for Surgery of the Foot (JSSF) RA foot and ankle scale, the JSSF hallux scale, and a self-administered foot evaluation questionnaire (SAFE-Q) were determined along with preoperative and postoperative radiographic parameters.There was a significant improvement, from preoperatively to final follow-up, in the mean JSSF RA foot and ankle score (from 52.2 to 76.9 points) and the mean JSSF hallux score (from 38.2 to 74.5 points). There was a recurrence (hallux valgus angle [HVA] of >20°) in 12 feet (16%). The preoperative DAS28-CRP score (disease activity score [based on 28 joints in the body]-C-reactive protein score) and intermetatarsal angles between the first and second metatarsals (M1M2A) and between the first and fifth metatarsals (M1M5A) were significantly greater in the recurrence group, as were the HVA, M1M2A, M1M5A, and Hardy grade at 3 months after surgery. There was a significant negative correlation between the preoperative DAS28-CRP score and the JSSF RA foot and ankle score at final follow-up (β = -0.39, p = 0.02) and a significant positive correlation between the preoperative DAS28-CRP score and the HVA at final follow-up (β = 0.44, p = 0.001).The modified Scarf osteotomy with medial capsule interposition for hallux valgus deformity improved clinical and radiographic outcomes in RA cases with severe destruction of the first MTP joint. Increased preoperative M1M2A and M1M5A; incomplete reduction of the sesamoid bone; and the HVA, M1M2A, and M1M5A at 3 months after surgery should be evaluated as they are associated with recurrence of the deformity. The preoperative DAS28-CRP score was associated with the clinical and radiographic outcomes after surgery.Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

  View details for DOI 10.2106/JBJS.17.00436

  View details for Web of Science ID 000437292100016

  View details for PubMedID 29715225

• Utility of intravenous tranexamic acid in single-level posterior lumbar interbody fusions Response JOURNAL OF NEUROSURGERY-SPINE Kushioka, J., Yamashita, T., Okuda, S., Maeno, T., Matsumoto, T., Iwasaki, M., Yamasaki, R. 2017; 27 (5): 616

  View details for DOI 10.3171/2017.4.SPINE17388

  View details for Web of Science ID 000414357100019

• High-dose tranexamic acid reduces intraoperative and postoperative blood loss in posterior lumbar interbody fusion JOURNAL OF NEUROSURGERY-SPINE Kushioka, J., Yamashita, T., Okuda, S., Maeno, T., Matsumoto, T., Yamasaki, R., Iwasaki, M. 2017; 26 (3): 363–67

  Abstract

  OBJECTIVE Tranexamic acid (TXA), a synthetic antifibrinolytic drug, has been reported to reduce blood loss in orthopedic surgery, but there have been few reports of its use in spine surgery. Previous studies included limitations in terms of different TXA dose regimens, different levels and numbers of fused segments, and different surgical techniques. Therefore, the authors decided to strictly limit TXA dose regimens, surgical techniques, and fused segments in this study. There have been no reports of using TXA for prevention of intraoperative and postoperative blood loss in posterior lumbar interbody fusion (PLIF). The purpose of the study was to evaluate the efficacy of high-dose TXA in reducing blood loss and its safety during single-level PLIF. METHODS The study was a nonrandomized, case-controlled trial. Sixty consecutive patients underwent single-level PLIF at a single institution. The first 30 patients did not receive TXA. The next 30 patients received 2000 mg of intravenous TXA 15 minutes before the skin incision was performed and received the same dose again 16 hours after the surgery. Intra- and postoperative blood loss was compared between the groups. RESULTS There were no statistically significant differences in preoperative parameters of age, sex, body mass index, preoperative diagnosis, or operating time. The TXA group experienced significantly less intraoperative blood loss (mean 253 ml) compared with the control group (mean 415 ml; p < 0.01). The TXA group also had significantly less postoperative blood loss over 40 hours (mean 321 ml) compared with the control group (mean 668 ml; p < 0.01). Total blood loss in the TXA group (mean 574 ml) was significantly lower than in the control group (mean 1080 ml; p < 0.01). From 2 hours to 40 hours, postoperative blood loss in the TXA group was consistently significantly lower. There were no perioperative complications, including thromboembolic events. CONCLUSIONS High-dose TXA significantly reduced both intra- and postoperative blood loss without causing any complications during or after single-level PLIF.

  View details for DOI 10.3171/2016.8.SPINE16528

  View details for Web of Science ID 000394925900013

  View details for PubMedID 27885960

• Outcomes of modified metatarsal shortening offset osteotomy for forefoot deformity in patients with rheumatoid arthritis: Short to mid-term follow-up MODERN RHEUMATOLOGY Hirao, M., Ebina, K., Tsuboi, H., Nampei, A., Kushioka, J., Noguchi, T., Tsuji, S., Owaki, H., Hashimoto, J., Yoshikawa, H. 2017; 27 (6): 981–89

  Abstract

  Advances in drug therapy for rheumatoid arthritis (RA) have been encouraging us to preserve the metatarsopharangeal (MTP) joint in correction of forefoot deformities, and original metatarsal shortening offset osteotomy was recommended as one of the conventional surgical options for forefoot deformities in RA cases. The objective of this study was to evaluate short- to mid-term outcomes of modified metatarsal shortening offset osteotomy.A retrospective observational study was completed for 80 RA cases (mean follow-up period: 3.2 years) who underwent modified metatarsal shortening offset osteotomy. Both lesser toe scales and RA foot ankle scales were administered using the Japanese Society for Surgery of the Foot (JSSF) standard rating system, and a postoperative self-administered foot evaluation questionnaire (SAFE-Q) at final follow-up was also checked to evaluate clinical outcomes.This procedure significantly improved clinical scores of both the JSSF [lesser toes and RA foot and ankle] scales. Of 80 feet, 24 (30%) showed recurrence of MTP joint subluxation/dislocation. Furthermore, the feet in the recurrence group showed significant varus hindfoot. On the other hand, valgus foot in the recurrence group more frequently included midfoot bony ankyloses. All of the affected feet showed the limitation of MTP joints (<70°) after surgery.Modified metatarsal shortening offset osteotomy was recommended for RA forefoot disorders as one of the joint preservation surgeries in short- to mid-term follow-up. However, some modifications to avoid limitation of ROM in the MTP joint are required. It must be borne in mind that varus hindfoot and/or bony ankyloses in the mid-hindfoot can cause recurrence of dorsal dislocation/subluxation of the lesser toe MTP joint.

  View details for DOI 10.1080/14397595.2016.1276512

  View details for Web of Science ID 000415965200010

  View details for PubMedID 28142302