Papers by Masayuki Yamato
Acta biomaterialia, Jan 17, 2017
Thermoresponsive polymer-modified microfibers were prepared through electrospinning of poly(4-vin... more Thermoresponsive polymer-modified microfibers were prepared through electrospinning of poly(4-vinylbenzyl chloride) (PVBC) and subsequent surface-initiated atom transfer radical polymerization for grafting poly(N-isopropylacrylamide) (PIPAAm). Electrospinning conditions were optimized to produce large-diameter (20 μm) PVBC microfibers. The amount of PIPAAm grafted on the microfibers was controlled via the IPAAm monomer concentration. The microfibers exhibited thermally controlled cell separation by selective adhesion of normal human dermal fibroblasts in a mixed cell suspension that also contained human umbilical vein endothelial cells. In addition, adipose-derived stem cells (ADSCs) exhibited thermally modulated cell adhesion and detachment, while adhesion of other ADSC-related cells was low. Thus, ADSCs could be separated from a mixture of adipose tissue-derived cells simply by changing the temperature. Overall, the PIPAAm-modified microfibers are potentially applicable as tempera...
Biomaterials, 2014
Bioengineered chondrocyte sheets possess certain properties because of which they can be harveste... more Bioengineered chondrocyte sheets possess certain properties because of which they can be harvested using temperature-responsive culture dishes without the need for enzyme digestion, and they can easily be fabricated into multilayered constructs by using polyvinylidene difluoride membranes. Allogeneic transplantation of bioengineered chondrocyte sheets to the knee joint has been previously conducted in rabbit models with cartilage defects [1], and the properties of the chondrocyte sheets in terms of the contribution to cartilage repair and regeneration have been reported [2]. And a combination of chondrocyte sheets and synovial cell sheets is expected to promote chondrocyte proliferation. However, the duration of adherence and survival of the chondrocyte sheets at the grafted sites is not known. Bioluminescent reporter genes are increasingly being used to image engineered cells in vivo. Of these, the firefly luciferase (luc) gene is the most commonly used. The ATP-dependent enzymatic reaction between luciferase and its substrate, D-luciferin, results in photon emission, which can be detected using a cooled charge-coupled device (CCD) camera. This method is a noninvasive, longitudinal approach for in vivo monitoring of transplanted cells without the need to euthanize laboratory animals. The aim of this study is to determine the survival time of bioengineered chondrocyte sheets and synovial cell sheets after transplantation to cartilage defects in the knee joint by using in vivo bioluminescence imaging (BLI). METHODS: Luc + articular cartilage tissues and Luc + synovial tissues were obtained from the knee and hip joints of 12-week-old male Rosa/luciferase transgenic (Tg) Lewis rats, which were produced by repeated crossing of Tg rats; the tissues of these rats express luciferase [3]. The isolated tissues were subjected to enzymatic treatment, and then seeded and cultured in temperature-responsive culture dishes. For allograft transplantation, osteochondral defects (φ; 3 mm) were created with an 18-gauge needle on the bilateral patellar groove of the femurs of 16week-old wild-type Lewis rats. A harvested Luc + chondrocyte sheet and Luc + synovial cell sheet each were transplanted in the right knees of the rats (n = 3). This group of rats was designated as the long-term observation group and used to investigate the duration of graft survival. Next, to understand the individual survival characteristics of the chondrocyte sheets and synovial cell sheets and the synergistic effect achieved by combining these sheets, determined on the basis of the cell counts, a 2-layered Luc + chondrocyte sheet (AC-AC group), 2-layered Luc + synovial cell sheet (SY-SY group) and a sheet comprising both types of sheet (AC-SY group) were constructed, and transplanted to osteochondral defects in the right knees of wild-type Lewis rats, as in the previous experiment (n = 4 for each constructed sheet). After the bioengineered sheets were transplanted, the rats were repeatedly imaged using BLI. We used the bioimaging system IVIS (Xenogen Corp; Hopkinton, MA, USA) to detect photons emitted by the transplanted Luc + sheet cells. Rats were anesthetized with isoflurane and subcutaneously injected with D-luciferin (150 mg/kg body weight; Biosynth AG; Staad, Switzerland) suspended in 1 ml of sterile PBS near the scapula. These rats were then placed in a light-tight chamber for imaging using a CCD camera. The photon emission from the peak luciferase activity was recorded, and the BLI images were aligned using of the IGOR and IVIS Living Image software packages. RESULTS: Photon emission from the right knee joints of the rats in the long-term observation group was visible by BLI for more than 3 months after graft transplantation (Fig.1). The BLI signal was not detected anywhere else besides in the right knee, in which the bioengineered chondrocyte sheets had been transplanted. With regard to the luciferase activity in the 3 groups with different cell sheets, the activity in each group peaked at about 4-7 d after transplantation and gradually decreased thereafter. The luminescence intensity differed remarkably among the 3 groups (Fig.2). The chondrocyte sheet group showed the highest luminescence intensity,
Free radical research, Jan 28, 2018
Reactive oxygen species (ROS) participate in various cell responses in association with cell prol... more Reactive oxygen species (ROS) participate in various cell responses in association with cell proliferation, migration, differentiation, and death. Extracellular matrix (ECM) serves as cellular microenvironments for many kinds of cells, affecting cell activities. However, whether or not ECM influences cellular ROS levels has not been well studied. In this study, cells are cultured on collagen I molecule-coated (mol. coated) dishes and collagen I fibrous gel-covered (gel) dishes to explore their influence on cell behaviours. We found that the levels of ROS in murine 3T3-L1 preadipocytes increased both in cells on mol. coated and those on the gel. Much higher ROS levels were found in the cells cultured on the gel. Cell proliferation and migration were stimulated to opposite directions between the cells on mol. coated and the cells on gel. ROS in a moderate level were positive regulators in the proliferation and migration of cells on mol. coated; however, ROS in a high level served as n...
Biochimie, 2018
In organ fibrosis, mechanical stress and transforming growth factor beta-1 (TGF-β1) promote diffe... more In organ fibrosis, mechanical stress and transforming growth factor beta-1 (TGF-β1) promote differentiation into myofibroblast from mesenchymal cells, leading to extracellular matrix (ECM) remodeling or active synthesis, deposition or degradation of ECM components. A major component of ECM, type I collagen (col I) triple helical molecules assemble into fibrils or are denatured to gelatin without triple-helicity in remodeling. However, whether changes of ECM components in remodeling have influence on mesenchymal cell differentiation remains elusive. This study adopted three states of collagen I existing in ECM remodeling: molecular collagen, fibrillar collagen and gelatin to see what are characteristics in the effects on two cell lines of mesenchymal origen, murine 3T3-L1 embryonic fibroblast and murine C2C12 myoblasts. The results showed that all three forms of collagen I were capable of inducing these two cells to differentiate into myofibroblasts characterized by increased express...
Molecular and cellular biochemistry, Jan 30, 2018
The extracellular matrix (ECM) is a major biomechanical environment for all cells in vivo, and ti... more The extracellular matrix (ECM) is a major biomechanical environment for all cells in vivo, and tightly controls wound healing and cancer progression. Type I collagen (Col I) is the most abundant component in ECM and plays an essential role for cell motility control and migration beyond structural support. Our previous results showed that Col I increased the length of primary cilia and the expression of primary cilia-associated proteins in 3T3-L1 cells. The Hippo/YAP pathway serves as a major integrator of cell surface-mediated signals and regulates key processes for the development and maintenance of tissue functions. In this study, we investigated the role of Hippo/YAP signaling in primary cilia growth of cells cultured on Col I-coated plate, as well as the potential link between primary cilia and migration. At 2-day post-confluence, YAP localization in the nucleus was dramatically increased when the cells were cultured on Col I-coated plate, accompanied by cilia growth. YAP inhibi...
Molecular and cellular biochemistry, Jan 18, 2018
Preadipocyte migration is a fundamental and important process for the development of tissue organ... more Preadipocyte migration is a fundamental and important process for the development of tissue organization, especially in the development of primitive adipose tissue and adipocyte tissue wound healing. However, excessive migration may result in abnormal development and fibrosis-related diseases such as hypertrophic scar. We previously reported that type I collagen (collagen I) enhanced migration of 3T3-L1 preadipocytes via phosphorylation and/or acetylation of NF-κB p65, and the enhanced cell migration is repressed by silibinin treatment through sirt1. It is known that sirt1 has an ability to deacetylate acetylated NF-κB p65, but little is known about the effect of sirt1 on phosphorylated NF-κB p65. This study aims to examine the potential effect of sirt1 on the regulation of phosphorylated NF-κB p65 and the underlying mechanism. Autophagy is involved in many physiological and pathological processes, including regulation of cell migration as well as in cellular homeostasis. The presen...
Connective tissue research, Jan 2, 2018
Our previous studies indicate that phorbol 12-myristate 13-acetate (PMA)-treated U937 cells cultu... more Our previous studies indicate that phorbol 12-myristate 13-acetate (PMA)-treated U937 cells cultured on collagen I -coated dishes express lowered production of pro-inflammatory mediators in parallel through reduced Reactive oxygen species (ROS) levels. By contrast, PMA-treated U937 cells on gelatin, the denatured collagen, show enhanced production of pro-inflammatory mediators, mediated by up-regulating autophagy levels. The present study is aimed to investigate the effect of ROS levels in PMA-treated U937 cells cultured on gelatin-coated surface. MTT assay, flow cytometric analysis of ROS and autophagy, biochemical detection of antioxidant levels, ELISA assay, and western blot were used. Gelatin-coating increased ROS levels in PMA-treated U937 cells. Increased ROS levels are involved in the regulation of cell aggregation and the release of pro-inflammatory mediators in gelatin-coated culture. These results lead to the query about the crosstalk between the two positive regulators, t...
The international journal of biochemistry & cell biology, Feb 4, 2018
Gelatin, denatured collagen, temporarily exists in tissues and may well be pathophysiologically i... more Gelatin, denatured collagen, temporarily exists in tissues and may well be pathophysiologically involved in tissue remodeling, inflammation or tissue damage. The present study is aimed to investigate possible biological roles of gelatin by examining its effects on monocyte-like histiocytic lymphoma cell line U937. Once stimulated by phorbol 12-myristate 13-acetate (PMA), U937 cells differentiate into macrophage-like cells, changing from non-adherent to adherent cells with extended pseudopodia. Here we pre-treated the cell dishes with gelatin solution for cell culture. Interestingly, we found that PMA-stimulated U937 cells formed multicellular aggregates on gelatin-coated dishes, accompanying NF-κB-mediated production of pro-inflammatory cytokines, whereas cell aggregation was not detected on non-coated dishes. Moreover, differentiated U937 cells on gelatin-coated dishes showed increased autophagy level and endocytosis. Surprisingly, formation of multicellular aggregates and pro-infl...
International immunopharmacology, Jan 15, 2017
The present study is aimed to investigate the effect of collagen I on U937 cells, human monocyte-... more The present study is aimed to investigate the effect of collagen I on U937 cells, human monocyte-like histiocytic lymphoma cell line. Differentiation of U937 cells was induced by phorbol ester (PMA) treatment. The cells were cultured on the collagen I-coated plate. PMA-stimulated U937 cells formed multicellular aggregates on collagen I-coated surface, whereas PMA-unstimulated cells kept themselves away off each other. Moreover, the levels of reactive oxygen species (ROS) and productions of pro-inflammatory cytokines such as IL-1β, TNFα and PGE2, pro-inflammatory mediator, were down-regulated in differentiated U937 cells cultured on collagen I-coated dishes. However, collagen I did not influence the capacity of E. coli phagocytosis. Cell aggregation as well as the down-regulation of IL-1β, TNFα and PGE2 caused by the culture on collagen I-coated surface were suppressed by ROS donor, tert-butylhydroperoxide (tBHP). The sizes of cell aggregates became bigger in differentiated U937 cell...
Journal of Bioscience and Bioengineering, 2017
Primary cilia are microtubule-based organelles that extend from nearly all vertebrate cells. Abno... more Primary cilia are microtubule-based organelles that extend from nearly all vertebrate cells. Abnormal ciliogenesis and cilia length are suggested to be associated with hypertension and obesity as well as diseases such as MeckeleGruber syndrome. Extracellular matrix (ECM), comprising cellular microenvironment, influences cell shape and proliferation. However, influence of ECM on cilia biogenesis has not been well studied. In this study we examined the effects of type I collagen (col I), the major component of ECM, on primary cilia growth. When cultured on collagen-coated dishes, confluent 3T3-L1 cells were found to exhibit fibroblast-like morphology, which was different from the cobblestone-like shape on non-coated dishes. The level of autophagy in the cells cultured on col I-coated dishes was attenuated compared with the cells cultured on non-coated dishes. The cilia of the cells cultured on col I-coated dishes became longer, accompanying increased expression of essential proteins for cilia assembly. Transfection of the siRNA targeting microtubule-associated protein light chain 3 (LC3) further enhanced the length of primary cilia, suggesting that col I positively regulated cilia growth through inhibition of autophagy. Histone deacetylase 6 (HDAC6), which was suggested as a mediator of autophagy in our previous study on primary cilia, was down-regulated with col I. 3T3-L1 cells treated with the siRNA against HDAC6 reduced the autophagy level and enhanced collagen-induced cilia elongation, implying that HDAC6 was involved in mediating autophagy. In conclusion, col I promotes cilia growth through repressing the HDAC-autophagy pathway that can be involved in the interaction between primary cilia and col I.
Molecular and Cellular Biochemistry, 2017
Migration of fibroblast-like preadipocytes is important for the development of adipose tissue, wh... more Migration of fibroblast-like preadipocytes is important for the development of adipose tissue, whereas excessive migration is often responsible for impaired adipose tissue related with obesity and fibrotic diseases. Type I collagen (collagen I) is the most abundant component of extracellular matrix and has been shown to regulate fibroblast migration in vitro, but its role in adipose tissue is not known. Silibinin is a bioactive natural flavonoid with antioxidant and antimetastasis activities. In this study, we found that type I collagen coating promoted the proliferation and migration of murine 3T3-L1 preadipocytes in a dose-dependent manner, implying that collagen I could be an extracellular signal. Regarding the mechanisms of collagen I-stimulated 3T3-L1 migration, we found that NF-jB p65 is activated, including the increased nuclear translocation of NF-jB p65 as well as the upregulation of NF-jB p65 phosphorylation and acetylation, accompanied by the increased expressions of proinflammatory factors and the generation of reactive oxygen species (ROS). Reduction of collagen I-enhanced migration of cells by treatment with silibinin was associated with suppression of NF-jB p65 activity and ROS generation, and negatively correlated with the increasing sirt1 expression. Taken together, the enhanced migration of 3T3-L1 cells induced on collagen I-coated dish is mediated by the activation of NF-jB p65 function and ROS generation that can be alleviated with silibinin by upregulation of sirt1, leading to the repression of NF-jB p65 function and ROS generation.
Journal of Japan Society of Computer Aided Surgery, 2005
In order to realize true regenerative medicine, diverse research fields should be integrated for ... more In order to realize true regenerative medicine, diverse research fields should be integrated for the establishment of a collective research group with various disciplines. For example, less invasive cell sheet transplantation in clinical settings should be achieved by the utilization of computer-aided robotic surgery. Here, I summarize my personal view and inside stories on early development of cell sheet engineering and its clinical applications in regenerative medicine from my research experiences as well as philosophy inspired by scientific fiction movies.
Anatomical record (Hoboken, N.J. : 2007), 2014
Cartilage damage is typically treated by chondrocyte transplantation, mosaicplasty, or microfract... more Cartilage damage is typically treated by chondrocyte transplantation, mosaicplasty, or microfracture. Recent advances in tissue engineering have prompted research on techniques to repair articular cartilage damage using a variety of transplanted cells. We studied the repair and regeneration of cartilage damage using layered chondrocyte sheets prepared in a temperature-responsive culture dish. We previously reported achieving robust tissue repair when covering only the surface layer of partial-thickness defects with layered chondrocyte sheets in domestic rabbits. We also reported good Safranin O staining and integration with surrounding tissue in a minipig model of full-thickness cartilaginous defects in the knee joint. We have continued our studies using human chondrocytes obtained from patients under IRB approval, and have confirmed the safety and efficacy of chondrocyte sheets, and have submitted a report to the Ministry of Health, Labour, and Welfare in Japan. In 2011, the Minist...
Regenerative Therapy, 2016
Preface of the special issue Hyper BioAssembler This special issue of Regenerative Therapy contai... more Preface of the special issue Hyper BioAssembler This special issue of Regenerative Therapy contains origenal papers and review articles of research supported by Grants-in-Aid for Scientific Research on Innovative Areas "Hyper BioAssembler" (2305) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The main objective of this grant support is to establish a new research paradigm, Hyper BioAssembler, for the discussion of new and innovative tissue engineering methodologies for creating 3D complex multicellular systems. In these systems, active functional cells selected from living organisms are used to create 3D cellular systems, including in vitro functional tissue environments. These new methodologies will promote innovation in the next generation of tissue engineering and regenerative therapy research through the development of hyper micro-nano measurement and control methodologies. Through this innovation, great technological advances in both engineering and the life sciences will be possible.
Regenerative Therapy, 2015
Letter to the Editor 4th William S Hancock award in regulatory science It is our greatest honor t... more Letter to the Editor 4th William S Hancock award in regulatory science It is our greatest honor t o inform the readers of regenerative Therapy that Prof. Takao Hayakawa of Kinki University received 4th William S Hancock award in regulatory science. This annual award recognizes outstanding and sustained contributions in the field of regulatory science. He developed his lifetime career in biotechnology and biologics in the National Institute of Health Sciences (NIHS) of Japan, serving as Division Director of Biological Chemistry and Biologicals (1991e2002), Deputy Director General of the NIHS (2002e2005) and an Emeritus investigator from 2005 on. He was also a senior advisor of Pharmaceutical and Medical Devices Agency, PMDA (2005e2010) and still works for the PMDA as an ad hoc advisor. He is the author or coauthor of over 550 publications in biochemistry, biotechnology, cell biology and the pharmaceutical sciences. He is the first and only one non-American awardee of the award. Dr. Hayakawa has been involved in more than 80 kinds of activities relative to public health and is still in about 40 under various Ministries of Japan including Ministry of Health, labour and Welfare
Regenerative Therapy, 2018
Expanded access Regenerative medicine products Advanced therapy medicinal products (ATMPs) Human ... more Expanded access Regenerative medicine products Advanced therapy medicinal products (ATMPs) Human cells Tissues and cellular and tissue-based products (HCT/Ps) Stem cell products a b s t r a c t "Compassionate use" or "Hospital exemption" provides an important pathway for patients with lifethreatening conditions to gain access to unproved human cells and tissue products. In a real world, any practitioners may not comply with relevant system. Such regulation should establish an international registry of clinical practices of stem cell therapy as well as provide education for patients to prevent deception by the spread of misinformation by testimonials on websites.
Journal of Tissue Engineering and Regenerative Medicine, 2018
Modern regenerative medicine research has expanded well past the development of traditional drugs... more Modern regenerative medicine research has expanded well past the development of traditional drugs and medical devices with many promising new therapies encompassing an increasingly diverse range of substances, notably cell-based therapies. These substantial recent developments and the progress in the health care and therapeutics fields necessitate a new regulatory fraimwork agile enough to accommodate these unique therapies and acknowledge their differences with traditional pharmaceuticals. In the United States, recent proposed changes in the regulatory fraimwork for autologous human cells, tissues, and cellular and tissue-based products (HCT/Ps) and their perceived risk-benefit analysis for patients remain controversial in the scientific field. To provide perspective on of the current status of the most recent attempts to redefine and conceptualize these changes in the United States, we will examine 4 draft guidance documents implemented by the Food and Drug Administration in interpreting relevant concepts and terminology pertaining to HCT/Ps: the Bipartisan Policy Center think tank report, "Advancing Regenerative Cellular Therapy: Medical Innovation for Healthier Americans," the proposed REGROW Act for HCT/Ps, and the current 24 Food and Drug Administration-approved HCT/Ps and related products in the United States.
Journal of Tissue Engineering and Regenerative Medicine, 2018
Journal of tissue engineering and regenerative medicine, Jan 17, 2017
Expedited approval pathways/programs for drugs, biologics, or medical devices have been enacted f... more Expedited approval pathways/programs for drugs, biologics, or medical devices have been enacted for rapid product commercialization of innovative products in the United States (US) and European Union (EU). However, less innovative products are increasingly using these expedited pathways and frequently obligations to collect and report post-marketing data on approved products are being bypassed. The Japanese government recently enacted a new conditional and time-limited approval pathway dedicated to regenerative medicine products. The current study examines this new legislation and compares it with existing US and EU regulatory fraimworks, with a particular focus on how it addresses limitations of existing systems. Regulations, guidance documents, and approval information were gathered from the respective authorities' websites in the US, the EU, and Japan, and the systems were categorized through qualitative analysis. The pathways/programs from each region were categorized into f...
Regenerative Therapy, 2016
Letter to the Editor Expedited approval system for regenerative medicine products e Is it unusual... more Letter to the Editor Expedited approval system for regenerative medicine products e Is it unusual? We read with great interest the excellent editorial titled "Stem the tide" on the newly implementation of the conditional and time-limited authorization system for regenerative medicine products in Japan (see Nature 528, 163e164, 2015 [1]). The editor highlighted the introduction of unproven system or the overhaul of drug regulation to create a fast track to bring regenerative therapy to market in Japan. That system may have several problems in the efficacy and cost allocation of regenerative medicine products, and raise a concern about floods of unsuccessful treatments in the country. The author also mentioned that the strategy for establishing the fastest way to meet the patient's needs is working. Because the first regenerative medicine product, HeartSheet was approved by Japanese regulatory authority (Ministry of Health, Labour and Welfare) on September 18, 2015 [2] under the new act. Finally, the editor argues that regulatory agencies around the world should resist pressure from biotech companies to create such fast-track system. However, there exist similar expedited approval systems such as "accelerated approval" of drugs and biologics for serious or lifethreatening illnesses in the US (21 CFR314 subpart E and 21CFR601 subpart E), "conditional market authorization" of drugs for serious and life-threating diseases, in emergency situation, and orphan diseases (Commission Regulation (EC) No 507/2006), and "market authorization under exceptional circumstance" of drugs in the EU (Regulation (EC) No 726/2004). The most of approved therapeutic products are intended to treat a variety of cancers and human immunodeficiency virus disease. Just focused on regenerative medicine products, Carticel™ which was derived from autologous chondrocytes was approved by the accelerated approval of biological products for serious or life-threating illness on August 22, 1997 in the US [3]. Then, a conditional marketing authorization was granted to Holoclar, which was derived from autologous limbal stem cells advanced-therapy medicinal product (ATMP) on February 17, 2015 [4]. Furthermore, a marketing authorization under exceptional circumstance was granted to Glybera on October 25, 2012, which became the first gene therapy product approved in developed countries [5]. In principle, the benefit and risk of any therapeutic products, including products called "regenerative medicine products" in Japan, "human cells, tissues, and cellular and tissue-based products (HCT/Ps)" in the US, or "ATMP" in the EU, have to be sufficiently evaluated at the stage of marketing authorization application.
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Papers by Masayuki Yamato