Hyaluronan and chondroitin sulfate in chicken-vegetable bone broth delay osteoporosis progression.

Bone broth has recently gained worldwide recognition as a superfood that supplements several nutrients lacking in modern human diets; however, little is known of its efficacy on osteoporosis. Therefore, we aimed to identify the components of chicken-vegetable bone broth (CVBB) that are associated with osteoporosis prevention and verified the efficacy of these components using in vivo studies. In biochemical and cell biological experiments, CVBB was fractionated using ion exchange chromatography (IEC), and the effect of each IEC fraction on osteoclast differentiation was evaluated based on tartrate-resistant acid phosphatase (TRAP) activity, TRAP staining, and quantitative polymerase chain reaction analysis using mouse macrophage-like cells (RAW264 cell). In animal experiments, an ovariectomized (OVX) rat model was generated, followed by whole bone broth (OVX/CVBB) or IEC fraction (OVX/CVBB-Ext) administration and bone structural parameter characterization of OVX rat tibia based on micro-CT. Four CVBB fractions were obtained using IEC, and the fraction containing both hyaluronan and chondroitin sulfate (CVBB-Ext) led to the maximum inhibition of RAW264 cell differentiation. CVBB-Ext downregulated the expression of osteoclast differentiation marker genes. In animal experiments, the OVX group showed a clear decrease in bone density compared to that in the Sham operation group. The OVX/CVBB and OVX/CVBB-Ext groups showed increased bone mineral density and bone volume/tissue volume values compared to those in the OVX/control group. These results suggested that CVBB and CVBB-Ext slowed osteoporosis progression. Therefore, we conclude that hyaluronan and chondroitin sulfate in CVBB are key substances that impede osteoporosis progression. PRACTICAL APPLICATION: This study provides practical information on the effects of bone broth ingredients on osteoporosis to expand the current knowledge on the efficacy of bone broth, which is a widely consumed food. These results may help in the future development of bone broth as a dietary supplement for managing osteoporosis.

Synergistic effect of FGF-2 and TGF-ß1 on the mineralization of human umbilical cord perivascular cells.

OBJECTIVE: Human umbilical cord perivascular cells (HUCPVCs) are derived from the human umbilical cord perivascular tissue and are expected to replace mesenchymal stromal cells in the future. We investigated the synergistic effects of fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 1 (TGF-ß1) on HUCPVC mineralization. DESIGN: We prepared HUCPVCs with (FGF(+)HUCPVCs) or without FGF-2 (FGF(-)HUCPVCs) in the presence of activated vitamin D3, a bone morphogenic protein inhibitor, and TGF-ß1. We examined the cell proliferative capacity, expression of various hard tissue-forming cell gene markers, and mineralization induction ability and identified the crystalline phases of the mineralized nodules. RESULTS: FGF(+)HUCPVCs exhibited higher intracellular alkaline phosphatase (ALP) gene expression and ALP activity, and their cell proliferation rate was higher than that of FGF(-)HUCPVCs. The expression levels of osteoblast marker genes increased in FGF(+)HUCPVCs, whereas those of elastic fiber and muscle cell markers increased in FGF(-)HUCPVCs. The expression of genes related to matrix vesicle-mediated mineralization was increased in FGF(+)HUCPVCs. While FGF(-)HUCPVCs displayed myofibroblast-like properties and could not induce mineralization, FGF(+)HUCPVCs demonstrated the ability to produce mineralized nodules. The resulting mineralized nodules consisted of hydroxyapatite as the major phase and minor amounts of octacalcium phosphate. The mineralized nodules exhibited the morphological characteristics of bone hydroxyapatite, composed of fibrous hydroxyapatite nanorods and polycrystalline sheets. CONCLUSION: We found that FGF-2 synergizes with TGF-ß1 and is a key factor in the differentiation of HUCPVCs into osteoblast-like cells. Thus, HUCPVCs can potentially serve as a new stem cell source for future bone regeneration and dental treatments.

Enzyme replacement with transferrin receptor-targeted α-L-iduronidase rescues brain pathology in mucopolysaccharidosis I mice.

Mucopolysaccharidosis I (MPS I), a lysosomal storage disease caused by dysfunction of α-L-iduronidase (IDUA), is characterized by the deposition of dermatan sulfate (DS) and heparan sulfate (HS) throughout the body, which causes several somatic and central nervous symptoms. Although enzyme-replacement therapy (ERT) is currently available to treat MPS I, it does not alleviate central nervous disorders, as it cannot penetrate the blood-brain barrier. Here we evaluate the brain delivery, efficacy, and safety of JR-171, a fusion protein comprising humanized anti-human transferrin receptor antibody Fab and IDUA, using monkeys and MPS I mice. Intravenously administered JR-171 was distributed in major organs, including the brain, and reduced DS and HS concentrations in the central nervous system and peripheral tissues. JR-171 exerted similar effects on peripheral disorders similar to conventional ERT and further reversed brain pathology in MPS I mice. We found that JR-171 improved spatial learning ability, which was seen to deteriorate in the vehicle-treated mice. Further, no safety concerns were noted in repeat-dose toxicity studies in monkeys. This study provides nonclinical evidence that JR-171 might potentially prevent and even improve disease conditions in patients with neuronopathic MPS I without serious safety concerns.

Characterization of bioactive substances involved in the induction of bone augmentation using demineralized bone sheets.

PURPOSE: To investigate the bone augmentation ability of demineralized bone sheets mixed with allogeneic bone with protein fractions containing bioactive substances and the interaction between coexisting bioactive substances and proteins. METHODS: Four types of demineralized bone sheets mixed with allogeneic bone in the presence or absence of bone proteins were created. Transplantation experiments using each demineralized bone sheet were performed in rats, and their ability to induce bone augmentation was analysed by microcomputed tomography images. Bioactive substances in bone proteins were isolated by heparin affinity chromatography and detected by the measurement of alkaline phosphatase activity in human periodontal ligament cells and dual luciferase assays. Noncollagenous proteins (NCPs) coexisting with the bioactive substances were identified by mass spectrometry, and their interaction with bioactive substances was investigated by in vitro binding experiments. RESULTS: Demineralized bone sheets containing bone proteins possessed the ability to induce bone augmentation. Bone proteins were isolated into five fractions by heparin affinity chromatography, and transforming growth factor-beta (TGF-ß) was detected in the third fraction (Hep-c). Dentin matrix protein 1 (DMP1), matrix extracellular phosphoglycoprotein (MEPE), and biglycan (BGN) also coexisted in Hep-c, and the binding of these proteins to TGF-ß increased TGF-ß activity by approximately 14.7% to 32.7%. CONCLUSIONS: Demineralized bone sheets are capable of inducing bone augmentation, and this ability is mainly due to TGF-ß in the bone protein mixed with the sheets. The activity of TGF-ß is maintained when binding to bone NCPs such as DMP1, MEPE, and BGN in the sheets.

Pathogenic Roles of Heparan Sulfate and Its Use as a Biomarker in Mucopolysaccharidoses.

Heparan sulfate (HS) is an essential glycosaminoglycan (GAG) as a component of proteoglycans, which are present on the cell surface and in the extracellular matrix. HS-containing proteoglycans not only function as structural constituents of the basal lamina but also play versatile roles in various physiological processes, including cell signaling and organ development. Thus, inherited mutations of genes associated with the biosynthesis or degradation of HS can cause various diseases, particularly those involving the bones and central nervous system (CNS). Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders involving GAG accumulation throughout the body caused by a deficiency of GAG-degrading enzymes. GAGs are stored differently in different types of MPSs. Particularly, HS deposition is observed in patients with MPS types I, II, III, and VII, all which involve progressive neuropathy with multiple CNS system symptoms. While therapies are available for certain symptoms in some types of MPSs, significant unmet medical needs remain, such as neurocognitive impairment. This review presents recent knowledge on the pathophysiological roles of HS focusing on the pathogenesis of MPSs. We also discuss the possible use and significance of HS as a biomarker for disease severity and therapeutic response in MPSs.

Pharmacokinetics and pharmacodynamics of once-weekly administration of JR-142, a long-acting albumin-fused human growth hormone: A rondemized, placebo-controlled phase 1 study.

OBJECTIVE: Under clinical development for patients with growth hormone deficiency, JR-142 is a long-acting growth hormone with a half-life extended by fusion with modified serum albumin. We conducted a Phase 1 study to investigate the safety, tolerability, and pharmacokinetic (PK) and pharmacodynamic (PD) profiles of once-weekly subcutaneous administrations of JR-142. The study consisted of two parts: an open-label single ascending dosing study (Part 1), and a randomized, placebo-controlled, assessor-blinded multiple ascending dosing study (Part 2). DESIGN: A total of 31 healthy Japanese male participants were enrolled. In Part 1, seven of them received a single subcutaneous injection of JR-142 each at dosages of 0.15 mg/kg (n = 1), 0.25 mg/kg (n = 2), 0.5 mg/kg (n = 2), or 1.0 mg/kg (n = 2). In Part 2, one weekly subcutaneous injection of JR-142 at 0.25 mg/kg, 0.5 mg/kg, 1.0 mg/kg or a placebo were given for four weeks to each of the other 24 participants (six in each group). Plasma JR-142 and serum insulin-like growth factor-1 (IGF-1) concentrations were measured for PK and PD assessments. Safety was evaluated on the basis of adverse events (AEs), laboratory tests, and other measures. RESULTS: JR-142 induced dose-dependent increases in the maximum plasma JR-142 concentration (Cmax) and the area under the plasma concentration-time curve from time 0 to τ (AUC0-τ). A similar dose-response relationship was observed in serum IGF-1 concentrations. All trough IGF-1 levels were well sustained one week after the final administrations of JR-142 at the three dosages, while the peak concentrations of IGF-1 remained mildly elevated. No serious AEs were observed, and laboratory tests, including assessment of anti-drug antibodies, uncovered no significant safety issues. CONCLUSIONS: Once-weekly subcutaneous injections of JR-142 produced positive dose-dependent PK and PD profiles over the dosage range. Drug accumulation was observed after the four-week administration period but did not raise safety concerns, indicating that JR-142 is well-tolerated in healthy participants. The PD profiles observed in terms of IGF-1 concentrations were also positive, and we believe the encouraging results of this study warrant substantiation in further clinical trials in patients with GHD. ETHICS: This clinical study was conducted at one investigational site in Osaka, Japan, where the clinical study and the non-clinical data of JR-142 were reviewed and approved by its Institutional Review Board on 9th May 2019. The study was conducted in compliance with the approved study protocol, the Declaration of Helsinki, 1964, as revised in 2013, and Good Clinical Practice.

Response of TGF-ß isoforms in epithelial-mesenchymal transition of enamel epithelial cells.

OBJECTIVE: During enamel formation, transforming growth factor-beta (TGF-ß) isoforms exhibit different activities for gene expression, apoptosis, and endocytosis. This study aimed to investigate the differential response of TGF-ß isoforms to epithelial-mesenchymal transition (EMT) in enamel epithelial cells. DESIGN: Using a mouse enamel epithelial cell line (mHAT9d) cultured in the presence of each TGF-ß isoform, (1) the morphological changes in EMT were explored, (2) EMT-related genes were analyzed by next-generation sequencing (NGS), (3) TGF-ß pathway for EMT was identified by inhibition experiments, and (4) the expression of the TGF-ß receptor gene in response to the binding affinity of the TGF-ß isoform were analyzed. RESULTS: EMT was observed in mHAT9d cultured in the presence of TGF-ß1 and ß3 but not TGF-ß2. The expression of both epithelial and mesenchymal marker genes was observed in mHAT9d exhibiting EMT. NGS analysis suggested extracellular signal-regulated kinase (ERK) and Rho pathways as TGF-ß signaling pathways associated with EMT. However, EMT in mHAT9d cultured in the presence of TGF-ß1 or ß3 occurred even in presence of an ERK1/2 inhibitor and was suppressed by Rho-kinase inhibitor. The expression of co-receptors for TGF-ß signaling in mHAT9d cells reduced following stimulation with each TGF-ß isoform. In contrast, endoglin levels increased following TGF-ß1 or ß3 stimulation, but no change was noted in response to TGF-ß2. CONCLUSIONS: We propose that in TGF-ß-stimulated enamel epithelial cells, EMT mainly occurred via the Rho signaling pathway, and the differences in response across TGF-ß isoforms were due to their endoglin-mediated binding affinity for the TGF-ß receptor.

Dose-dependent effects of a brain-penetrating iduronate-2-sulfatase on neurobehavioral impairments in mucopolysaccharidosis II mice.

Deposition of heparan sulfate (HS) in the brain of patients with mucopolysaccharidosis II (MPS II) is believed to be the leading cause of neurodegeneration, resulting in several neurological signs and symptoms, including neurocognitive impairment. We recently showed that pabinafusp alfa, a blood-brain-barrier-penetrating fusion protein consisting of iduronate-2-sulfatase and anti-human transferrin receptor antibody, stabilized learning ability by preventing the deposition of HS in the CNS of MPS II mice. We further examined the dose-dependent effect of pabinafusp alfa on neurological function in relation to its HS-reducing efficacy in a mouse model of MPS II. Long-term intravenous treatment with low (0.1 mg/kg), middle (0.5 mg/kg), and high (2.0 mg/kg) doses of the drug dose-dependently decreased HS concentration in the brain and cerebrospinal fluid (CSF). A comparable dose-dependent effect in the prevention of neuronal damage in the CNS, and dose-dependent improvements in neurobehavioral performance tests, such as gait analysis, pole test, Y maze, and Morris water maze, were also observed. Notably, the water maze test performance was inversely correlated with the HS levels in the brain and CSF. This study provides nonclinical evidence substantiating a quantitative dose-dependent relationship between HS reduction in the CNS and neurological improvements in MPS II.

[Pharmacological property, mechanism of action and clinical study results of Pabinafusp Alfa (Genetical Recombination) (IZCARGO® I.V. Infusion 10†mg) as the therapeutic for Mucopolysaccharidosis type-II (Hunter syndrome)].

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal storage disease with the accumulation of glycosaminoglycans in tissues and organs throughout the body caused by dysfunction or loss of iduronate-2-sulfatase (IDS), resulting in somatic and central nervous system (CNS) disorders. Although enzyme replacement therapy (ERT) with recombinant human IDS is the current first-line therapy for MPS II, it is not effective for the CNS because intravenously administered enzyme cannot cross the blood-brain barrier (BBB) and thereby does not reach the brain parenchyma. Pabinafusp alfa, approved in March 2021 in Japan, is a recombinant fusion protein composed of human IDS and humanized anti-human transferrin receptor (hTfR) antibody, utilizing the BBB-penetrating technology "J-Brain Cargo®" established by JCR Pharmaceuticals. Nonclinical studies showed that pabinafusp alfa was distributed in the brain of hTfR knock-in mice and monkeys after intravenous administration, and dose-dependently decreased heparan sulfate (HS) glycosaminoglycan deposited in major organs including the brain of MPS II mice. Pabinafusp alfa also suppressed neurodegeneration in cerebellum and hippocampus, leading to the maintenance of spatial learning ability. Phase II/III clinical study conducted in Japan showed that pabinafusp alfa decreased HS concentration in the cerebrospinal fluid, which serves as an efficacy biomarker for central nervous symptoms, and improved or stabilized the developmental age of the patients. Moreover, pabinafusp alfa exerted comparable effects to current ERT in terms of improvement of somatic manifestations. Therefore, pabinafusp alfa is a promising therapeutic option as a BBB-penetrating enzyme for the treatment of patients with neuronopathic MPS II.

Repurposing MDZ as a tool for tissue regeneration in dental cells.

BACKGROUND: Several recent studies have focused on the utility of drug repurposing to expand clinical application of approved therapeutics. Here, we investigate the efficacy of midazolam (MDZ) and cytokines for regenerating calcified tissue, using immortalized porcine dental pulp (PPU7) and mouse skeletal muscle derived myoblast (C2C12) cells, with the goal of repurposing MDZ as a new treatment to facilitate calcified tissue regeneration. HIGHLIGHTS: We noted that PPU7 and C2C12 cells cultured with various MDZ regimens displayed increased bone morphogenic protein (BMP-2), transforming growth factor beta (TGF-ß), and alkaline phosphatase activity. These increases were highest in PPU7 cells cultured with MDZ alone, and in C2C12 cells cultured with MDZ and BMP-2. PPU7 cells cultured under these conditions demonstrated markedly elevated expression of odontoblastic gene markers, indicating their likely differentiation into odontoblasts. Expression levels of osteoblastic gene markers also increased in C2C12 cells, suggesting that MDZ potentiates the effect of BMP-2, inducing osteoblast differentiation in these cells. Newly formed calcified deposits in both PPU7 and C2C12 cells were identified as hydroxyapatite via crystallographic and crystal engineering analyses. CONCLUSION: MDZ increases ALP activity, inducing expression of specific marker genes for both odontoblasts and osteoblasts while promoting hydroxyapatite production in both PPU7 and C2C12 cells. These responses were cell type specific. MDZ treatment alone could induce these changes in PPU7 cells, but C2C12 cell differentiation required BMP-2 addition.

Development and Characterization of Alkaline Phosphatase-Positive Human Umbilical Cord Perivascular Cells.

Human umbilical cord perivascular cells (HUCPVCs), harvested from human umbilical cord perivascular tissue, show potential for future use as an alternative to mesenchymal stromal cells. Here, we present the results for the characterization of the properties alkaline phosphatase-positive HUCPVCs (ALP(+)-HUCPVCs). These ALP(+)-HUCPVCs were created from HUCPVCs in this study by culturing in the presence of activated vitamin D3, an inhibitor of bone morphogenetic protein signaling and transforming growth factor-beta1 (TGF-ß1). The morphological characteristics, cell proliferation, gene expression, and mineralization-inducing ability of ALP(+)-HUCPVCs were investigated at the morphological, biological, and genetic levels. ALP(+)-HUCPVCs possess high ALP gene expression and activity in cells and a slow rate of cell growth. The morphology of ALP(+)-HUCPVCs is fibroblast-like, with an increase in actin filaments containing alpha-smooth muscle actin. In addition to ALP expression, the gene expression levels of type I collagen, osteopontin, elastin, fibrillin-1, and cluster of differentiation 90 are increased in ALP(+)-HUCPVCs. ALP(+)-HUCPVCs do not have the ability to induce mineralization nodules, which may be due to the restriction of phosphate uptake into matrix vesicles. Moreover, ALP(+)-HUCPVCs may produce anti-mineralization substances. We conclude that ALP(+)-HUCPVCs induced from HUCPVCs by a TGF-ß1 stimulation possess myofibroblast-like properties that have little mineralization-inducing ability.

Usability of detecting delivery errors during treatment of prostate VMAT with a gantry-mounted transmission detector.

Volumetric-modulated arc therapy (VMAT) requires highly accurate control of multileaf collimator (MLC) movement, rotation speed of linear accelerator gantry, and monitor units during irradiation. Pretreatment validation and monitoring of these factors during irradiation are necessary for appropriate VMAT treatment. Recently, a gantry mounted transmission detector "Delta4 Discover® (D4D)" was developed to detect errors in delivering doses and dose distribution immediately after treatment. In this study, the performance of D4D was evaluated. Simulation plans, in which the MLC position was displaced by 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mm from the clinically used original plans, were created for ten patients who received VMAT treatment for prostate cancer. Dose deviation (DD), distance-to-agreement (DTA), and gamma index analysis (GA) for each plan were evaluated by D4D. These results were compared to the results (DD, DTA and GA) measured by Delta4 Phantom + (D4P). We compared the deviations between the planned and measured values of the MLC stop positions A-side and B-side in five clinical cases of prostate VMAT during treatment and measured the GA values. For D4D, when the acceptable errors for DD, DTA, and GA were determined to be ≤3%, ≤2 mm, and ≤3%/2 mm, respectively, the minimum detectable errors in the MLC position were 2.0, 1.5, and 1.5 mm based on DD, DTA, and GA respectively. The corresponding minimum detectable MLC position errors were 2.0, 1.0, and 1.5 mm, respectively, for D4P. The deviation between the planned and measured position of MLC stopping point of prostate VMAT during treatment was stable at an average of -0.09 ± 0.05 mm, and all GA values were above 99.86%. In terms of delivering doses and dose distribution of VMAT, error detectability of D4D was comparable to that of D4P. The transmission-type detector "D4D" is thus suitable for detecting delivery errors during irradiation.

Nonclinical safety evaluation of pabinafusp alfa, an anti-human transferrin receptor antibody and iduronate-2-sulfatase fusion protein, for the treatment of neuronopathic mucopolysaccharidosis type II.

Pabinafusp alfa is a fusion protein comprising a humanized anti-human transferrin receptor (TfR) antibody and human iduronate-2-sulfatase. It was developed as a novel modality to target central nervous system-related symptoms observed in patients with mucopolysaccharidosis type II (MPS II, also known as Hunter syndrome). As the fusion protein contains an entire IgG1 molecule that binds TfR, there may be specific safety concerns, such as unexpected cellular toxicity due to its effector functions or its ability to inhibit iron metabolism, in addition to general safety concerns. Here, we present the comprehensive results of a nonclinical safety assessment of pabinafusp alfa. Pabinafusp alfa did not exhibit effector functions, as assessed by antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity studies in TfR-expressing hematopoietic cells. Repeat-dose toxicity studies in cynomolgus monkeys showed that pabinafusp alfa did not induce any significant toxicological changes at doses up to 30 mg/kg/week upon intravenous administration for up to 26 weeks. Interaction of transferrin with TfR was not inhibited by pabinafusp alfa, suggesting that the effect of pabinafusp alfa on the physiological iron transport system is minimal, which was confirmed by toxicity studies in cynomolgus monkeys. These findings suggest that pabinafusp alfa is expected to be safe for long-term use in individuals with MPS II.

New insights into bioactivity of ceria-stabilized zirconia: Direct bonding to bone-like hydroxyapatite at nanoscale.

Osseointegration resulting from biomineralization means tight bone-implant attachment, which is clinically essential for successful dental implant treatment. The osseointegration ability of ceria-stabilized zirconia, a promising implant material, has been questionable and is unclear despite its clinical use due to zirconia's bioinert nature. The purpose of this research was to investigate the osseointegration ability of ceria-stabilized zirconia by clarifying its bioactivity. Here we show that ceria-stabilized zirconia is highly bioactive, contrary to the general consensus. Transmission electron microscopy observation revealed that the zirconia nanocrystals of a ceria-stabilized zirconia substrate directly bonded to osteoblastic cell-precipitated hydroxyapatite crystals at lattice fringe scale. This bonding was achieved without chemical treatment of the substrate surface before use. Hydroxyapatite crystals exhibited a morphology of flexible nanofibers less than 10 nm wide with nanometer-thick plates filling the spaces between nanofibers. Elemental analysis of the hydroxyapatites showed that they contained alkaline metal cations (Na, Mg, and K) as minor elements and that their average Ca/P atomic % ratio was ~1.40, similar to those of bone apatite. High bioactivity of ceria-stabilized zirconia resulted in direct bonding to bone-like hydroxyapatite, suggesting nanoscale direct osseointegration with bone in vivo that contributes to improving the success rate of dental implant treatment.

Clearance of heparan sulfate in the brain prevents neurodegeneration and neurocognitive impairment in MPS II mice.

Mucopolysaccharidosis II (MPS II), a lysosomal storage disease caused by mutations in iduronate-2-sulfatase (IDS), is characterized by a wide variety of somatic and neurologic symptoms. The currently approved intravenous enzyme replacement therapy with recombinant IDS (idursulfase) is ineffective for CNS manifestations due to its inability to cross the blood-brain barrier (BBB). Here, we demonstrate that the clearance of heparan sulfate (HS) deposited in the brain by a BBB-penetrable antibody-enzyme fusion protein prevents neurodegeneration and neurocognitive dysfunctions in MPS II mice. The fusion protein pabinafusp alfa was chronically administered intravenously to MPS II mice. The drug reduced HS and attenuated histopathological changes in the brain, as well as in peripheral tissues. The loss of spatial learning abilities was completely suppressed by pabinafusp alfa, but not by idursulfase, indicating an association between HS deposition in the brain, neurodegeneration, and CNS manifestations in these mice. Furthermore, HS concentrations in the brain and reduction thereof by pabinafusp alpha correlated with those in the cerebrospinal fluid (CSF). Thus, repeated intravenous administration of pabinafusp alfa to MPS II mice decreased HS deposition in the brain, leading to prevention of neurodegeneration and maintenance of neurocognitive function, which may be predicted from HS concentrations in CSF.

Characterization of Living Dental Pulp Cells in Direct Contact with Mineral Trioxide Aggregate.

Mineral trioxide aggregate (MTA) was introduced as a material for dental endodontic regenerative therapy. Here, we show the dynamics of living dental pulp cells in direct contact with an MTA disk. A red fluorescence protein (DsRed) was introduced into immortalized porcine dental pulp cells (PPU7) and cloned. DsRed-PPU7 cells were cultured on the MTA disk and cell proliferation, chemotaxis, the effects of growth factors and the gene expression of cells were investigated at the biological, histomorphological and genetic cell levels. Mineralized precipitates formed in the DsRed-PPU7 cells were characterized with crystal structural analysis. DsRed-PPU7 cells proliferated in the central part of the MTA disk until Day 6 and displayed a tendency to move to the outer circumference. Both transforming growth factor beta and bone morphogenetic protein promoted the proliferation and movement of DsRed-PPU7 cells and also enhanced the expression levels of odontoblastic gene differentiation markers. Mineralized precipitates formed in DsRed-PPU7 were composed of calcium and phosphate but its crystals were different in each position. Our investigation showed that DsRed-PPU7 cells in direct contact with the MTA disk could differentiate into odontoblasts by controlling cell-cell and cell-substrate interactions depending on cell adhesion and the surrounding environment of the MTA.

Pharmacokinetics and pharmacodynamics of JR-051, a biosimilar of agalsidase beta, in healthy adults and patients with Fabry disease: Phase I and II/III clinical studies.

Fabry disease is a rare X-linked lysosomal disease, in which mutations in the gene encoding α-galactosidase A result in progressive cellular accumulation of globotriaosylceramide (GL-3) in various organs including the skin, kidney, and heart, often leading to life-threatening conditions. Enzyme replacement therapy is currently the standard therapy for the disease, to which two α-galactosidase A formulations have been approved: agalsidase α (Replagal®, Shire) and agalsidase ß (Fabrazyme®, Sanofi). We have recently developed a biosimilar of agalsidase ß, JR-051, and investigated its pharmacokinetics and pharmacodynamics to assess its bioequivalence to agalsidase ß. In a randomized phase I study, healthy adult male volunteers were treated with JR-051 or agalsidase ß and the pharmacokinetics of the drugs were compared. The ratio of geometric means (90% confidence interval [CI]) of the AUC0-24 and Cmax for JR-051 over agalsidase ß were 0.91 (0.8294, 1.0082) and 0.90 (0.7992, 1.0125), respectively. In a 52-week, single-arm, phase II/III study, patients with Fabry disease switched therapy from agalsidase ß to JR-051 to evaluate its pharmacodynamics. The mean (95% CI) plasma GL-3 concentrations at weeks 26 and 52 relative to pre-JR-051 administration were 1.03 (0.91, 1.15) and 0.96 (0.86, 1.06), respectively, which were within the pre-determined bioequivalence acceptance range (0.70, 1.43). The mean (95% CI) plasma globotriaosylsphingosine (lyso-GL-3) concentrations at weeks 26 and 52 relative to pre-JR-051 administration were 1.07 (0.92, 1.23) and 1.13 (1.03, 1.22), respectively. Estimated glomerular filtration rate and left ventricular mass index, as renal and cardiac function indicators, showed no notable changes from baseline throughout the study period, and no new safety concerns were identified. In conclusion, these studies demonstrated bioequivalence of JR-051 to agalsidase ß in terms of its pharmacokinetics and pharmacodynamics. JR-051 offers a potential new treatment option for patients with Fabry disease.

Physicochemical and biological evaluation of JR-131 as a biosimilar to a long-acting erythropoiesis-stimulating agent darbepoetin alfa.

Renal anemia is predominantly caused by a relative deficiency in erythropoietin (EPO). Conventional treatment for renal anemia includes the use of recombinant human EPO (rhEPO) or a long-acting erythropoiesis-activating agent named darbepoetin alfa, which is a modified rhEPO with a carbohydrate chain structure that differs from native hEPO. We have developed a biosimilar to darbepoetin alfa designated JR-131. Here, we comprehensively compare the physicochemical and biological characteristics of JR-131 to darbepoetin alfa. JR-131 demonstrated similar protein structure to the originator, darbepoetin alfa, by peptide mapping and circular dichroism spectroscopy. Additionally, mass spectroscopic analyses and capillary zone electrophoresis revealed similar glycosylation patterns between the two products. Human bone marrow-derived erythroblasts differentiated and proliferated to form colonies with JR-131 to a similar degree as darbepoetin alfa. Finally, JR-131 stimulated erythropoiesis and improved anemia in rats similarly to darbepoetin alfa. Our data show the similarity in physicochemical and biological properties of JR-131 to those of darbepoetin alfa, and JR-131 therefore represents a biosimilar for use in the treatment of renal anemia.

Combined Effect of Midazolam and Bone Morphogenetic Protein-2 for Differentiation Induction from C2C12 Myoblast Cells to Osteoblasts.

In drug repositioning research, a new concept in drug discovery and new therapeutic opportunities have been identified for existing drugs. Midazolam (MDZ) is an anesthetic inducer used for general anesthesia. Here, we demonstrate the combined effects of bone morphogenetic protein-2 (BMP-2) and MDZ on osteogenic differentiation. An immortalized mouse myoblast cell line (C2C12 cell) was cultured in the combination of BMP-2 and MDZ (BMP-2+MDZ). The differentiation and signal transduction of C2C12 cells into osteoblasts were investigated at biological, immunohistochemical, and genetic cell levels. Mineralized nodules formed in C2C12 cells were characterized at the crystal engineering level. BMP-2+MDZ treatment decreased the myotube cell formation of C2C12 cells, and enhanced alkaline phosphatase activity and expression levels of osteoblastic differentiation marker genes. The precipitated nodules consisted of randomly oriented hydroxyapatite nanorods and nanoparticles. BMP-2+MDZ treatment reduced the immunostaining for both α1 and γ2 subunits antigens on the gamma-aminobutyric acid type A (GABAA) receptor in C2C12 cells, but enhanced that for BMP signal transducers. Our investigation showed that BMP-2+MDZ has a strong ability to induce the differentiation of C2C12 cells into osteoblasts and has the potential for drug repositioning in bone regeneration.

Porcine keratin 75 in developing enamel.

OBJECTIVE: To provide in vivo biochemical evidence for the isolation, identification, and characterization of porcine keratin 75 (K75) in developing enamel. METHODS: Immunolocalization of K75 was observed in mandibles from mice at postnatal days 5 and 11. K75 gene expression was analyzed by quantitative reverse transcription-polymerase chain reaction using enamel organ epithelium (EOE) of incisors from pigs at 5 months of age. Enamel protein was extracted and isolated from both immature and mature enamel of second molars from 5-month-old pigs, and the K75 antibody-positive fraction was analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). In vitro protease digestion of K75-antibody-positive fraction was carried out using porcine kallikrein 4 (pKLK4) or recombinant human enamelysin (rhMMP20) and their degradation patterns were characterized by both SDS-PAGE and western blotting. RESULTS: Specific immunostaining for K75 was restricted to the layers of stratum intermedium and the enamel side of ameloblasts in mice at postnatal day 5, and to the papillary layer at postnatal day 11. Porcine K75 was expressed throughout enamel formation, but its transcript levels were significantly higher in the transition EOE than in the secretory- and maturation-stage EOE. Porcine K75 was extracted from the neutral soluble fraction from both immature and mature enamel. It was identified by LC-MS/MS analysis, and was found not to be degraded by either pKLK4 or rhMMP20. CONCLUSION: We propose that K75 is present in the developing enamel and undergoes different processing/degradation compared to other enamel proteins.