ABSTRACT
A population pharmacokinetic/pharmacodynamic (popPK/PD) model for BIIB059 (anti-blood dendritic cell antigen 2 [anti-BDCA2]), a humanized immunoglobulin G1 monoclonal antibody currently under development for the treatment of SLE and CLE, is presented. BIIB059 binds BDCA2, a plasmacytoid dendritic cell (pDC)-specific receptor that inhibits the production of IFN-I and other inflammatory mediators when ligated. Phase 1 PK and PD data of healthy adult volunteers (HV, n = 87) and SLE subjects (n = 22) were utilized for the development of the popPK/PD model. The data included single and multiple dosing of intravenous and subcutaneous BIIB059. BDCA2 internalization (PD marker) was measured for all subjects by monitoring reduction of BDCA2 on pDC cell surface and used for development of the popPD model. A two-compartment popPK model with linear plus non-linear elimination was found to best describe BIIB059 PK. BDCA2 levels were best captured using an indirect response model with stimulation of the elimination of BDCA2. Clearance in SLE subjects was 25% higher compared to HV (6.87 vs 5.52 mL/h). Bodyweight was identified as only other covariate on clearance and central volume. The estimates of EC50 and Emax were 0.35 µg/mL and 8.92, respectively. No difference in EC50 and Emax was observed between SLE and HV. The popPK/PD model described the data accurately, as evaluated by pcVPCs and bootstrap. The presented popPK/PD model for BIIB059 provides valuable insight into the dynamics and dose-response relationship of BIIB059 for the treatment of SLE and CLE and was used to guide dose selection for the Phase 2 clinical study (NCT02847598).
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacokinetics , Lectins, C-Type/antagonists & inhibitors , Lupus Erythematosus, Cutaneous/drug therapy , Lupus Erythematosus, Systemic/drug therapy , Membrane Glycoproteins/antagonists & inhibitors , Models, Biological , Receptors, Immunologic/antagonists & inhibitors , Administration, Intravenous , Adult , Aged , Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Monoclonal, Humanized/adverse effects , Area Under Curve , Biological Availability , Dose-Response Relationship, Drug , Drug Administration Schedule , Female , Half-Life , Humans , Injections, Subcutaneous , Lectins, C-Type/immunology , Lupus Erythematosus, Cutaneous/blood , Lupus Erythematosus, Cutaneous/immunology , Lupus Erythematosus, Systemic/blood , Lupus Erythematosus, Systemic/immunology , Male , Membrane Glycoproteins/immunology , Metabolic Clearance Rate , Middle Aged , Receptors, Immunologic/immunologyABSTRACT
BACKGROUND: To oppose the disadvantages of autologous osteochondral transplantation in the treatment of deep osteochondral defects such as donor site morbidity, size limitation, and insufficient chondral integration, we developed two biphasic scaffolds of either hydroxylapatite/collagen (scaffold A) or allogenous sterilized bone/collagen (scaffold B) and tested their integration in a sheep model. METHODS: We collected chondral biopsies from 12 sheep for the isolation of chondroblasts and cultured them for 4 wk. We created defects at the femoral condyle and implanted either scaffold A or B with chondrocytes or cell free. After 6 wk, animals were euthanized, we explanted the condyles, and evaluated them using histological, immunohistochemical, molecular biological, and histomorphometrical methods. RESULTS: Specimens with scaffold A showed severe lowering of the surface, and the defect size was larger than for scaffold B. We found more immune-competent cells around scaffold A. Chondrocytes were scarcely detected on the surface of both scaffolds. Histomorphometry of the interface between scaffold and recipient showed no significant difference regarding tissue of chondral, osseous, fibrous or implant origin or tartrate-resistant acid phosphatase-positive cells. Real-time reverse transcriptase-polymerase chain reaction analysis revealed significant up-regulation for collagen II and SOX-9 messenger ribonucleic acid expression on the surface of scaffold B compared with scaffold A. CONCLUSIONS: Scaffold B proved to be stable and sufficiently integrated in the short term compared with scaffold A. More extensive evaluations with scaffold B appear to be expedient.
Subject(s)
Arthroplasty, Subchondral , Chondrocytes/transplantation , Tissue Scaffolds , Animals , Female , Immunohistochemistry , Sheep , Transplantation, AutologousABSTRACT
Bruton's tyrosine kinase (BTK), expressed in B cells and cells of innate immunity, including microglia, is an essential signaling element downstream of the B-cell receptor and Fc-receptors. Tolebrutinib (PRN2246, SAR442168) is a potent BTK inhibitor that covalently binds the kinase, resulting in durable inhibition with the potential to target inflammation in the periphery and central nervous system (CNS). Tolebrutinib crosses the blood-brain barrier and potently inhibits BTK in microglial cells isolated from the CNS. A first-in-human randomized, double-blind, placebo-controlled study of tolebrutinib was conducted. The trial design consisted of five single ascending dose arms with oral administration of a single dose of 5, 15, 30, 60, and 120 mg (n = 6 per arm, n = 2 placebo), five multiple ascending dose arms with oral administration of 7.5, 15, 30, 60, and 90 mg (n = 8 per arm, n = 2 placebo) over 10 days, and one arm (n = 4) in which cerebral spinal fluid (CSF) exposure was measured 2 h after a single 120 mg dose. Tolebrutinib was well-tolerated in the study and all treatment-related treatment emergent adverse events were mild. Tolebrutinib was rapidly absorbed following oral administration with a rapid half-life of ~ 2 h. Peripheral BTK occupancy was assessed at various timepoints by an enzyme-linked immunosorbent assay-based readout using an irreversible probe. Assessments demonstrated extensive and prolonged peripheral BTK occupancy at steady-state with once daily doses as low as 7.5 mg. Further, CSF exposure was demonstrated 2 h after administration at 120 mg.
Subject(s)
Protein Kinase Inhibitors , Agammaglobulinaemia Tyrosine Kinase , Dose-Response Relationship, Drug , Double-Blind Method , Half-Life , Humans , Protein Kinase Inhibitors/adverse effectsABSTRACT
BACKGROUND: Tight monitoring of efficacy and safety of anticoagulants such as warfarin is imperative to optimize the benefit-risk ratio of anticoagulants in patients. The standard tests used are measurements of prothrombin time (PT), usually expressed as international normalized ratio (INR), and activated partial thromboplastin time (aPTT). OBJECTIVE: To leverage a previously developed quantitative systems pharmacology (QSP) model of the human coagulation network to predict INR and aPTT for warfarin and rivaroxaban, respectively. METHODS: A modeling and simulation approach was used to predict INR and aPTT measurements of patients receiving steady-state anticoagulation therapy. A previously developed QSP model was leveraged for the present analysis. The effect of genetic polymorphisms known to influence dose response of warfarin (CYP2C9, VKORC1) were implemented into the model by modifying warfarin clearance (CYP2C9 *1: 0.2 L/h; *2: 0.14 L/h, *3: 0.04 L/h) and the concentration of available vitamin K (VKORC1 GA: -22% from normal vitamin K concentration; AA: -44% from normal vitamin K concentration). Virtual patient populations were used to assess the ability of the model to accurately predict routine INR and aPTT measurements from patients under long-term anticoagulant therapy. RESULTS: The introduced model accurately described the observed INR of patients receiving long-term warfarin treatment. The model was able to demonstrate the influence of genetic polymorphisms of CYP2C9 and VKORC1 on the INR measurements. Additionally, the model was successfully used to predict aPTT measurements for patients receiving long-term rivaroxaban therapy. CONCLUSION: The QSP model accurately predicted INR and aPTT measurements observed during routine therapeutic drug monitoring. This is an exemplar of how a QSP model can be adapted and used as a model-based precision dosing tool during clinical practice and drug development to predict efficacy and safety of anticoagulants to ultimately help optimize anti-thrombotic therapy.
ABSTRACT
Orally administered riluzole extends survival in patients with amyotrophic lateral sclerosis, although it has significant shortcomings (eg, adverse events, dysphagic patients) that limit its utility. BHV-0223 is a Zydis-based orally disintegrating formulation of riluzole designed for sublingual administration that addresses the limitations of conventional tablets. This study assessed the bioequivalence between 40-mg BHV-0223 and standard 50-mg oral riluzole tablets, and the food effect on BHV-0223 pharmacokinetics in healthy volunteers. Overall, 133 healthy subjects received BHV-0223 and riluzole tablets under fasted conditions. Geometric mean ratios for the area under the plasma concentration-time curve (AUC) from time zero to time of last nonzero concentration (AUC0-t ) (89.9%; confidence interval [CI], 87.3%-92.5%), AUC from time zero to infinity (AUC0-∞ ) (89.8%; CI, 87.3%-92.4%), and maximum observed concentration (112.7%; CI, 105.5%-120.4%) all met bioequivalence criteria (80%-125%). Subsequently, 67 subjects received BHV-0223 under fed conditions. The geometric mean ratios of AUC0-t (91.2%; CI, 88.1-94.3%), and AUC0-∞ (92.0%; CI, 89.0-95.1%) were similar, but maximum observed concentration ratios were not within bioequivalence criteria. BHV-0223 was well tolerated. This study demonstrated that 40-mg sublingual BHV-0223 is bioequivalent to 50-mg oral riluzole tablets.
Subject(s)
Food-Drug Interactions , Neuroprotective Agents/administration & dosage , Riluzole/administration & dosage , Administration, Oral , Administration, Sublingual , Adolescent , Adult , Area Under Curve , Female , Humans , Male , Middle Aged , Neuroprotective Agents/pharmacokinetics , Riluzole/pharmacokinetics , Tablets , Therapeutic Equivalency , Young AdultABSTRACT
Acetylcholine (ACh) is detected in a variety of non-neuronal cells where it acts as a para/autocrine signaling molecule controlling basic cell functions such as proliferation, differentation, and maintenance of cell-cell contacts. ACh-synthesizing enzymes include choline acetyltransferase and carnitine acetyltransferase (CarAT). ACh is released through vesicular exocytosis or directly from the cytoplasm via organic cation transporters (OCT). Extracellular ACh binds to nicotinic (nAChR) and muscarinic receptors (MR). Degradation of ACh is performed by acetylcholinesterase and butyrylcholinesterase (BChE). Here, we have determined whether these molecules are expressed in osteoblast-like cells, by means of reverse transcription polymerase chain reaction and immunohistochemistry, focusing on nAChR subunits alpha3 and alpha5. RNA for CarAT, OCT-1, M2R, M5R, nAChR subunits alpha3, alpha5, alpha9, alpha10, beta2, beta3, and BChE were detected in human (SAOS-2) and murine (MC3T3-E1) osteoblast-like cells. Other cholinergic components were only expressed species-specifically, e.g., M3R and nAChR subunit alpha7. Immunhistochemistry localized the nAChR subunits alpha3 and alpha5 in osteoblasts in vitro and in vivo where they were up-regulated after application of bone morphogenetic protein-2 (BMP-2) during fracture healing in a rat model. Thus, the cholinergic system of osteoblast-like cells might be regulated by BMP-2 during bone remodeling. Osteoblast-like cells express all necessary enzymes, transporters, and receptors for ACh synthesis and recycling.
Subject(s)
Butyrylcholinesterase/genetics , Carnitine Acyltransferases/genetics , Organic Cation Transport Proteins/genetics , Osteoblasts/metabolism , Osteogenesis , Receptors, Muscarinic/genetics , Receptors, Nicotinic/genetics , Acetylcholine , Animals , Bone Morphogenetic Protein 2/metabolism , Bone Remodeling , Butyrylcholinesterase/metabolism , Carnitine Acyltransferases/metabolism , Cell Line , Fracture Healing , Humans , Mice , Organic Cation Transport Proteins/metabolism , Osteoblasts/cytology , Rats , Receptors, Muscarinic/metabolism , Receptors, Nicotinic/metabolism , Up-RegulationABSTRACT
In bone a role of connexin 43 has been implicated with the fusion of mononuclear precursors of the monocyte/macrophage lineage into multinucleated cells. In order to investigate the putative role of connexin 43 in formation of bone osteoclast-like foreign body giant cells which are formed in response to implantation of biomaterials, nanoparticulate hydroxyapatite had been implanted into defects of minipig femura. After 20 days the defect areas were harvested and connexin 43 expression and synthesis were investigated by using immunohistochemistry, Western Blot, and in situ hybridization within macrophages and osteoclast-like foreign body giant cells. Morphological analysis of gap junctions is performed ultrastructurally. As shown on protein and mRNA level numerous connexin 43 positive macrophages and foreign body giant cells (FBGC) were localized within the granulation tissue and along the surfaces of the implanted hydroxyapatite (HA). Besides, the formation of FBGC by fusion of macrophages could be shown ultrastructurally. Connexin 43 labeling observed on the protein and mRNA level could be attributed to gap junctions identified ultrastructurally between macrophages, between FBGC, and between FBGC and macrophages. Annular gap junctions in the cytoplasm of FBGC pointed to degradation of the channels, and the ubiquination that had occurred in the course of degradation was confirmed by Western blot analysis. All in all, the presently observed pattern of connexin 43 labeling refers to an functional role of gap junctional communication in the formation of osteoclast-like foreign body giant cells formed in response to implantation of the nanoparticulate HA.
Subject(s)
Biocompatible Materials/chemistry , Connexin 43/biosynthesis , Durapatite/chemistry , Gap Junctions/metabolism , Giant Cells, Foreign-Body/metabolism , Nanoparticles/chemistry , Acid Phosphatase/chemistry , Animals , Bone and Bones/metabolism , In Situ Hybridization , Isoenzymes/chemistry , Macrophages/metabolism , Microscopy, Electron, Transmission , Osteoclasts/metabolism , Swine , Swine, Miniature , Tartrate-Resistant Acid PhosphataseABSTRACT
The Transient Receptor Potential Channel Subunit 4 (TRPC4) has been considered as a crucial Ca2+ component in cardiomyocytes promoting structural and functional remodeling in the course of pathological cardiac hypertrophy. TRPC4 assembles as homo or hetero-tetramer in the plasma membrane, allowing a non-selective Na+ and Ca2+ influx. Gαq protein-coupled receptor (GPCR) stimulation is known to increase TRPC4 channel activity and a TRPC4-mediated Ca2+ influx which has been regarded as ideal Ca2+ source for calcineurin and subsequent nuclear factor of activated T-cells (NFAT) activation. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4ß. Aim of the present study was to analyze cytosolic Ca2+ signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4ß. The analysis of Ca2+ transients in neonatal rat cardiomyocytes (NRCs) showed that TRPC4α and TRPC4ß affected Ca2+ cycling in beating cardiomyocytes with both splice variants inducing an elevation of the Ca2+ transient amplitude at baseline and TRPC4ß increasing the Ca2+ peak during angiotensin II (Ang II) stimulation. NRCs infected with TRPC4ß (Ad-C4ß) also responded with a sustained Ca2+ influx when treated with Ang II under non-pacing conditions. Consistent with the Ca2+ data, NRCs infected with TRPC4α (Ad-C4α) showed an elevated calcineurin/NFAT activity and a baseline hypertrophic phenotype but did not further develop hypertrophy during chronic Ang II/phenylephrine stimulation. Down-regulation of endogenous TRPC4α reversed these effects, resulting in less hypertrophy of NRCs at baseline but a markedly increased hypertrophic enlargement after chronic agonist stimulation. Ad-C4ß NRCs did not exhibit baseline calcineurin/NFAT activity or hypertrophy but responded with an increased calcineurin/NFAT activity after GPCR stimulation. However, this effect was not translated into an increased propensity towards hypertrophy but rather less hypertrophy during GPCR stimulation. Further analyses revealed that, although hypertrophy was preserved in Ad-C4α NRCs and even attenuated in Ad-C4ß NRCs, cardiomyocytes had an increased apoptosis rate and thus were less viable after chronic GPCR stimulation. These findings suggest that TRPC4α and TRPC4ß differentially affect Ca2+ signals, calcineurin/NFAT signaling and hypertrophy but similarly impair cardiomyocyte viability during GPCR stimulation.
Subject(s)
Cardiomegaly/metabolism , Myocytes, Cardiac/cytology , TRPC Cation Channels/genetics , TRPC Cation Channels/metabolism , Alternative Splicing , Animals , Animals, Newborn , Calcineurin/metabolism , Calcium/metabolism , Cardiomegaly/genetics , Cell Survival , Cells, Cultured , Disease Models, Animal , Mice , Myocytes, Cardiac/metabolism , NFATC Transcription Factors/metabolism , Rats , Signal TransductionABSTRACT
Gap junctional intercellular communication between Sertoli cells and between Sertoli cells and spermatogonia is considered to play a key role in the regulation of both proliferation and differentiation of germ cells. A member of the gap junction protein family, Connexin 33 (cx33), probably has an inhibitory effect on the formation of gap junctions and so far it is the only cx that has been exclusively found in rat and mouse testes. Thus, this connexin seems to be a special member of the cx family. Using immunohistochemistry, Western blot analysis, polymerase chain reaction, and reverse transcription (RT)-PCR (tissue homogenate and microdissected cells), we studied the possible occurrence of cx33 at the protein, the DNA, and the RNA level in human testis. Whereas immunohistochemistry using the only commercially available anti-cx33 antibody showed similar labeling to the rat within the seminiferous epithelium, we could not find any further evidence for the existence of cx33 using Western blot analysis, PCR, and RT-PCR in human testis. Based on the demonstration of the staining pattern of mitochondria in human germ cells and on preabsorption studies, we could demonstrate anti-cx33 antibody cross-reacting with mitochondrial ferritin, a protein localized in the mitochondria of human testicular spermatids. Therefore, we were not able to abide by the suspicion that cx33 is present in human testis. Additionally, it was not possible to demonstrate cx33 via PCR and immunohistochemistry in the testis of different mammals (dog, cattle, pig, horse, and marmoset monkey) with normal spermatogenesis. These data indicate that cx33 seems to be the first rodent-specific testicular cx.
Subject(s)
Connexins/biosynthesis , Gap Junctions/metabolism , Testis/metabolism , Amino Acid Sequence , Animals , Blotting, Western , Cattle , Connexins/genetics , Cross Reactions , Dogs , Ferritins/metabolism , Humans , Immunohistochemistry , Male , Mice , Mitochondria/metabolism , Molecular Sequence Data , Rats , Reverse Transcriptase Polymerase Chain Reaction , Sertoli Cells/metabolism , Species SpecificityABSTRACT
Acetylcholine (ACh) is an important signaling molecule in non-neuronal systems where it is involved in regulation of viability, proliferation, differentiation and migration of mesenchymal stem cells (MSC) that are capable to differentiate into osteoblasts, chondrocytes and adipocytes. Patients with the systemic disease osteoporosis show altered MSC properties, reduced bone formation and mineral density followed by increased bone fragility and high fracture incidence. Here we asked whether nicotinic and muscarinic acetylcholine receptors (AChR) are expressed in osteoblasts, adipocytes and chondrocytes differentiated from bone MSC extracted from human reaming debris (RDMSC) that was harvested during surgery of long bone diaphyseal fractures. Using RT-PCR, AChR were detected in RDMSC, osteoblasts, chondrocytes and adipocytes of male and female bone-healthy and of female osteoporotic donors. An up-regulation in multiplicity and occurrence of AChR subtypes was found in female compared to male donors and in osteoblast of male donors compared to adipocytes. Real-time RT-PCR analysis resulted in a significant increase in relative expression of nAChR α9 in chondrocytes compared to adipocytes of healthy female donors. The nAChR subunit α10 was significantly up-regulated in osteoblasts of healthy compared to osteoporotic donors as well as the mAChR M5 that is additionally decreased in osteoporotic osteoblasts compared to MSC and chondrocytes of osteoporotic donors. In summary, the gene expression of AChR during differentiation of RDMSC and its regulation in cells of osteoporotic donors lead to the assumption that AChR signaling is involved in bone formation and might be utilized to stimulate bone remodeling processes.
Subject(s)
Mesenchymal Stem Cells/physiology , Receptors, Muscarinic/metabolism , Receptors, Nicotinic/metabolism , Adipocytes/metabolism , Adult , Aged , Aged, 80 and over , Cell Differentiation , Cells, Cultured , Female , Gene Expression Regulation , Humans , Male , Mesenchymal Stem Cells/cytology , Middle Aged , Osteoblasts/metabolism , Osteoporosis/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Receptors, Muscarinic/genetics , Receptors, Nicotinic/genetics , Reverse Transcriptase Polymerase Chain Reaction , Young AdultABSTRACT
Acetylcholinesterase (AChE) hydrolyzes acetylcholine (ACh) to acetate and choline and thereby terminates nerve impulse transmission. ACh is also expressed in bone tissue and enhances here proliferation and differentiation of osteoblasts, which makes it interesting to investigate effects of AChE deficiency on bone. To our knowledge, this is the first study that analyzed bone of heterozygous acetylcholinesterase-knockout (AChE-KO) mice. Tibia, femur, thoracic and lumbar vertebrae of 16-week-old female heterozygous AChE-KO mice and their corresponding wildtypes (WT) were analyzed using real-time RT-PCR, dual-energy X-ray absorptiometry, biomechanics, micro-computed tomography, histology and histomorphometry. Our data revealed that heterozygous AChE-KO did not cause negative effects upon bone parameters analyzed. In contrast, the number of osteoclasts per perimeter was significantly reduced in lumbar vertebrae. In addition, we found a significant decrease in trabecular perimeter of lumbar vertebrae and cortical area fraction (Ct.Ar/Tt.Ar) in the mid-diaphysis of femurs of AChE-KO mice compared to their WT. Therefore, presumably a local homozygous knockout of AChE or AChE-inhibitor administration might be beneficial for bone formation due to ACh accumulation. However, many other bone parameters analyzed did not differ statistically significantly between AChE-KO and WT mice. That might be reasoned by the compensating effect of butyrylcholinesterase (BChE).
Subject(s)
Acetylcholinesterase/metabolism , Bone Density/physiology , Gene Expression Regulation, Enzymologic/physiology , Absorptiometry, Photon , Acetylcholinesterase/genetics , Animals , Biomechanical Phenomena , Bone Density/genetics , Female , Mice , Mice, Knockout , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
High frequency of osteoporosis is found in postmenopausal women where several molecular components were identified to be involved in bone loss that subsequently leads to an increased fracture risk. Bone loss has already been determined in male mice with gene deficiency of muscarinic acetylcholine receptor M3 (M3R-KO). Here we asked whether bone properties of female 16-week old M3R-KO present similarities to osteoporotic bone loss by means of biomechanical, radiological, electron microscopic, cell- and molecular biological methods. Reduced biomechanical strength of M3R-KO correlated with cortical thickness and decreased bone mineral density (BMD). Femur and vertebrae of M3R-KO demonstrated a declined trabecular bone volume, surface, and a higher trabecular pattern factor and structure model index (SMI) compared to wild type (WT) mice. In M3R-KO, the number of osteoclasts as well as the cathepsin K mRNA expression was increased. Osteoclasts of M3R-KO showed an estimated increase in cytoplasmic vesicles. Further, histomorphometrical analysis revealed up-regulation of alkaline phosphatase. Osteoblasts and osteocytes showed a swollen cytoplasm with an estimated increase in the amount of rough endoplasmatic reticulum and in case of osteocytes a reduced pericellular space. Thus, current results on bone properties of 16-week old female M3R-KO are related to postmenopausal osteoporotic phenotype. Stimulation and up-regulation of muscarinic acetylcholine receptor subtype M3 expression in osteoblasts might be a possible new option for prevention and therapy of osteoporotic fractures. Pharmacological interventions and the risk of side effects have to be determined in upcoming studies.
Subject(s)
Bone Density/physiology , Bone and Bones/ultrastructure , Cathepsin K/metabolism , Gene Expression Regulation/physiology , Receptor, Muscarinic M3/metabolism , Animals , Biomechanical Phenomena , Bone and Bones/metabolism , Cathepsin K/genetics , Female , Male , Mice , Mice, Knockout , Receptor, Muscarinic M3/genetics , Sex FactorsABSTRACT
Peri-prosthetic infections caused by multidrug resistant bacteria have become a serious problem in surgery and orthopedics. The aim is to introduce biomaterials that avoid implant-related infections caused by multiresistant bacteria. The efficacy of silver nanoparticles (AgNP) against a broad spectrum of bacteria and against multiresistant pathogens has been repeatedly described. In the present study polymethylmethacrylate (PMMA) bone cement functionalized with AgNP and/or gentamicin were tested regarding their biocompatibility with bone forming cells. Therefore, influences on viability, cell number and differentiation of primary human mesenchymal stem cells (MSCs) and MSCs cultured in osteogenic differentiation media (MSC-OM) caused by the implant materials were studied. Furthermore, the growth behavior and the morphology of the cells on the testing material were observed. Finally, we examined the induction of cell stress, regarding antioxidative defense and endoplasmatic reticulum stress. We demonstrated similar cytocompatibility of PMMA loaded with AgNP compared to plain PMMA or PMMA loaded with gentamicin. There was no decrease in cell number, viability and osteogenic differentiation and no induction of cell stress for all three PMMA variants after 21 days. Addition of gentamicin to AgNP-loaded PMMA led to a slight decrease in osteogenic differentiation. Also an increase in cell stress was detectable for PMMA loaded with gentamicin and AgNP. In conclusion, supplementation of PMMA bone cement with gentamicin, AgNP, and both results in bone implants with an antibacterial potency and suitable cytocompatibility in MSCs and MSC-OM.
Subject(s)
Bone Cements/chemistry , Cell Differentiation/drug effects , Mesenchymal Stem Cells/cytology , Nanoparticles/chemistry , Osteoblasts/cytology , Polymethyl Methacrylate/chemistry , Silver/chemistry , Anti-Bacterial Agents/pharmacology , Apoptosis/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Gentamicins/pharmacology , Humans , In Vitro Techniques , Materials Testing , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Osteoblasts/drug effects , Osteoblasts/metabolism , Osteogenesis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
A correlation between obesity and bone metabolism is strongly assumed because adipocytes and osteoblasts originate from the same precursor cells and their differentiation is conversely regulated by the same factors. It is controversially discussed if obesity protects bone or leads to loss of bone mass. Thus, the aim of the present study was to investigate the influence of diet-induced mild obesity (11% increased body weight compared to control) on bone microstructure in mice. Four-week-old male C57BL/6J mice received a high-fat diet (HFD, 60% kcal from fat) and were analyzed by means of dual X-ray absorptiometry, histological methods, real-time RT-PCR, and transmission electron microscopy in comparison to control animals (10% kcal from fat). The cancellous bone mass, collagen 1α1 expression, amount of osteoid, and cohesion of cells via cell-to-cell contacts decreased in HFD mice whereas the bone mineral density and the amount of osteoblasts and osteoclasts were not modified. The amount of apoptotic osteocytes was increased in HFD mice in comparison to controls. We conclude that moderately increased body weight does not protect bone architecture from age-dependent degeneration. By contrast, bone microstructure is negatively affected and reduced maintenance of cell-cell contacts may be one of the underlying mechanisms.
ABSTRACT
The prevention of implant-related infections is an important issue in medical research. The aim is to exploit the strong antimicrobial effect of silver nanoparticles (AgNP) to develop new antibacterial coatings for implants. However, there is still a serious lack of information on the influence of AgNP on bone metabolism. In the present study we have evaluated the influence of AgNP on cell stress, viability, proliferation and differentiation of primary human mesenchymal stem cells (MSC) and osteoblasts (OB). Finally, cellular uptake of the AgNP was examined. After 21 days impairment of cell viability of MSC and OB occurred at a concentration of 10 µg/g of AgNP. Cytotoxicity and inhibition of proliferation was highly time and dose dependent. No influence on cell differentiation, but an increase in cell stress, was observed. Uptake of AgNP into MSC and OB could be confirmed. In summary, these results demonstrate AgNP-mediated cytotoxicity at higher concentrations. Therefore, a therapeutical window for the application of AgNP in medical products might exist. However, the antibacterial benefits and potential health risks of AgNP need to be weighed in further studies.
Subject(s)
Biocompatible Materials/pharmacology , Materials Testing , Mesenchymal Stem Cells/cytology , Metal Nanoparticles/chemistry , Osteoblasts/cytology , Silver/pharmacology , Adult , Alkaline Phosphatase/metabolism , Cell Count , Cell Proliferation/drug effects , Cell Shape/drug effects , Cell Survival/drug effects , Cell Survival/genetics , Gene Expression Regulation/drug effects , Humans , Ions , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/enzymology , Mesenchymal Stem Cells/ultrastructure , Metal Nanoparticles/ultrastructure , Osteoblasts/drug effects , Osteoblasts/enzymology , Osteoblasts/ultrastructure , RNA, Messenger/genetics , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Spectrophotometry, UltravioletABSTRACT
Fracture healing is a physiological process of repair which proceeds in stages, each characterized by a different predominant tissue in the fracture gap. Matrix reorganization is regulated by cytokines and growth factors. Neurotrophins and their receptors might be of importance to osteoblasts and endothelial cells during fracture healing. The aim of this study was to examine the presence of brain-derived neurotrophic factor (BDNF) and its tropomyosin-related kinase B receptor (TrkB) during human fracture healing. BDNF and TrkB were investigated in samples from human fracture gaps and cultured cells using RT-PCR, Western blot, and immunohistochemistry. Endothelial cells and osteoblastic cell lines demonstrated a cytoplasmic staining pattern of BDNF and TrkB in vitro. At the mRNA level, BDNF and TrkB were expressed in the initial and osteoid formation phase of human fracture healing. In the granulation tissue of fracture gap, both proteins--BDNF and TrkB--are concentrated in endothelial and osteoblastic cells at the margins of woven bone suggesting their involvement in the formation of new vessels. There was no evidence of BDNF or TrkB during fracture healing in chondrocytes of human enchondral tissue. Furthermore, BDNF is absent in mature bone. Taken together, BDNF and TrkB are involved in vessel formation and osteogenic processes during human fracture healing. The detection of BDNF and its TrkB receptor during various stages of the bone formation process in human fracture gap tissue were shown for the first time. The current study reveals that both proteins are up-regulated in human osteoblasts and endothelial cells in fracture healing.
Subject(s)
Bone and Bones/physiology , Brain-Derived Neurotrophic Factor/physiology , Fracture Healing/physiology , Receptor, trkB/physiology , Adult , Aged , Bone Development/physiology , Bone and Bones/metabolism , Brain-Derived Neurotrophic Factor/metabolism , Cell Line , Female , Humans , Ilium/physiology , Male , Middle Aged , Osteoblasts/physiology , Receptor, trkB/metabolism , Young AdultABSTRACT
Insertion of bone substitution materials accelerates healing of osteoporotic fractures. Biodegradable materials are preferred for application in osteoporotic patients to avoid a second surgery for implant replacement. Degraded implant fragments are often absorbed by macrophages that are removed from the fracture side via passage through veins or lymphatic vessels. We investigated if lymphatic vessels occur in osteoporotic bone defects and whether they are regulated by the use of different materials. To address this issue osteoporosis was induced in rats using the classical method of bilateral ovariectomy and additional calcium and vitamin deficient diet. In addition, wedge-shaped defects of 3, 4, or 5 mm were generated in the distal metaphyseal area of femur via osteotomy. The 4 mm defects were subsequently used for implantation studies where bone substitution materials of calcium phosphate cement, composites of collagen and silica, and iron foams with interconnecting pores were inserted. Different materials were partly additionally functionalized by strontium or bisphosphonate whose positive effects in osteoporosis treatment are well known. The lymphatic vessels were identified by immunohistochemistry using an antibody against podoplanin. Podoplanin immunopositive lymphatic vessels were detected in the granulation tissue filling the fracture gap, surrounding the implant and growing into the iron foam through its interconnected pores. Significant more lymphatic capillaries were counted at the implant interface of composite, strontium and bisphosphonate functionalized iron foam. A significant increase was also observed in the number of lymphatics situated in the pores of strontium coated iron foam. In conclusion, our results indicate the occurrence of lymphatic vessels in osteoporotic bone. Our results show that lymphatic vessels are localized at the implant interface and in the fracture gap where they might be involved in the removal of lymphocytes, macrophages, debris and the implants degradation products. Therefore the lymphatic vessels are involved in implant integration and fracture healing.
Subject(s)
Absorbable Implants , Bone Substitutes/therapeutic use , Femur/pathology , Lymphatic Vessels/pathology , Membrane Glycoproteins/analysis , Osteoporotic Fractures/pathology , Osteoporotic Fractures/surgery , Animals , Bone Substitutes/chemistry , Collagen/chemistry , Collagen/therapeutic use , Diphosphonates/chemistry , Diphosphonates/therapeutic use , Disease Models, Animal , Female , Femur/surgery , Iron Compounds/chemistry , Iron Compounds/therapeutic use , Rats , Rats, Sprague-Dawley , Silicon Dioxide/chemistry , Silicon Dioxide/therapeutic use , Strontium/chemistry , Strontium/therapeutic useABSTRACT
AIMS: As the stimulation of the α7-nicotinic acetylcholine receptor (nAChR), which is present in the synovium of patients with rheumatoid arthritis (RA), leads to a decrease in pro-inflammatory cytokines, the α7-nAChR is being discussed as a new therapeutic target. On this background we addressed the question whether α7-nAChR mRNA was differentially expressed in RA compared to osteoarthritis (OA) synovial samples and whether other components of the non-neuronal cholinergic system were also present and differentially expressed in the synovium of patients with RA in comparison to OA. MAIN METHODS: The expression of nicotinic and muscarinic acetylcholine receptors (mAChRs), choline and acetylcholine transporters, synthesising and degrading enzymes was determined in human samples of synovial tissue from patients with RA and OA using RT-PCR and immunofluorescence labelling. KEY FINDINGS: Compared to OA, patients with RA showed increased expression of nAChR subunit ß4 while a decline in subunits α2 and α4 as well as in mAChR M1R was observed. For all other nAChR subunits and mAChRs however there was no significant difference between RA and OA patients. With regard to the ACh transporters and enzymes no expressional changes were observed between OA and RA patients, except for the choline acetyltransferase (ChAT) which was only detected in OA but not in RA synovium. SIGNIFICANCE: Our results indicate that besides α7-nAChR other components of the non-neuronal cholinergic system are present and differentially expressed in the synovium of RA and OA patients, which makes them interesting alternative targets in the development of new strategies for RA therapy.
Subject(s)
Arthritis, Rheumatoid/genetics , Knee Joint/metabolism , Osteoarthritis/genetics , Receptors, Muscarinic/genetics , Receptors, Nicotinic/genetics , Synovial Membrane/metabolism , Acetylcholine/metabolism , Adult , Aged , Arthritis, Rheumatoid/metabolism , Gene Expression , Humans , Middle Aged , Osteoarthritis/metabolism , RNA, Messenger/geneticsABSTRACT
Antimicrobial coatings are of interest as a means to improve infection prophylaxis in cementless joint arthroplasty. However, those coatings must not interfere with the essential bony integration of the implants. Gentamicin-hydroxyapatite (gentamicin-HA) and gentamicin-RGD (arginine-glycine-aspartate)-HA coatings have recently been shown to significantly reduce infection rates in a rabbit infection prophylaxis model. The purpose of the current study was to investigate the in vitro elution kinetics and in vivo effects of gentamicin-HA and gentamicin-RGD-HA coatings on new bone formation, implant integration and biocompatibility in a rabbit model. In vitro elution testing showed that 95% and 99% of the gentamicin was released after 12 and 24 h, respectively. The in vivo study comprised 45 rabbits in total, with six animals for each of the gentamicin-HA, gentamicin-RGD-HA group and control pure HA coating groups for the 4 week time period, and nine animals for each of the three groups for the 12 week observation period. A 2.0 mm steel K-wire with one of the coatings under test was placed in the intramedullary canal of the tibia. After 4 and 12 weeks the tibiae were harvested and three different areas (proximal metaphysis, shaft area, distal metaphysis) were assessed by quantitative and qualitative histology for new bone formation, direct implant-bone contact and the formation of multinucleated giant cells. The results exhibited high standard deviations in all subgroups. There was a trend towards better bone formation and better direct implant contact in the pure HA coating group compared with both gentamicin coatings after 4 and 12 weeks, which was, however, not statistically significant. The number of multinucleated giant cells did not differ significantly between the three groups at both time points. In summary, both gentamicin coatings with 99% release of gentamicin within 24 h revealed good biocompatibility and bony integration, which was not statistically significant different compared with pure HA coating. Limitations of the study are the high standard deviation of the results and the limited number of animals per time point.
Subject(s)
Anti-Bacterial Agents/pharmacology , Biocompatible Materials , Bone Development/drug effects , Gentamicins/pharmacology , Oligopeptides/chemistry , Animals , Anti-Bacterial Agents/chemistry , Gentamicins/chemistry , Gentamicins/pharmacokinetics , In Vitro Techniques , Models, Animal , RabbitsABSTRACT
This is the first work to report on additional Arginin-Glycin-Aspartat (RGD) coating on precoated hydroxyapatite (HA) surfaces regarding new bone formation, implant bone contact, and biocompatibility compared to pure HA coating and uncoated stainless K-wires. There were 39 rabbits in total with 6 animals for the RGD-HA and HA group for the 4 week time period and 9 animals for each of the 3 implant groups for the 12 week observation. A 2.0 K-wire either with RGD-HA or with pure HA coating or uncoated was placed into the intramedullary canal of the tibia. After 4 and 12 weeks, the tibiae were harvested and three different areas of the tibia were assessed for quantitative and qualitative histology for new bone formation, direct implant bone contact, and formation of multinucleated giant cells. Both RGD-HA and pure HA coating showed statistically higher new bone formation and implant bone contact after 12 weeks than the uncoated K-wire. There were no significant differences between the RGD-HA and the pure HA coating in new bone formation and direct implant bone contact after 4 and 12 weeks. The number of multinucleated giant did not differ significantly between the RGD-HA and HA group after both time points. Overall, no significant effects of an additional RGD coating on HA surfaces were detected in this model after 12 weeks.