Reproductive and fetal toxicity studies of histamine H3 receptor antagonist DL76 used in mice to prevent maximal electroshock-induced seizure.

Introduction: Brain histamine is considered an endogenous anticonvulsant and histamine H1 receptor. H1R antagonists have, in earlier studies, been found to induce convulsions. Moreover, research during the last two decades has provided more information concerning the anticonvulsant activities of histamine H3R (H3R) antagonists investigated in a variety of animal epilepsy models. Methods: Therefore, the in vivo anticonvulsant effect of the H3R antagonist DL76, with proven high in vitro affinity, in vitro selectivity profile, and high in vivo antagonist potency in mice against maximal electroshock (MES)-induced seizures in mice, was assessed. Valproic acid (VPA) was used as a reference antiepileptic drug (AED). In addition, DL76 was tested for its reproductive and fetal toxicity in the same animal species. Results and discussion: Our observations showed that acute systemic administration (intraperitoneal; i.p.) of DL76 (7.5 mg/kg, 15 mg/kg, 30 mg/kg, and 60 mg/kg, i.p.) provided significant and dose-dependent protection against MES-induced seizures in female and male mice. Moreover, the DL76-provided protective effects were comparable to those offered by the VPA and were reversed when animals were co-administered the CNS-penetrant selective H3R agonist R-(α)-methylhistamine (RAM, 10 mg/kg, i.p.). Furthermore, the administration of single (7.5 mg/kg, 15 mg/kg, 30 mg/kg, or 60 mg/kg, i.p.) or multiple doses (3 × 15 mg/kg, i.p.) of H3R antagonist DL76 on gestation days (GD) 8 or 13 failed to affect the maternal body weight of mice when compared with the control mice group. No significant alterations were detected in the average number of implantations and resorptions between the control and DL76-treated groups at the early stages of gestation and the organogenesis period. In addition, no significant differences in the occurrence of skeletal abnormalities, urogenital abnormalities, exencephaly, exomphalos, facial clefts, and caudal malformations were observed. The only significant abnormalities witnessed in the treated groups of mice were in the length of long bones and body length. In conclusion, the novel H3R antagonist DL76 protected test animals against MES-induced seizures and had a low incidence of reproductive and fetal malformation with decreased long bone lengths in vivo, signifying the potential therapeutic value of H3R antagonist DL76 for future preclinical as well as clinical development for use in the management of epilepsy.

Harnessing the Potential of PLGA Nanoparticles for Enhanced Bone Regeneration.

Recently, nanotechnologies have become increasingly prominent in the field of bone tissue engineering (BTE), offering substantial potential to advance the field forward. These advancements manifest in two primary ways: the localized application of nanoengineered materials to enhance bone regeneration and their use as nanovehicles for delivering bioactive compounds. Despite significant progress in the development of bone substitutes over the past few decades, it is worth noting that the quest to identify the optimal biomaterial for bone regeneration remains a subject of intense debate. Ever since its initial discovery, poly(lactic-co-glycolic acid) (PLGA) has found widespread use in BTE due to its favorable biocompatibility and customizable biodegradability. This review provides an overview of contemporary advancements in the development of bone regeneration materials using PLGA polymers. The review covers some of the properties of PLGA, with a special focus on modifications of these properties towards bone regeneration. Furthermore, we delve into the techniques for synthesizing PLGA nanoparticles (NPs), the diverse forms in which these NPs can be fabricated, and the bioactive molecules that exhibit therapeutic potential for promoting bone regeneration. Additionally, we addressed some of the current concerns regarding the safety of PLGA NPs and PLGA-based products available on the market. Finally, we briefly discussed some of the current challenges and proposed some strategies to functionally enhance the fabrication of PLGA NPs towards BTE. We envisage that the utilization of PLGA NP holds significant potential as a potent tool in advancing therapies for intractable bone diseases.

High-Density Lipoprotein Is Located Alongside Insulin in the Islets of Langerhans of Normal and Rodent Models of Diabetes.

Recent studies have implicated pre-beta and beta lipoproteins (VLDL and LDL) in the etiopathogenesis of complications of diabetes mellitus (DM). In contrast, alpha lipoprotein (HDL) is protective of the beta cells of the pancreas. This study examined the distribution of HDL in the islets of Langerhans of murine models of type 1 diabetic rats (streptozotocin (STZ)-induced DM in Wistar rats) and type 2 models of DM rats (Goto-Kakizaki (GK), non-diabetic Zucker lean (ZL), and Zucker diabetic and fatty (ZDF)). The extent by which HDL co-localizes with insulin or glucagon in the islets of the pancreas was also investigated. Pancreatic tissues of Wistar non-diabetic, diabetic Wistar, GK, ZL, and ZDF rats were processed for immunohistochemistry. Pancreatic samples of GK rats fed with either a low-fat or a high-fat diet were prepared for transmission immune-electron microscopy (TIEM) to establish the cytoplasmic localization of HDL in islet cells. HDL was detected in the core and periphery of pancreatic islets of Wistar non-diabetic and diabetic, GK, ZL, and ZDF rats. The average total of islet cells immune positive for HDL was markedly (<0.05) reduced in GK and ZDF rats in comparison to Wistar controls. The number of islet cells containing HDL was also remarkably (p < 0.05) reduced in Wistar diabetic rats and GK models fed on high-fat food. The co-localization study using immunofluorescence and TIEM techniques showed that HDL is detected alongside insulin within the secretory granules of ß-cells. HDL did not co-localize with glucagon. This observation implies that HDL may contribute to the metabolism of insulin.

A pilot study: effect of irisin on trabecular bone in a streptozotocin-induced animal model of type 1 diabetic osteopathy utilizing a micro-CT.

Background: Osteoporosis is a significant co-morbidity of type 1 diabetes mellitus (DM1) leading to increased fracture risk. Exercise-induced hormone 'irisin' in low dosage has been shown to have a beneficial effect on bone metabolism by increasing osteoblast differentiation and reducing osteoclast maturation, and inhibiting apoptosis and inflammation. We investigated the role of irisin in treating diabetic osteopathy by observing its effect on trabecular bone. Methods: DM1 was induced by intraperitoneal injection of streptozotocin 60 mg/kg body weight. Irisin in low dosage (5 µg twice a week for 6 weeks I/P) was injected into half of the control and 4-week diabetic male Wistar rats. Animals were sacrificed six months after induction of diabetes. The trabecular bone in the femoral head and neck was analyzed using a micro-CT technique. Bone turnover markers were measured using ELISA, Western blot, and RT-PCR techniques. Results: It was found that DM1 deteriorates the trabecular bone microstructure by increasing trabecular separation (Tb-Sp) and decreasing trabecular thickness (Tb-Th), bone volume fraction (BV/TV), and bone mineral density (BMD). Irisin treatment positively affects bone quality by increasing trabecular number p < 0.05 and improves the BMD, Tb-Sp, and BV/TV by 21-28%. The deterioration in bone microarchitecture is mainly attributed to decreased bone formation observed as low osteocalcin and high sclerostin levels in diabetic bone samples p < 0.001. The irisin treatment significantly suppressed the serum and bone sclerostin levels p < 0.001, increased the serum CTX1 levels p < 0.05, and also showed non-significant improvement in osteocalcin levels. Conclusions: This is the first pilot study to our knowledge that shows that a low dose of irisin marginally improves the trabecular bone in DM1 and is an effective peptide in reducing sclerostin levels.

Bacterial Inhibition and Osteogenic Potentials of Sr/Zn Co-Doped Nano-Hydroxyapatite-PLGA Composite Scaffold for Bone Tissue Engineering Applications.

Bacterial infection associated with bone grafts is one of the major challenges that can lead to implant failure. Treatment of these infections is a costly endeavor; therefore, an ideal bone scaffold should merge both biocompatibility and antibacterial activity. Antibiotic-impregnated scaffolds may prevent bacterial colonization but exacerbate the global antibiotic resistance problem. Recent approaches combined scaffolds with metal ions that have antimicrobial properties. In our study, a unique strontium/zinc (Sr/Zn) co-doped nanohydroxyapatite (nHAp) and Poly (lactic-co-glycolic acid) -(PLGA) composite scaffold was fabricated using a chemical precipitation method with different ratios of Sr/Zn ions (1%, 2.5%, and 4%). The scaffolds' antibacterial activity against Staphylococcus aureus were evaluated by counting bacterial colony-forming unit (CFU) numbers after direct contact with the scaffolds. The results showed a dose-dependent reduction in CFU numbers as the Zn concentration increased, with 4% Zn showing the best antibacterial properties of all the Zn-containing scaffolds. PLGA incorporation in Sr/Zn-nHAp did not affect the Zn antibacterial activity and the 4% Sr/Zn-nHAp-PLGA scaffold showed a 99.7% bacterial growth inhibition. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay showed that Sr/Zn co-doping supported osteoblast cell proliferation with no apparent cytotoxicity and the highest doping percentage in the 4% Sr/Zn-nHAp-PLGA was found to be ideal for cell growth. In conclusion, these findings demonstrate the potential for a 4% Sr/Zn-nHAp-PLGA scaffold with enhanced antibacterial activity and cytocompatibility as a suitable candidate for bone regeneration.

Diabetes Mellitus Alters the Immuno-Expression of Neuronal Nitric Oxide Synthase in the Rat Pancreas.

Nitric oxide is generated from nitric oxide synthase following hyperglycemia-induced oxidative stress during the course of diabetes mellitus (DM). We examined the temporal immuno-expression of neuronal nitric oxide synthase (nNOS) in the pancreas of diabetic and non-diabetic rats using immunohistochemical, immunofluorescence and western blot techniques 12 h, 24 h, 1 week, 2 weeks, 1, 8 and 15 months after induction of DM. nNOS co-localized with pancreatic beta cells but disappears 12 h after the onset of DM. In contrast, the nNOS content of pancreatic nerves increased significantly (p < 0.001) 24 h after the induction of DM, and decreased sharply thereafter. However, nNOS-positive ganglion cells were observed even 15 months post-diabetes. ROS increased by more than 100% two months after the onset of DM compared to non-diabetic control but was significantly (p < 0.000001) reduced at 9 months after the induction of DM. The pancreatic content of GSH increased significantly (p < 0.02) after 9 months of DM. Although, TBARS content was significantly (p < 0.009; p < 0.002) lower in aged (9 months) non-diabetic and DM rats, TBARS rate was markedly (p < 0.02) higher 9 months after the induction of DM when compared to younger age group. In conclusion, nNOS is present in pancreatic beta cell, but disappears 12 h after the onset of diabetes. In contrast, the tissue level of nNOS of pancreatic nerves increased in the first week of diabetes, followed by a sharp reduction. nNOS may play important roles in the metabolism of pancreatic beta cell.

Biomimetic PLGA/Strontium-Zinc Nano Hydroxyapatite Composite Scaffolds for Bone Regeneration.

Synthetic bone graft substitutes have attracted increasing attention in tissue engineering. This study aimed to fabricate a novel, bioactive, porous scaffold that can be used as a bone substitute. Strontium and zinc doped nano-hydroxyapatite (Sr/Zn n-HAp) were synthesized by a water-based sol-gel technique. Sr/Zn n-HAp and poly (lactide-co-glycolide) (PLGA) were used to fabricate composite scaffolds by supercritical carbon dioxide technique. FTIR, XRD, TEM, SEM, and TGA were used to characterize Sr/Zn n-HAp and the composite scaffolds. The synthesized scaffolds were adequately porous with an average pore size range between 189 to 406 µm. The scaffolds demonstrated bioactive behavior by forming crystals when immersed in the simulated body fluid. The scaffolds after immersing in Tris/HCl buffer increased the pH value of the medium, establishing their favorable biodegradable behavior. ICP-MS study for the scaffolds detected the presence of Sr, Ca, and Zn ions in the SBF within the first week, which would augment osseointegration if implanted in the body. nHAp and their composites (PLGA-nHAp) showed ultimate compressive strength ranging between 0.4-19.8 MPa. A 2.5% Sr/Zn substituted nHAp-PLGA composite showed a compressive behavior resembling that of cancellous bone indicating it as a good candidate for cancellous bone substitute.

Vimentin Is at the Heart of Epithelial Mesenchymal Transition (EMT) Mediated Metastasis.

Epithelial-mesenchymal transition (EMT) is a reversible plethora of molecular events where epithelial cells gain the phenotype of mesenchymal cells to invade the surrounding tissues. EMT is a physiological event during embryogenesis (type I) but also happens during fibrosis (type II) and cancer metastasis (type III). It is a multifaceted phenomenon governed by the activation of genes associated with cell migration, extracellular matrix degradation, DNA repair, and angiogenesis. The cancer cells employ EMT to acquire the ability to migrate, resist therapeutic agents and escape immunity. One of the key biomarkers of EMT is vimentin, a type III intermediate filament that is normally expressed in mesenchymal cells but is upregulated during cancer metastasis. This review highlights the pivotal role of vimentin in the key events during EMT and explains its role as a downstream as well as an upstream regulator in this highly complex process. This review also highlights the areas that require further research in exploring the role of vimentin in EMT. As a cytoskeletal protein, vimentin filaments support mechanical integrity of the migratory machinery, generation of directional force, focal adhesion modulation and extracellular attachment. As a viscoelastic scaffold, it gives stress-bearing ability and flexible support to the cell and its organelles. However, during EMT it modulates genes for EMT inducers such as Snail, Slug, Twist and ZEB1/2, as well as the key epigenetic factors. In addition, it suppresses cellular differentiation and upregulates their pluripotent potential by inducing genes associated with self-renewability, thus increasing the stemness of cancer stem cells, facilitating the tumour spread and making them more resistant to treatments. Several missense and frameshift mutations reported in vimentin in human cancers may also contribute towards the metastatic spread. Therefore, we propose that vimentin should be a therapeutic target using molecular technologies that will curb cancer growth and spread with reduced mortality and morbidity.

Exogenous Ghrelin Increases Plasma Insulin Level in Diabetic Rats.

Ghrelin, a 28-amino acid peptide, is a strong growth hormone secretagogue and a regulator of food intake. In addition, ghrelin is thought to play a role in insulin secretion and in glucose homeostasis. A lot of contradictory data have been reported in the literature regarding the co-localization of ghrelin with other hormones in the islet of Langerhans, its role in insulin secretion and attenuation of type 2 diabetes mellitus. In this study, we investigate the effect of chronic ghrelin treatment on glucose, body weight and insulin level in normal and streptozotocin-induced diabetic male Wistar rats. We have also examined the distribution pattern and co-localization of ghrelin with insulin in pancreatic islet cells using immunohistochemistry and immune-electron microscopy and the ability of ghrelin to stimulate insulin release from the CRL11065 beta cell line. Control, non-diabetic groups received intraperitoneal injection of normal saline, while treated groups received intraperitoneal injection of 5 µg/kg body weight of ghrelin (amino acid chain 24-51) on a daily basis for a duration of four weeks. Our results show that the administration of ghrelin increases the number of insulin-secreting beta cells and serum insulin level in both normal and diabetic rats. We also demonstrated that ghrelin co-localizes with insulin in pancreatic islet cells and that the pattern of ghrelin distribution is altered after the onset of diabetes. Moreover, ghrelin at a dose of 10-6M and 10-12M increased insulin release from the CRL11065 beta cell line. In summary, ghrelin co-localizes with insulin in the secretory granules of pancreatic beta cells and enhances insulin production.

Strontium- and Zinc-Containing Bioactive Glass and Alginates Scaffolds.

With an increasingly elderly population, there is a proportionate increase in bone injuries requiring hospitalization. Clinicians are increasingly adopting tissue-engineering methods for treatment due to limitations in the use of autogenous and autologous grafts. The aim of this study was to synthesize a novel, bioactive, porous, mechanically stable bone graft substitute/scaffold. Strontium- and zinc-containing bioactive glasses were synthesized and used with varying amounts of alginate to form scaffolds. Differential scanning calorimetric analysis (DSC), FTIR, XRD, and NMR techniques were used for the characterization of scaffolds. SEM confirmed the adequate porous structure of the scaffolds required for osteoconductivity. The incorporation of the bioactive glass with alginate has improved the compressive strength of the scaffolds. The bioactivity of the scaffolds was demonstrated by an increase in the pH of the medium after the immersion of the scaffolds in a Tris/HCl buffer and by the formation of orthophosphate precipitate on scaffolds. The scaffolds were able to release calcium, strontium and zinc ions in the Tris/HCl buffer, which would have a positive impact on osteogenesis if tested in vivo.

Type 2 Diabetes Mellitus Increases the Risk to Hip Fracture in Postmenopausal Osteoporosis by Deteriorating the Trabecular Bone Microarchitecture and Bone Mass.

T2DM is linked to an increase in the fracture rate as compared to the nondiabetic population even with normal or raised bone mineral density (BMD). Hence, bone quality plays an important role in the pathogenesis of skeletal fragility due to T2DM. This study analyzed the changes in the trabecular bone microstructure due to T2DM at various time points in ovariectomized and nonovariectomized rats. Animals were divided into four groups: (I) control (sham), (II) diabetic (sham), (III) ovariectomized, and (IV) ovariectomized with diabetes. The trabecular microarchitecture of the femoral head was characterized using a micro-CT. The differences between the groups were analyzed at 8, 10, and 14 weeks of the onset of T2DM using a two-way analysis of variance and by post hoc multiple comparisons. The diabetic group with and without ovariectomies demonstrated a significant increase in trabecular separation and a decrease in bone volume fraction, trabecular number, and thickness. BMD decreased in ovariectomized diabetic animals at 14 weeks of the onset of T2DM. No significant change was found in connectivity density and degree of anisotropy among groups. The structural model index suggested a change towards a weaker rod-like microstructure in diabetic animals. The data obtained suggested that T2DM affects the trabecular structure within a rat's femoral heads negatively and changes are most significant at a longer duration of T2DM, increasing the risk to hip fractures.

In vivo Labeling of Bone Microdamage in an Animal Model of Type 1 Diabetes Mellitus.

Type 1 diabetes mellitus (DM1) is linked to a decrease in bone strength. Bone strength entails both bone mineral density and bone quality. Limited data are available regarding diabetes-induced microdamage, which can severely influence bone quality. This study has investigated bone microdamage as a measure of bone quality in an animal model of DM1. Microdamage in the neck of the femur was labelled in vivo using multiple fluorochromes at 4, 12 and 24 weeks after the onset of DM1. Microcracks were quantified and their morphology analyzed using microscopy techniques. The mean length of microcracks at 24 weeks, and crack numerical and surface densities were significantly higher (p < 0.05) 4 weeks after the onset of DM1 when compared with control. Diffuse damage density was highest at 12 weeks after the onset of DM1. The arrangement of the collagen fibrils became progressively more irregular from 4 to 24 weeks of DM. This is the first study to analyze microdamage in vivo at different time points of DM1. DM1is associated with microcracks from the early stage, however bone microstructure shows toughening mechanisms that arrest their growth but disease progression further deteriorates bone quality resulting in longer microcracks which may increase fracture risk.

An update of SGLT1 and SGLT2 inhibitors in early phase diabetes-type 2 clinical trials.

Introduction: More than 424 million adults have diabetes mellitus (DM). This number is expected to increase to 626 million by 2045. The majority (90-95%) of people with DM has type 2-diabetes (T2DM). The continued prevalence of DM and associated complications has prompted investigators to find new therapies. One of the most recent additions to the anti-diabetic armamentarium are inhibitors of sodium-glucose co-transporters 1 and 2 (SGLT1, SGLT2). Areas covered: The authors review the status of SGLT2 inhibitors for the treatment of T2DM and place an emphasis on those agents in early phase clinical trials. Data and information were retrieved from American Diabetes Association, Diabetes UK, ClinicalTrials.gov, PubMed, and Scopus websites. The keywords used in the search were T2DM, SGLT1, SGLT2, and clinical trials. Expert opinion: The benefits of SGLT inhibitors include reductions in serum glycated hemoglobin (HbA1c), body weight, blood pressure and cardiovascular and renal events. However, SGLT inhibitors increase the risk of genitourinary tract infections, diabetic ketoacidosis, and bone fractures. The development of SGLT inhibitors with fewer side effects and as combination therapies are the key to maximizing the therapeutic effects of this important class of anti-diabetic drug.

An update on therapies for the treatment of diabetes-induced osteoporosis.

Introduction: Currently, 424 million people aged between 20 and 79 years worldwide are diabetic. More than 25% of adults aged over 65 years in North America have Type 2 diabetes mellitus (DM). Diabetes-induced osteoporosis (DM-OS) is caused by chronic hyperglycemia, advanced glycated end products and oxidative stress. The increase in the prevalence of DM-OS has prompted researchers to develop new biological therapies for the management of DM-OS. Areas covered: This review covered the current and novel biological agents used in the management of DM-OS. Data were retrieved from PubMed, Scopus, American Diabetes Association and International Osteoporosis Foundation websites, and ClinicalTrials.gov. The keywords for the search included: DM, osteoporosis, and management. Expert opinion: Several biological molecules have been examined in order to find efficient drugs for the treatment of DM-OS. These biological agents include anti-osteoporosis drugs: net anabolics (parathyroid hormone/analogs, androgens, calcilytics, anti-sclerostin antibody), net anti-resorptive osteoporosis drugs (calcitonin, estrogen, selective estrogen receptor modulators, bisphosphonates, RANKL antibody) and anti-diabetic drugs (alpha glucosidase inhibitors, sulfonylureas, biguanides, meglitinides, thiazolidinediones, GLP-1 receptor agonists, dipeptidylpeptidase-4 inhibitors, sodium glucose co-transporter-2 inhibitors, insulin). Biological medications that effectively decrease hyperglycemia and, at the same time, maintain bone health would be an ideal drug/drug combination for the treatment of DM-OS.

Fabrication of Porous Bone Scaffolds Using Alginate and Bioactive Glass.

Porous composite scaffold using an alginate and bioactive glass ICIE16M was synthesized by a simple freeze-drying technique. The scaffold was characterized using compression testing, Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), X-ray diffraction (XRD), X-ray microtomography (XMT) and scanning electron microscopy (SEM). The bioactivity of the scaffold was evaluated by its ability to form apatite on its surface in simulated body fluid (SBF). The data collected showed evidence that the novel material produced had an appropriate pore size for osteoconduction, with an average pore size of 110 µm and maximum pore size of 309 µm. Statistical analysis confirmed that the glass filler significantly (P < 0.05) increased the collapse yield of the scaffolds compared with pure alginate scaffolds. The ICIE16M glass had an amorphous structure, favorable for bioactivity.

The behaviour of microcracks in compact bone.

This paper summarises four separate studies carried out by our group over the past number of years in the area of bone microdamage. The first study investigated the manner by which microcracks accumulate and interact with bone microstructure during fatigue testing of compact bone specimens. In a series of fatigue tests carried out at four different stress ranges between 50 and 80 MPA, crack density increased with loading cycles at a rate determined by the applied stress. Variations in the patterns of microdamage accumulation suggest that that at low stress levels, larger amounts of damage can build up without failure occurring. In a second study using a series of four-pont bending tests carried out on ovine bone samples, it was shown that bone microstructure influenced the ability of microcracks to propagate, with secondary osteons acting as barriers to crack growth. In a third study, the manner by which crack growth disrupts the canalicular processes connecting osteocytes was investigated. Analysis of individual cracks showed that disruption of the canalicular processes connecting osteocytes occurred due to shear displacement at the face of propagating microcracks, suggesting that this may play some role in the mechanism that signals bone remodelling. In a fourth in vivo study, it was shown that altering the mechanical load applied to the long bones of growing rats causes microcrack formation. In vivo microdamage was present in rats subjected to hindlimb suspension with a higher microcrack density found in the humeri than the femora. Microdamage was also found in control animals. This is the first study to demonstrate in vivo microcracks in normally loaded bones in a rat model.