Exploring the osteogenic potential of chitosan-quercetin bio-conjugate: In vitro and in vivo investigations in osteoporosis models.

Anti-osteoporotic agents are clinically employed to improve bone health and prevent osteoporotic fractures. In the current study, we investigated the potential of chitosan-quercetin bio-conjugate as an anti-osteoporotic agent. The conjugate was prepared and characterized by FTIR and found notable interactions between chitosan and quercetin. Treating mouse MSCs with the bioconjugate in osteogenic conditions for a week led to elevated expression of differentiation markers Runx2, ALP, and Col-I, as determined by real-time PCR analysis. Evaluation at the cellular level using alizarin red staining demonstrated enhanced calcium deposition in MSCs following treatment with the bioconjugate. Likewise, ELISA analysis showed significantly elevated levels of secretory osteocalcin and osteonectin in groups treated with the conjugate. To broaden our comprehension, we utilized a zebrafish-based in vivo model of dexamethasone-induced osteoporosis to investigate bone regeneration. Toxicity profiling with zebrafish larvae confirmed the bio-conjugate's compatibility at a concentration of 25 µg/ml, underscoring the significance of finding the right dosage. Furthermore, in zebrafish models of osteoporosis, the bio-conjugate demonstrated significant potential for bone regeneration, as indicated by improved bone calcification, callus formation, and overall bone healing in a tail fin fracture model. Additionally, the study revealed that the bio-conjugate inhibited osteoclastic activity, leading to reduced TRAP activity and hydroxyproline release, suggesting its effectiveness in mitigating bone resorption. In conclusion, our research provides compelling evidence for the osteogenic capabilities of the chitosan-quercetin bio-conjugate, highlighting its promising applications in regenerative medicine and the treatment of conditions like osteoporosis.

Tooth-derived stem cells integrated biomaterials for bone and dental tissue engineering.

Recent years have seen the emergence of tissue engineering strategies as a means to overcome some of the limits of conventional medical treatment. A biomaterial with tailored physio-chemical characteristics is used in this sophisticated method to transport stem cells and growth factors/bioactive substances, or to attract local endogenous cells, enabling new tissue formation. Biomaterials might serve as a biomimetic structure inspired by the natural milieu, assisting the cells in establishing their natural relationships. Such a method would benefit from having ready access to an abundant reservoir of stem cells with strong tissue regeneration capacity, in addition to using biological compatible material to promote new tissue creation. Teeth may have a plethora of self-renewing, multipotent mesenchymal stem cell (MSC) populations. Recent advancements and promising directions for cell transplantation and homing techniques using dental MSCs for tissue regeneration are discussed in this review paper. Overall, this research paints a picture of the present landscape of new approaches to using tooth-derived MSCs in conjunction with biomaterials and bioactive substances for tissue regeneration.

Glucagon-like peptide-1 receptor promotes osteoblast differentiation of dental pulp stem cells and bone formation in a zebrafish scale regeneration model.

Bone cells express the glucagon-like peptide 1 receptor (GLP-1R). However, its presence and role in human dental pulp derived stem cells (hDPSCs) remains elusive. Hence, in the current study, we isolated hDPSCs and differentiated them into osteoblasts, where GLP-1R expression was found to be upregulated during osteoblast differentiation. GLP-1 receptor agonist, liraglutide peptide treatment, increased osteoblast differentiation in hDPSCs by increasing calcium deposition, ALP activity, and osteoblast marker genes, Runx2, type 1 col, osteonectin, and osteocalcin. Furthermore, activation of long non-coding RNA (LncRNA) LINC00968 and microRNA-3658 signalling increased Runx2 expression. Specifically, liraglutide increased LncRNA-LINC00968 expression while decreasing miR-3658 expression. LINC00968 targets miR-3658, and miR-3658 targets Runx2. Additionally, in an in-vivo study, zebrafish scale regeneration model, liraglutide promoted calcium deposition, osteoblastic cell count, collagen 1α, osteonectin, osteocalcin, runx2a MASNA isoform expression (transcribed from promoter P1), and Ca/P ratio in scales. Overall, GLP-1R activation promotes osteoblast differentiation via Runx2/LncRNA-LINC00968/miR-3658 signalling in hDPSCs and promotes bone formation in zebrafish scale regeneration.

Rutin-Zn(II) complex promotes bone formation - A concise assessment in human dental pulp stem cells and zebrafish.

We have assessed the molecular role of Rutin and rutin-Zn(II) complex on osteoblast differentiation and mineralization in human dental pulp cells and zebrafish model. The biocompatibility of the rutin-Zn(II) complex was determined using MTT and chick embryotoxicity assays. Alizarin red staining and ALP measurements were performed to study the osteogenic role of Rutin and rutin-Zn(II) complex at the cellular level in hDPSCs. At molecular level, following rutin and rutin-Zn(II) exposure, the mRNA expression profile of osteoblast markers such Runx2, type 1 col, OC, and ON were investigated. In addition to this, the expression of negative regulators of osteoblast development such Smad7, Smurf1, and HDAC7 waere studied by Real time RT-PCR analysis. The osteogenic role of prepared complex under in vivo was studied by an in-house zebrafish scale model followed by osteoblast differentiation markers expression profiling and Ca:P level measurement by ICP-MS. Rutin and the rutin-Zn(II) complex were found to be non-toxic till 10 µM and increased the expression of osteoblast differentiation marker genes. It also enhanced calcium deposition in both in vitro and in vivo models. Osteogenic property of rutin-Zn(II) in hDPSCs was found be mediated by Smad7, Smurf1, and HDAC7 and enhancing Runx2 expression. Our study warrants the possible use of rutin-Zn(II) as naïve agent or in combination with other bone scaffolding systems/materials for bone tissue engineering applications.

MicroRNA-432-5p regulates sprouting and intussusceptive angiogenesis in osteosarcoma microenvironment by targeting PDGFB.

Osteosarcoma (OS) is a type of bone tumor conferred with high metastatic potential. Attainable growth of tumors necessitates functional vasculature mediated by sprouting angiogenesis (SA) and intussusceptive angiogenesis (IA). However, the regulation of IA and SA is still unclear in OS. To understand the mechanisms adopted by OS to induce angiogenesis, initially, we assessed the expression profile of a set of miRNAs' in both OS cells (SaOS2 and MG63) and normal bone cells. Amongst them, miR-432-5p was found to be highly downregulated in OS. The functional role of miR-432-5p in OS was further analyzed using miR-432-5p mimic/inhibitor. Platelet-derived growth factor-B (PDGFB) was found to be a putative target of miR-432-5p and it was further confirmed that the PDGFB 3'UTR is directly targeted by miR-432-5p using the luciferase reporter gene system. PDGFB was found to be secreted by OS to regulate angiogenesis by targeting the cells in its microenvironment. The conditioned medium obtained from miR-432-5p mimic transfected MG63 and SaOS2 cells decreased cell viability, proliferation, migration, and aorta ring formation in endothelial cells. The miRNA mimic/inhibitor transfected MG63 and SaOS2 cells were placed on SA (day 6) and IA (day 9) phase of CAM development to analyze SA and IA mechanisms. It was found that miR-432-5p mimic transfection in OS promotes the transition of SA to IA which was documented by the angiogenic parameters and SA and IA-associated gene expression. Interestingly, this outcome was also supported by the zebrafish tumor xenograft model. Corroborating these results, it is clear that miR-432-5p expression in OS cells regulates SA and IA by targeting PDGFB genes. We conclude that targeting miR-432-5p/PDGFB signaling can be a potential therapeutic strategy to treat OS along with other existing strategies.

Melatonin regulates tumor angiogenesis via miR-424-5p/VEGFA signaling pathway in osteosarcoma.

Melatonin is recognized as an anti-angiogenic agent, but its function in the tumor microenvironment especially in osteosarcoma remains uncertain. Among the selected miRNAs, miR-205, miR-424, miR-140, miR-106, and miR-519 were upregulated by melatonin in osteosarcoma cells. The functional role of miR-424-5p in osteosarcoma was further analyzed using miR-424-5p mimic/inhibitor. VEGFA mRNA and protein expression were altered by miR-424-5p mimic/inhibitor transfection with and without melatonin treatment and it was further identified that the VEGFA 3'UTR is directly targeted by miR-424-5p using the luciferase reporter gene system. The conditioned medium from SaOS2 and MG63 cells treated with melatonin and/or transfected with miR-424-5p mimic/inhibitor was exposed to endothelial cells, and cell proliferation and migration was analyzed. MG-63 and SaOS2 cells are also transfected with miR-424-5p inhibitors and positioned on CAM vascular bed to study the angiogenic activity at both morphological and molecular level under melatonin treatment. Our observations demonstrate for the first time that, melatonin upregulated the expression of miR-424-5p in osteosarcoma inhibiting VEGFA. Furthermore, it suppresses tumor angiogenesis, modulating surrounding endothelial cell proliferation and migration as well as the morphology of blood vessels, and angiogenic growth factors. These findings suggest that melatonin could play a pivotal role in tumor suppression via miR-424-5p/VEGFA axis.

Intussusceptive angiogenesis as a key therapeutic target for cancer therapy.

Deregulation of angiogenesis is a key reason for tumor growth and progression. Several anti-angiogenic drugs in clinical practice attempt to normalize abnormal tumor vasculature. Unfortunately, these drugs are ineffective due to the development of resistance in patients after drug holidays. A sizable literature suggests that resistance to these anti-angiogenic drugs occurs due to various compensatory mechanisms of tumor angiogenesis. Therefore, we describe different compensatory mechanisms of tumor angiogenesis, and explain why intussusceptive angiogenesis (IA), is a crucial mechanism of compensatory angiogenesis in tumors which resist anti-VEGF (vascular endothelial growth factor) therapies. IA is often overlooked due to the scarcity of experimental models. Therefore, we examine data from existing experimental models and our novel ex-ovo model of angiogenesis in chick embryos, and explain the important genes and signaling pathways driving IA. Using bio-informatic analyses of major genes regulating conventional sprouting angiogenesis (SA) and intussusceptive angiogenesis, we provide fresh insights on the 'angiogenic switch' which regulates the transition from SA to IA. Finally, we examine the interplay between molecules regulating SA, IA, and molecules known to promote tumor progression. Based on these analyses, we conclude that intussusceptive angiogenesis (IA) is a promising therapeutic target for developing effective anti-cancer treatment regimes.

Inflammation in myocardial injury: mesenchymal stem cells as potential immunomodulators.

Ischemic heart disease is a growing worldwide epidemic. Improvements in medical and surgical therapies have reduced early mortality after acute myocardial infarction and increased the number of patients living with chronic heart failure. The irreversible loss of functional cardiomyocytes puts these patients at significant risk of ongoing morbidity and mortality after their index event. Recent evidence suggests that inflammation is a key mediator of postinfarction adverse remodeling in the heart. In this review, we discuss the cardioprotective and deleterious effects of inflammation and its mediators during acute myocardial infarction. We also explore the role of mesenchymal stem cell therapy to limit secondary injury and promote myocardial healing after myocardial infarction.

Models to investigate intussusceptive angiogenesis: A special note on CRISPR/Cas9 based system in zebrafish.

Angiogenesis is a distinct process which follows sprouting angiogenesis (SA) and intussusceptive angiogenesis (IA) forming the basis for various physiological and pathological scenarios. Angiogenesis is a double edged sword exerting both desirable and discernible effects owing to the referred microenvironment. Therapeutic interventions to promote angiogenesis in regenerative medicine is essential to achieve functional syncytium of tissue constructs while, angiogenic inhibition is a key therapeutic target to suppress tumor growth. In the recent years, clustered regularly interspaced short palindromic repeats associated 9 (CRISPR-Cas9) based gene editing approaches have been gaining considerable attention in the field of biomedical research owing to its ease in tailoring targeted genome in living organisms. The Zebrafish model, with adequately high-throughput fitness, is a likely option for genome editing and angiogenesis research. In this review, we focus on the implication of Zebrafish as a model to study IA and furthermore enumerate CRISPR/Cas9 based genome editing in Zebrafish as a candidate for modeling different types of angiogenesis and support its candidature as a model organism.

Chitosan based thermoresponsive hydrogel containing graphene oxide for bone tissue repair.

Chitosan (CS), glycerophosphate (GP) based injectable hydrogels are explored for its implications in bone defect healing and regeneration. Both acellular and cell laden CS based hydrogels are widely investigated and improved through the inclusion of various nanoparticles, polymers and bioactive molecules. In order to improve its applicability for bone tissue repair, we developed an injectable, thermosensitive CS hydrogel containing graphene oxide (GO) and investigated its properties. The hydrogels were investigated for its porous architecture using scanning electron microscopy (SEM), swelling property, protein adsorption ability, degradation rate and exogenous biomineralization. GO addition improved the physico-chemical properties with notable betterment. The CS/GP/GO hydrogel was biocompatible to mesenchymal stem cells and they were metabolically active upon encapsulation. The hydrogel promoted osteogenic differentiation of mouse mesenchymal stem cells by upregualtion of Runt-related transcription factor 2 (Runx2), Alkaline phosphatase (ALP), Type -1 collagen (COL-1), and osteocalcin (OC) under osteogenic conditions. The hydrogel proves to be an amenable platform for carrying cells and exhibited suitable properties to be a potential candidate for bone tissue regeneration.

Biogenic gold nanoparticles synthesis mediated by Mangifera indica seed aqueous extracts exhibits antibacterial, anticancer and anti-angiogenic properties.

During the last few decades, gold nanoparticles (AuNP's) have gained considerable attention in nanomedicine and expanded its application in clinical diagnosis and as therapeutics. Employing plant extract for synthesising gold nanoparticles proves to be an eco-friendly technology for large scale production. It is highly economical and suitable for biological applications by negating the use of chemicals involved in conventional route. In this study, AuNP's was prepared by a simple one step method of employing aqueous Mangifera indica seed extract as a reducing agent. Scanning electron microscopy and transmission electron microscopy revealed spherical shaped nanoparticles and dynamic light scattering analysis indicated the AuNP's to be approximately 46.8 nm in size. AuNP's efficiently inhibited the growth of E. coli and S. aureus by its inherent ability to generate reactive oxygen species (ROS) and exhibited detrimental effects towards the tested bacterial species. Biocompatibility assessment indicated the non-toxic nature of AuNP's towards mesenchymal stem cells at 25 µg/ml and interestingly, suppressed the growth of human gastric cancer cells under in vitro culture conditions. AuNP's significantly exhibited anti-angiogenic property in chick chorioallantoic membrane model (CAM) by downregulating Ang-1/Tie2 pathway. Overall, the synthesized AuNP's exhibited antibacterial and anti-angiogenic properties with high biocompatibility thereby supporting its candidature for various biomedical applications. It can be employed in suppressing tumor growth, combat inflammatory diseases that necessitate the involvement of angiogenesis suppression, and antibacterial activity is suitable for its clinical translation to negate surgery associated infections.

Synthesis and characterization of silibinin/phenanthroline/neocuproine copper(II) complexes for augmenting bone tissue regeneration: an in vitro analysis.

In the recent decades, flavonoid metal complexes have been widely investigated for their multifaceted role in enabling osteoblast differentiation and bone formation. Silibinin complexed with copper(II) ion has been synthesized along with two mixed ligand complexes, namely copper(II) silibinin-phenanthroline and neocuproine as co-ligands, and their positive role in promoting neovacularization and osteoblast differentiation was investigated. Silibinin mono complex [Cu(sil)(H2O)2] and [Cu(sil)(phen)] showed similar UV-visible absorption in the region of 315 and 222 nm, whereas Cu(silibinin)(neocuproine) [Cu(sil)(neo)] showed a blueshift in the 320 nm transition. The involvement of carbonyl group present in the C-ring in metal ion chelation was identified by FT-IR analysis. Thermal gravimetric analysis (TGA) depicted that [Cu(sil)(neo)] has higher thermal stability when compared with the control silibinin and Cu-silibinin mono, and phen complexes. Cu-silibinin complexes were found to be non-toxic to human MG-63 cells and mouse mesenchymal stem cells (MSCs). Our investigations identified the positive role of these complexes in promoting osteoblast differentiation by enhancing calcium deposition and alkaline phosphatase (ALP) activity at the cellular level and stimulation of osteoblastic marker genes such as Runx2, ALP, type 1 collagen, and OCN mRNAs expression at the molecular level. These complexes also supported angiogenesis by upregulation of VEGF and Ang 1 expression in mouse MSCs. Hence, our results suggest that the potential of these metal complexes along with mixed ligand complexes promoted osteoblast differentiation, thus warranting its candidature for bone tissue regeneration application.

Synthesis and characterization of zinc-silibinin complexes: A potential bioactive compound with angiogenic, and antibacterial activity for bone tissue engineering.

Zinc silibinin complex [Zn(sil)(H2O)2] and mixed ligand zinc complexes such as Zn(silibinin)(phenanthroline) [Zn(sil)(phen)], and Zn(silibinin)(neocuproine) [Zn(sil)(neo)] have been synthesized and characterized. The UV-vis spectra of the Zn(II) complexes showed a considerable shift in the intra-ligand transition. From the IR spectra, it is clear that carbonyl group in the C-ring is involved in the metal chelation besides A/C-ring hydroxyl group. Thermal gravimetric analysis showed that [Zn(sil)(neo)] has higher thermal stability compared to the other two Zn(II) complexes. The potential biological activities of the synthesized complexes were studied systematically. In osteoblast differentiation, silibinin and Zn-silibinin complexes enhanced osteoblast differentiation at the cellular level by increasing calcium deposition and ALP activity, and at molecular level increased osteoblast markers include Runx2, type 1 col, ALP and OC mRNAs expression. Additionally, Zn-silibinin complexes showed promising effects on osteoblast differentiation by regulating miR-590/Smad7 signaling pathway. Among the complexes, Zn(sil)(phen) showed more stimulatory effect on osteoblastic differentiation. These complexes also exhibited angiogenic property by increasing VEGF and Ang 1 expression in mouse MSCs and antibacterial activity against E. coli (Gram-negative) and S. aureus (Gram-positive) strains. Thus, the present study demonstrated that the Zn-silibinin complexes exhibit great potential as a pharmacological agent for bone tissue engineering.

Prophylactic supplementation of resveratrol is more effective than its therapeutic use against doxorubicin induced cardiotoxicity.

Resveratrol (RSV), a polyphenolic compound and naturally occurring phytoalexin, has been reported to exert cardio-protective effects in several animal studies. However, the outcome of initial clinical trials with RSV was less effective compared to pre-clinical studies. Therefore, RSV treatment protocols need to be optimized. In this study we evaluated prophylactic versus therapeutic effect of resveratrol (RSV) in mitigating doxorubicin (Dox)-induced cardiac toxicity in rats. To investigate prophylactic effects, RSV was supplemented for 2 weeks along with Dox administration. After 2 weeks, Dox treatment was stopped and RSV was continued for another 4 weeks. To study therapeutic effects, RSV treatment was initiated after 2 weeks of Dox administration and continued for 4 weeks. Both prophylactic and therapeutic use of RSV mitigated Dox induced deterioration of cardiac function as assessed by echocardiography. Also RSV treatment (prophylactic and therapeutic) prevented Dox induced myocardial damage as measured by cardiac enzymes (LDH and CK-MB) in serum. Which was associated with decrease in Dox induced myocardial apoptosis and fibrosis. Interestingly our study also reveals that prophylactic use of RSV was more effective than its therapeutic use in mitigating Dox induced apoptosis and fibrosis in the myocardium. Therefore, prophylactic use of resveratrol may be projected as a possible future adjuvant therapy to minimize cardiotoxic side effects of doxorubicin in cancer patients.

Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells as an Individual-Specific and Renewable Source of Adult Stem Cells.

This chapter deals with the employment of human-induced pluripotent stem cells (hiPSCs) as a candidate to differentiate into mesenchymal stem cells (MSCs). This would enable to help establish a regular source of human MSCs with the aim of avoiding the problems associated with procuring the MSCs either from different healthy individuals or patients, limited extraction potentials, batch-to-batch variations or from diverse sources such as bone marrow or adipose tissue. The procedures described herein allow for a guided and ensured approach for the regular maintenance of hiPSCs and their subsequent differentiation into MSCs using the prescribed medium. Subsequently, an easy protocol for the successive isolation and purification of the hiPSC-differentiated MSCs is outlined, which is carried out through passaging and can be further sorted through flow cytometry. Further, the maintenance and expansion of the resultant hiPSC-differentiated MSCs using appropriate characterization techniques, i.e., Reverse-transcription PCR and immunostaining is also elaborated. The course of action has been deliberated keeping in mind the awareness and the requisites available to even beginner researchers who mostly have access to regular consumables and medium components found in the general laboratory.

Preparation, characterization and antimicrobial activity of a bio-composite scaffold containing chitosan/nano-hydroxyapatite/nano-silver for bone tissue engineering.

In this study, a bio-composite scaffold containing chitosan/nano-hydroxyapatite/nano-silver particles (CS/nHAp/nAg) was developed by freeze drying technique, followed by introduction of silver ions in controlled amount through reduction phenomenon by functional groups of chitosan. The scaffolds were characterized using SEM, FT-IR, XRD, swelling, and biodegradation studies. The testing of the prepared scaffolds with Gram-positive and Gram-negative bacterial strains showed antibacterial activity. The scaffold materials were also found to be non-toxic to rat osteoprogenitor cells and human osteosarcoma cell line. Thus, these results suggested that CS/nHAp/nAg bio-composite scaffolds have the potential in controlling implant associated bacterial infection during reconstructive surgery of bone.