Osteogenic Lineage Research Articles

Biomaterial-Based Nanocomposite for Osteogenic Repurposing of Doxycycline.

BackgroundBesides its antimicrobial action, doxycycline (DX) has lately been repurposed as a small-molecule drug for osteogenic purposes. However, osteogenic DX application is impeded by its dose-dependent cytotoxicity. Further, high-dose DX impairs cell differentiation and mineralization.PurposeIntegrating DX into a biomaterial-based delivery system that can control its release would not only ameliorate its cytotoxic actions but also augment its osteogenic activity. In this work, we managed to engineer novel composite DX–hydroxyapatite–polycaprolactone nanoparticles (DX/HAp/PCL) to modify DX osteogenic potential.MethodsEmploying a 23-factorial design, we first optimized HApN for surface-area attributes to maximize DX loading. Composite DX/HAp/PCL were then realized using a simple emulsification technique, characterized using various in vitro methods, and evaluated for in vitro osteogenesis.ResultsThe developed HApN exhibited a favorable crystalline structure, Ca:P elemental ratio (1.67), mesoporous nature, and large surface area. DX/HAp/PCL achieved the highest reported entrapment efficiency (94.77%±1.23%) of DX in PCL-based particles. The developed composite system achieved controlled release of the water-soluble DX over 24 days. Moreover, the novel composite nanosystem managed to significantly ameliorate DX cytotoxicity on bone-marrow stem cells, as well as enhance its overall proliferation potential. Alkaline phosphatase and mineralization assays revealed superior osteodifferentiation potential of the composite system. Quantification of gene expression demonstrated that while DX solution was able to drive bone-marrow stem cells down the osteogenic lineage into immature osteoblasts after 10-day culture, the innovative composite system allowed maturation of osteodifferentiated cells. To the best of our knowledge, this is the first work to elaborate the impact of DX on the expression of osteogenic genes: RUNX2, OSP, and BSP. Further, the osteogenicity of a DX-loaded particulate-delivery system has not been previously investigated.ConclusionOur findings indicate that repurposing low-dose DX in complementary biomaterial-based nanosystems can offer a prominent osteogenic candidate for bone-regeneration purposes.

Liposomes embedded with differentiating factors as a new strategy for enhancing DPSC osteogenic commitment.

Human dental pulp stem cell (DPSC) differentiation toward the osteoblastic phenotype is enhanced when culture media are supplemented with differentiating factors, i.e. ascorbic acid, β-glycerophosphate and dexamethasone. Liposomes, spherical vesicles formed by a phospholipid bilayer, are frequently used as carriers for drugs, growth factors and hydrophobic molecules. The aim of this work was to speed up DPSC commitment to the osteogenic lineage by embedding differentiating factors within liposomes. Firstly, liposomes were prepared by rehydrating a phospholipidic thin film and characterised in terms of dimensions. Secondly, liposome-exposed DPSCs were characterised by their immunophenotypic profile. Levels of CD90 were significantly decreased in the presence of liposomes filled with ascorbic acid, β-glycerophosphate and dexamethasone (Lipo-Mix) with respect to normal differentiation medium (DM), while CD73 and CD29 expression were enhanced, suggesting osteogenic commitment. Additionally, an appreciable extracellular matrix deposition is detected. Thirdly, the Lipo-Mix formulation better increases alkaline phosphatase activity and levels of Collagen I secretion with respect to DM. In parallel, the new liposome formulation is capable of decreasing the release of H2O2 and of triggering a precocious antioxidant cell response, redressing the redox balance required upon mesenchymal stem cell commitment to osteogenesis. It can be therefore hypothesised that Lipo-Mix could represent a suitable tool for clinical regenerative purposes in the field of tissue engineering by speeding up DPSC osteogenic commitment, mineralised matrix deposition and remodelling.

Effective elastin-like recombinamers coating on poly(vinylidene) fluoride membranes for mesenchymal stem cell culture

Bone’s inherent piezoelectricity is a key factor in regulating bone growth and mesenchymal stem cells (MSCs) fate towards the osteogenic lineage. The piezoelectric polymer poly(vinylidene) fluoride (PVDF) was thus used to manufacture electroactive membranes by means of non-solvent induced phase separation (NIPS), producing porous membranes with approximately 90% of γ-phase for MSCs culture. The combination of the porous surface and PVDF hydrophobicity hinders cell adhesion and requires a coating to improve cell culture conditions. A layer-by-layer (LbL) method was used to deposit elastin-like recombinamers (ELRs) containing RGD sequences applying click cross-linking chemistry. ELRs potential was confirmed by comparing traditional fibronectin adsorption with ELRs LbL on PVDF electroactive membranes. Porcine bone marrow MSCs preferred ELRs-coated surfaces, which enhanced initial cell adhesion and improved proliferation after 7 days. These findings lead to new possibilities for regenerative therapies in the area of bone tissue engineering, offering the advantages of MSC commitment towards the osteogenic lineage by applying electro-mechanical stimulation on electroactive substrates.

Platelet-derived biomaterials-mediated improvement of bone injury through migratory ability of embryonic fibroblasts: in vitro and in vivo evidence.

Bony injuries lead to compromised skeletal functional ability which further increase in aging population due to decreased bone mineral density. Therefore, we aimed to investigate the therapeutic potential of platelet-derived biomaterials (PDB) against bone injury. Specifically, we assessed the impact of PDB on osteo-inductive characteristics and migration of mouse embryonic fibroblasts (MEFs). Osteogenic lineage, matrix mineralization and cell migration were determined by gene markers (RUNX2, OPN and OCN), alizarin Red S staining, and migration markers (FAK, pFAK and Src) and EMT markers, respectively. The therapeutic impact of TGF-β1, a key component of PDB, was confirmed by employing inhibitor of TGF-β receptor I (Ti). Molecular imaging-based in vivo cellular migration in mice was determined by establishing bone injury at right femurs. Results showed that PDB markedly increased expression of osteogenic markers, matrix mineralization, migration and EMT markers, revealing higher osteogenic and migratory potential of PDB-treated MEFs. In vivo cell migration was manifested by expression of migratory factors, SDF-1 and CXCR4. Compared to control, PDB-treated mice exhibited higher bone density and volume. Ti treatment inhibited both migration and osteogenic potential of MEFs, affirming impact of TGF-β1. Collectively, our study clearly indicated PDB-rescued bone injury through enhancing migratory potential of MEFs and osteogenesis.

A novel hydrogel scaffold contained bioactive glass nanowhisker (BGnW) for osteogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro

Employing hydrogels as an alternative strategy for repairing bone defects has received great attention in bone tissue engineering. In this study, hydrogel scaffold based on collagen, gelatin, and glutaraldehyde was combined with bioactive glass nanowhiskers (BGnW) to differentiate human mesenchymal stem cells (hMSCs) into the osteogenic lineage and inducing biomineralization. Pure Gel-Glu-Col and bioactive glass nanowhiskers were used as control throughout the paper. Chemical, physical and morphological characteristics of the nanocomposite scaffold were assessed meticulously using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), porosity measurement, water uptake ability, tensile test, and scanning electron microscopy (SEM). To determine the cytotoxicity and cell viability of the hydrogel, MTT assay and Acridine orange (AO) staining were performed. hMSCs seeded on Gel-Glu-Col/BGnW were then incubated with osteogenic differentiation media for 14 days. Biomineralization assays (alkaline phosphatase (ALP) activity, calcium content assay, von Kossa, and Alizarin red staining) were carried out, and osteogenic genes and protein markers were examined using real time-PCR and immunocytochemistry. Results showed that the components of the hydrogel were properly integrated. The mechanical property of hydrogel was enhanced following the addition of BGnW. Cell viability assays confirmed the biocompatibility of the scaffold and increasing the proliferation after incorporating BGnW into pure Ge1-Glu-Col. Our nanocomposite maintained an enhanced ability of biomineralization as compared to its pure counterparts. Molecular investigations revealed an elevated level of osteogenic markers as compared to Ge1-Glu-Col and BGnW. All in all, Gel-Glu-Col/BGnW seems to be a potential candidate for the regeneration of bone tissue.

Epigenetic Control of Osteogenic Lineage Commitment

Within the eukaryotic nucleus the genomic DNA is organized into chromatin by stably interacting with the histone proteins as well as with several other nuclear components including non-histone proteins and non-coding RNAs. Together these interactions distribute the genetic material into chromatin subdomains which can exhibit higher and lower compaction levels. This organization contributes to differentially control the access to genomic sequences encoding key regulatory genetic information. In this context, epigenetic mechanisms play a critical role in the regulation of gene expression as they modify the degree of chromatin compaction to facilitate both activation and repression of transcription. Among the most studied epigenetic mechanisms we find the methylation of DNA, ATP-dependent chromatin remodeling, and enzyme-mediated deposition and elimination of post-translational modifications at histone and non-histone proteins. In this mini review, we discuss evidence that supports the role of these epigenetic mechanisms during transcriptional control of osteoblast-related genes. Special attention is dedicated to mechanisms of epigenetic control operating at the Runx2 and Sp7 genes coding for the two principal master regulators of the osteogenic lineage during mesenchymal stem cell commitment.

New prospects in skin regeneration and repair using nanophased hydroxyapatite embedded in collagen nanofibers

This study reflects an exploitation of a composite matrix produced by electrospinning of collagen and electrospraying of nanophased hydroxyapatite (nanoHA), for skin regeneration applications. The main goal was to evaluate the effect of nanoHA, as source of localized calcium delivery, on human dermal fibroblasts, keratinocytes, and human mesenchymal stem cells (hMSCs) growth, proliferation, differentiation, and extracellular matrix production. This study revealed that calcium ions provided by nanoHA significantly enhanced cellular growth and proliferation rates and prevented adhesion of pathogenic bacteria strains typically found in human skin flora. Moreover, hMSCs were able to differentiate in both osteogenic and adipogenic lineages. Rat subcutaneous implantation of the membranes also revealed that no adverse reaction occurred. Therefore, the mechanically fit composite membrane presents a great potential to be used either as cell transplantation scaffold for skin wound regeneration or as wound dressing material in plastic surgery, burns treatment or skin diseases.

Identification of ALP+/CD73+ defining markers for enhanced osteogenic potential in human adipose-derived mesenchymal stromal cells by mass cytometry

BackgroundThe impressive progress in the field of stem cell research in the past decades has provided the ground for the development of cell-based therapy. Mesenchymal stromal cells obtained from adipose tissue (AD-MSCs) represent a viable source for the development of cell-based therapies. However, the heterogeneity and variable differentiation ability of AD-MSCs depend on the cellular composition and represent a strong limitation for their use in therapeutic applications. In order to fully understand the cellular composition of MSC preparations, it would be essential to analyze AD-MSCs at single-cell level.MethodRecent advances in single-cell technologies have opened the way for high-dimensional, high-throughput, and high-resolution measurements of biological systems. We made use of the cytometry by time-of-flight (CyTOF) technology to explore the cellular composition of 17 human AD-MSCs, interrogating 31 markers at single-cell level. Subcellular composition of the AD-MSCs was investigated in their naïve state as well as during osteogenic commitment, via unsupervised dimensionality reduction as well as supervised representation learning approaches.ResultThis study showed a high heterogeneity and variability in the subcellular composition of AD-MSCs upon isolation and prolonged culture. Algorithm-guided identification of emerging subpopulations during osteogenic differentiation of AD-MSCs allowed the identification of an ALP+/CD73+ subpopulation of cells with enhanced osteogenic differentiation potential. We could demonstrate in vitro that the sorted ALP+/CD73+ subpopulation exhibited enhanced osteogenic potential and is moreover fundamental for osteogenic lineage commitment. We finally showed that this subpopulation was present in freshly isolated human adipose-derived stromal vascular fractions (SVFs) and that could ultimately be used for cell therapies.ConclusionThe data obtained reveal, at single-cell level, the heterogeneity of AD-MSCs from several donors and highlight how cellular composition impacts the osteogenic differentiation capacity. The marker combination (ALP/CD73) can not only be used to assess the differentiation potential of undifferentiated AD-MSC preparations, but also could be employed to prospectively enrich AD-MSCs from the stromal vascular fraction of human adipose tissue for therapeutic applications.

Ionized jet deposition of antimicrobial and stem cell friendly silver-substituted tricalcium phosphate nanocoatings on titanium alloy

Orthopedic infections pose severe societal and economic burden and interfere with the capability of the implanted devices to integrate in the host bone, thus significantly increasing implants failure rate. To address infection and promote integration, here nanostructured antibacterial and bioactive thin films are proposed, obtained, for the first time, by Ionized Jet Deposition (IJD) of silver-substituted tricalcium phosphate (Ag-TCP) targets on titanium. Coatings morphology, composition and mechanical properties are characterized and proof-of-concept of biocompatibility is shown. Antimicrobial efficacy is investigated against four Gram positive and Gram negative bacterial strains and against C. albicans fungus, by investigating the modifications in planktonic bacterial growth in the absence and presence of silver. Then, for all bacterial strains, the capability of the film to inhibit bacterial adhesion is also tested. Results indicate that IJD permits a fine control over films composition and morphology and deposition of films with suitable mechanical properties. Biological studies show a good efficacy against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis and against fungus Candida albicans, with evidences of efficacy against planktonic growth and significant reduction of bacterial cell adhesion. No cytotoxic effects are evidenced for equine adipose tissue derived mesenchymal stem cells (ADMSCs), as no reductions are caused to cells viability and no interference is assessed in cells differentiation towards osteogenic lineage, in the presence of silver. Instead, thanks to nanostructuration and biomimetic composition, tricalcium phosphate (TCP) coatings favor cells viability, also when silver-substituted. These findings show that silver-substituted nanostructured coatings are promising for orthopedic implant applications.

Genome-wide association study implicates novel loci and reveals candidate effector genes for longitudinal pediatric bone accrual

BackgroundBone accrual impacts lifelong skeletal health, but genetic discovery has been primarily limited to cross-sectional study designs and hampered by uncertainty about target effector genes. Here, we capture this dynamic phenotype by modeling longitudinal bone accrual across 11,000 bone scans in a cohort of healthy children and adolescents, followed by genome-wide association studies (GWAS) and variant-to-gene mapping with functional follow-up.ResultsWe identify 40 loci, 35 not previously reported, with various degrees of supportive evidence, half residing in topological associated domains harboring known bone genes. Of several loci potentially associated with later-life fracture risk, a candidate SNP lookup provides the most compelling evidence for rs11195210 (SMC3). Variant-to-gene mapping combining ATAC-seq to assay open chromatin with high-resolution promoter-focused Capture C identifies contacts between GWAS loci and nearby gene promoters. siRNA knockdown of gene expression supports the putative effector gene at three specific loci in two osteoblast cell models. Finally, using CRISPR-Cas9 genome editing, we confirm that the immediate genomic region harboring the putative causal SNP influences PRPF38A expression, a location which is predicted to coincide with a set of binding sites for relevant transcription factors.ConclusionsUsing a new longitudinal approach, we expand the number of genetic loci putatively associated with pediatric bone gain. Functional follow-up in appropriate cell models finds novel candidate genes impacting bone accrual. Our data also raise the possibility that the cell fate decision between osteogenic and adipogenic lineages is important in normal bone accrual.

Tri-Lineage Differentiation of NTERA2 Clone D1 Cells towards Neural, Hepatic and Osteogenic Lineages in Vitro

Over the past decades, the in vitro use of pluripotent cell lines gained a crucial role in toxicology, preclinical drug testing and developmental biology. NTERA2 clone D1 cells were identified as pluripotent cells with high potential for neural differentiation. Although they are commonly used cellular sources in neuropharmacology and neurodevelopmental studies, their endodermal and mesodermal differentiation potential awaits further characterization. Here, we devised improved protocols for hepatogenic and osteogenic differentiation of NTERA2 clone D1 cells. Our in vitro differentiation assays showed significant up-regulation of multiple hepatogenic markers. We also observed robust mineralization and osteogenic marker expression of NTERA2 clone D1 cells upon in vitro osteogenic induction. These results suggest that NTERA2 clone D1 cells may be utilized as an in vitro model system to study various aspects of liver biology and osteogenesis. In addition, tri-lineage differentiation of NTERA2 clone D1 cells may serve as a simple experimental control system when validating pluripotency of other cell types.

Basal cell carcinoma stem cells exhibit osteogenic and chondrogenic differentiation potential

Specific cell subpopulations identified as cancer stem cells (CSCs) can be found in basal cell carcinoma (BCC). Generally, CSCs have a marked trans-differentiation potential that could potentially be used in differentiation therapies. However, there are no studies regarding BCC CSCs multipotency. The aim of the study was to analyze the characteristic of CSCs of BCC with emphasis on their differentiation potential upon specific induction. Specific staining and cell morphology were used for differentiation confirmation, along with the expression analysis of osteogenic (ALP, BSP, Runx2, OCN, BMP2), chondrogenic (COL1 and COL2A1), adipogenic (PPAR-γ) and neurogenic (Nestin and MAP2) markers. BCC CSCs differentiated into osteogenic and chondrogenic lineages, as judged by staining and high expression of specific markers (from 2-to 92-fold higher upon induction). Concomitantly with differentiation, the levels of cancer stem cell markers decreased in the cultures. Adipo-differentiation and neuro-differentiation were unsuccessful. In conclusion, BCC CSCs exhibit the capacity to trans-differentiate, a characteristic that may potentially be useful in the development of new strategies for the treatment of aggressive BCCs.

Melatonin restores osteoporosis-impaired osteogenic potential of bone marrow mesenchymal stem cells and alleviates bone loss through the HGF/PTEN/Wnt/β-catenin axis.

Background:Previous studies reported that melatonin exerts its effect on mesenchymal stem cell (MSC) survival and differentiation into osteogenic and adipogenic lineage. In the current study we aimed to explore the effect of melatonin on osteoporosis and relevant mechanisms.Methods:Real-time qualitative polymerase chain reaction (RT-qPCR) and Western blot analysis were conducted to determine expression of HGF, PTEN, and osteoblast differentiation-related genes in ovariectomy (OVX)-induced osteoporosis mice and the isolated bone marrow MSCs (BMSCs). Pre-conditioning with melatonin (1 μmol/l, 10 μmol/l and 100 μmol/l) was carried out in OVX mice BMSCs. Bone microstructure was analyzed using micro-computed tomography and the contents of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase 5b (TRAP5b) were detected by enzyme-linked immunosorbent assay in serum. BMSC proliferation was measured by cell-counting kit (CCK)-8 assay. Alizarin red S (ARS) staining and ALP activity assay were performed to assess BMSC mineralization and calcification. The activity of the Wnt/β-catenin pathway was evaluated by dual-luciferase reporter assay.Results:Melatonin prevented bone loss in OVX mice. Melatonin increased ALP expression and reduced TRAP5b expression. HGF and β-catenin were downregulated, while PTEN was upregulated in the femur of OVX mice. Melatonin elevated HGF expression and then stimulated BMSC proliferation and osteogenic differentiation. Additionally, HGF diminished the expression of PTEN, resulting in activated Wnt/β-catenin pathway both in vitro and in vivo. Furthermore, melatonin was shown to ameliorate osteoporosis in OVX mice via the HGF/PTEN/Wnt/β-catenin axis.Conclusion:Melatonin could potentially enhance osteogenic differentiation of BMSCs and retard bone loss through the HGF/PTEN/Wnt/β-catenin axis.

Identification of stemness and differentially expressed genes in human cementum-derived cells.

PurposePeriodontal treatment aims at complete regeneration of the periodontium, and developing strategies for periodontal regeneration requires a deep understanding of the tissues composing the periodontium. In the present study, the stemness characteristics and gene expression profiles of cementum-derived cells (CDCs) were investigated and compared with previously established human stem cells. Candidate marker proteins for CDCs were also explored.MethodsPeriodontal ligament stem cells (PDLSCs), pulp stem cells (PULPSCs), and CDCs were isolated and cultured from extracted human mandibular third molars. Human bone marrow stem cells (BMSCs) were used as a positive control. To identify the stemness of CDCs, cell differentiation (osteogenic, adipogenic, and chondrogenic) and surface antigens were evaluated through flow cytometry. The expression of cementum protein 1 (CEMP1) and cementum attachment protein (CAP) was investigated to explore marker proteins for CDCs through reverse-transcription polymerase chain reaction. To compare the gene expression profiles of the 4 cell types, mRNA and miRNA microarray analysis of 10 samples of BMSCs (n=1), PDLSCs (n=3), PULPSCs (n=3), and CDCs (n=3) were performed.ResultsThe expression of mesenchymal stem cell markers with a concomitant absence of hematopoietic markers was observed in PDLSCs, PULPSCs, CDCs and BMSCs. All 4 cell populations also showed differentiation into osteogenic, adipogenic, and chondrogenic lineages. CEMP1 was strongly expressed in CDCs, while it was weakly detected in the other 3 cell populations. Meanwhile, CAP was not found in any of the 4 cell populations. The mRNA and miRNA microarray analysis showed that 14 mRNA genes and 4 miRNA genes were differentially expressed in CDCs vs. PDLSCs and PULPSCs.ConclusionsWithin the limitations of the study, CDCs seem to have stemness and preferentially express CEMP1. Moreover, there were several up- or down-regulated genes in CDCs vs. PDLSCs, PULPSCs, and BMSCs and these genes could be candidate marker proteins of CDCs.

Regeneration in Experimental Alveolar Bone Defect Using Human Umbilical Cord Mesenchymal Stem Cells

Cleft lip and palate is a congenital disorder including cleft lip, and/or cleft palate, and/or alveolar cleft, with high incidence.The alveolar cleft causes morphological and functional abnormalities. To obtain bone bridge formation and continuous structure between alveolar clefts, surgical interventions are performed from infancy to childhood. However, desirable bone bridge formation is not obtained in many cases. Regenerative medicine using mesenchymal stem cells (MSCs) is expected to be a useful strategy to obtain sufficient bone bridge formation between alveolar clefts. In this study, we examined the effect of human umbilical cord-derived MSCs by transplantation into a rat experimental alveolar cleft model. Human umbilical cords were digested enzymatically and the isolated cells were collected (UC-EZ cells). Next, CD146-positive cells were enriched from UC-EZ cells by magnetic-activated cell sorting (UC-MACS cells). UC-EZ and UC-MACS cells showed MSC gene/protein expression, in vitro. Both cells had multipotency and could differentiate to osteogenic, chondrogenic, and adipogenic lineages under the differentiation-inducing media. However, UC-EZ cells lacked Sox2 expression and showed the lower ratio of MSCs than UC-MACS cells. Thus, UC-MACS cells were transplanted with hydroxyapatite and collagen (HA + Col) into alveolar cleft model to evaluate bone formation in vivo. The results of micro computed tomography and histological staining showed that UC-MACS cells with HA + Col induced more abundant bone formation between the experimental alveolar clefts than HA + Col implantation only. Cells immunopositive for osteopontin were accumulated along the bone surface and some of them were embedded in the bone. Cells immunopositive for human-specific mitochondria were aligned along the newly formed bone surface and in the new bone, suggesting that UC-MACS cells contributed to the bone bridge formation between alveolar clefts. These findings indicate that human umbilical cords are reliable bioresource and UC-MACS cells are useful for the alveolar cleft regeneration.

Sol\u2013gel based synthesis and biological properties of zinc integrated nano bioglass ceramics for bone tissue regeneration

Bone is a flexible and electro active tissue that is vulnerable to various traumatic injuries. The self-healing of damaged bone tissue towards reconstruction is limited due to the lack of proper niche compliances. Nevertheless, the classical grafting techniques like autograft/allograft for bone repair pose challenges like bacterial infections and donor-site morbidity with unsatisfactory outcomes. The use of appropriate biomaterial with osteogenic potential can meet these challenges. In this regard, bioactive glass ceramics is widely used as a bone filler or graft material because of its bonding affinity to bone leading towards bone reconstruction applications without the challenge of post implant infections. Hence, the current study is aimed at addressing this potentiality of zinc (Zn) for doped the bioglass at nano-scale advantages for bone tissue repair. Since, Zn has been demonstrated to have not only antibacterial property but also the stimulatory effect on osteoblasts differentiation, mineralization by enhancing the osteogenic genes expression. In view of these, the present study is focused on sol–gel synthesis and pysico-chemical characterization of Zinc-doped bioglass nanoparticles (Zn-nBGC) and also analyzing its biological implications. The surface morphological and physiochemical characterizations using SEM, EDX, FT-IR and XRD analysis has shown the increased surface area of Zn-nBGC particles providing a great platform for biomolecular interaction, cytocompatibility, cell proliferation and osteogenic differentiation. The obtaining hydroxy apatite groups have initiated in vitro mineralization towards osteogenic lineage formation. Zn has not only involved in enhancing cellular actions but also strengthen the ceramic nanoparticles towards antibacterial application. Hence the finding suggests a biomaterial synthesis of better biomaterial for bone tissue engineering application by preventing post-operative bacterial infection.

Osteogenic Differentiation from Mouse Embryonic Stem Cells.

Embryonic stem cells (ESCs) are a unique model that allows the study of molecular pathways underlying commitment and differentiation. We have studied signaling pathways and their contributions to osteogenic differentiation. In addition to our previously published protocol where we recommended the addition of retinoic acid with later addition of dexamethasone to boost osteogenic lineage cells, here we describe an optimized protocol for osteogenic differentiation from R1 ESCs with suggestions for inhibition of Src activity.

Emerging strategies in reprogramming and enhancing the fate of mesenchymal stem cells for bone and cartilage tissue engineering

Mesenchymal stem cells (MSCs) are used extensively in developing tissue engineered constructs for bone and cartilage regeneration. An important factor in designing such constructs is that the MSCs are appropriately primed to differentiate along osteogenic or chondrogenic lineage. In contrast to a top-down method of tissue engineering where the differentiation of cells is guided by the scaffold and signals, a bottom-up method involves direct modulation of stem cell behavior without relying on the environmental cues. In this review, we discuss several bottom-up strategies that have emerged in engineering MSC behavior for bone and cartilage tissue engineering, including gene delivery, gene editing, and subpopulation isolation.

Gene Profiles in the Early Stage of Neuronal Differentiation of Mouse Bone Marrow Stromal Cells Induced by Basic Fibroblast Growth Factor.

A stably established population of mouse bone marrow stromal cells (BMSCs) with self-renewal and multilineage differentiation potential was expanded in vitro for more than 50 passages. These cells express high levels of mesenchymal stem cell markers and can be differentiated into adipogenic, chondrogenic, and osteogenic lineages in vitro. Subjected to basic fibroblast growth factor (bFGF) treatment, a typical neuronal phenotype was induced in these cells, as supported by neuronal morphology, induction of neuronal markers, and relevant electrophysiological excitability. To identify the genes regulating neuronal differentiation, cDNA microarray analysis was conducted using mRNAs isolated from cells differentiated for different time periods (0, 4, 24, and 72 h) after bFGF treatment. Various expression patterns of neuronal genes were stimulated by bFGF. These gene profiles were shown to be involved in developmental, functional, and structural integration of the nervous system. The expression of representative genes stimulated by bFGF in each group was verified by RT-PCR. Amongst proneural genes, the mammalian achate-schute homolog 1 (Mash-1), a basic helix-loop-helix transcriptional factor, was further demonstrated to be significantly upregulated. Overexpression of Mash-1 in mouse BMSCs was shown to induce the expression of neuronal specific enolase (NSE) and terminal neuronal morphology, suggesting that Mash-1 plays an important role in the induction of neuronal differentiation of mouse BMSCs.

Metformin-Loaded PCL/PVA Fibrous Scaffold Preseeded with Human Endometrial Stem Cells for Effective Guided Bone Regeneration Membranes.

Many studies have been devoted to investigating the potential of guided bone regeneration (GBR) membranes for bone defect reconstruction. Regardless of approaches for treating damaged bone tissues, a beneficial therapeutic strategy has remained a challenge. In this study, a novel GBR membrane with polycaprolactone (PCL) and poly(vinyl alcohol) (PVA) containing different concentrations of metformin (Met) for improving osteogenic properties was developed. The membranes were evaluated for their hydrophilicity, degradation rate, swelling ratio, drug release, mechanical properties, and biological responses. The results showed a significant increase in hydrophilicity, swelling ratio, and degradation rate and no significant changes in mechanical properties of PCL/PVA membranes with Met concentration enhancement. A decrease in cell viability cultured on the surface of the PCL/PVA membrane was seen when the amount of Met was changed from 10 to 15 wt %. The results of the in vitro quantitative real-time polymerase chain reaction (qRT-PCR) also confirmed the higher secretion of osteogenic-related genes in a PCL/PVA/Cell/10 wt % Met scaffold than in the PCL/PVA/Cell sample. Therefore, further in vivo studies were conducted using the electrospun PCL/PVA membrane containing human endometrial stem cells (hEnSCs) and 10% Met. Histopathological and histomorphometric results confirmed that PCL/PVA/hEnSCs/10 wt % Met has excellent potential to differentiate hEnSCs into osteogenic lineages and bone regeneration in calvarial defects of rats. The results of this study confirm the high potential of the PCL/PVA/10 wt % Met fibrous membrane preseeded with hEnSCs in GBR applications.