Oxaloacetate as new inducer for osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells in vitro.

BACKGROUND: Mitochondrial organelles play a crucial role in cellular metabolism so different cell types exhibit diverse metabolic and energy demands. Therefore, alternations in the intracellular distribution, quantity, function, and structure of mitochondria are required for stem cell differentiation. Finding an effective inducer capable of modulating mitochondrial activity is critical for the differentiation of specific stem cells into osteo-like cells for addressing issues related to osteogenic disorders. This study aimed to investigate the effect of oxaloacetate (OAA) on the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs) in vitro. METHODS AND RESULTS: First, the most favorable OAA concentration was measured through MTT assay and subsequently confirmed using acridine orange staining. Human ADSCs were cultured in osteogenic medium supplemented with OAA and analyzed on days 7 and 14 of differentiation. Various assays including alkaline phosphatase assay (ALP), cellular calcium content assay, mineralized matrix staining with alizarin red, catalase (CAT) and superoxide dismutase (SOD) activity, and real-time RT-PCR analysis of three bone-specific markers (ALP, osteocalcin, and collagen type I) were conducted to characterize the differentiated cells. Following viability assessment, OAA at a concentration of 1 µM was considered the optimal dosage for further studies. The results of osteogenic differentiation assays showed that OAA at a concentration of 1 × 10- 6 M significantly increased ALP enzyme activity, mineralization, CAT and SOD activity and the expression of bone-specific genes in differentiated cells compared to control groups in vitro. CONCLUSIONS: In conclusion, the fundings from this study suggest that OAA possesses favorable properties that make it a potential candidate for application in medical bone regeneration.

Carbachol, along with calcium, indicates new strategy in neural differentiation of human adipose tissue-derived mesenchymal stem cells in vitro.

Introduction: Over the past few years, stem cells have represented a promising treatment in neurological disorders due to the well-defined characteristics of their capability to proliferate and differentiate into any cell type, both in vitro and in vivo. Additionally, previous studies have shown that calcium signaling modulates the proliferation and differentiation of neural progenitor cells. The present study investigated the effect of carbachol (CCh), a cholinergic agonist activating acetylcholine receptors, with and without calcium, on the neural differentiation of human adipose tissue-derived mesenchymal stem cells (hADSCs) in neural media, including forskolin and 3-isobutyl-1-methyl-xanthine and retinoic acid. Methods: For this purpose, first, the MTT assay and acridine orange staining were studied to obtain the optimal concentration of CCh. Next, the differentiation tests, such as cellular calcium assay as well as evaluation of qualitative and quantitative expression of neuronal index markers through immunofluorescence staining and gene expression analysis, respectively, were performed on days 7 and 14 of the differentiation period. Results: According to the results, CCh at 1 µM concentration had no cytotoxicity on hADSCs and also induced cell proliferation. Furthermore, CCh with and without calcium increased the expression of neural-specific genes (NSE, MAP2, ß-III-tubulin, and MAPK3) and proteins (γ-enolase, MAP2, and ß-III-tubulin) as well as the amount of calcium in differentiated hADSCs at 7 and 14 days after induction. Conclusions: In conclusion, the findings suggest that CCh acts as an influential therapeutic factor in the field of neural regenerative medicine and research.

Evaluation of osteogenic differentiation of human mesenchymal stem cells (hMSCs) on random and aligned polycaprolactone-polyaniline-gelatin scaffolds.

Introduction: Biocompatible and biodegradable scaffolds have gained tremendous attention because of their potential in tissue engineering. In this study, the aim was to reach a feasible setup from a ternary hybrid of polyaniline (PANI), gelatin (GEL), and polycaprolactone (PCL) to fabricate aligned and random nanofibrous scaffolds by electrospinning for tissue engineering purposes. Methods: Different setups of PANI, PCL, and GEL were electrospun. Then, the best aligned and random scaffolds were chosen. SEM imaging was done to observe nanoscaffolds before and after stem cell differentiation. Mechanical properties of the fibers were tested. Their hydrophilicity was measured using the sessile drop method. SNL Cells were then seeded onto the fiber, and MTT was performed to assess its toxicity. The cells were then differentiated. After osteogenic differentiation, alkaline phosphatase activity, calcium content assay, and alizarin red staining were done to check the validity of osteogenic differentiation. Results: The two chosen scaffolds had an average diameter of 300 ± 50 (random) and 200 ± 50 (aligned). MTT was performed and its results showed that the scaffolds were non-toxic to cells. After stem cell differentiation, alkaline phosphatase activity was performed, confirming differentiation on both types of scaffolds. Calcium content and alizarin red staining also confirmed stem cell differentiation. Morphological analysis showed no difference regarding differentiation on either type of scaffold. However, unlike on the random fibers, cells followed a specific direction and had a parallel-like growth pattern on aligned fibers. Conclusion: All in all, PCL-PANI-GEL fibers showed to be capable candidates for cell attachment and growth. Furthermore, they proved to be of excellent use in bone tissue differentiation.

A new hydrogel with fluorapatite nanoparticles for osteogenic differentiation of human adipose-derived stem cells in tissue engineering field.

Since scaffolds are engineered to support functional tissue formation, their design and materials play an essential role in medical fields by providing different mechanical function. The aim of this study was to investigate the synthesis and structural characterization of collagen-gelatin (COL-GEL) composite scaffolds containing fluorapatite (FA) nanoparticles as well as evaluation of the osteogenic differentiation of human adipose-derived stem cells (hADSCs). First, the composite scaffolds were evaluated using Fourier transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction. The cytotoxicity of scaffolds and various concentrations of FA nanoparticles was studied through MTT assay and acridine orange/ethidium bromide staining. Next, the differentiated hADSCs were analyzed using Alizarin red and von Kossa staining, calcium content assay, alkaline phosphatase (ALP) activity, real-time RT-PCR, and immunocytochemical analyses. According to the characterization analyses, the composite scaffolds were properly integrated. The results also illustrated that COL-GEL composite scaffolds in the presence of FA nanoparticles not only showed no cytotoxicity but also increased ALP activity and calcium deposition as well as the expression of osteogenic genes, including Runx2, Col-I, ALP, and osteocalcin and the synthesis of proteins such as osteocalcin and osteopontin in vitro. The obtained data were confirmed by Alizarin red and von Kossa staining. These results are very promising for further tissue engineering experiments, in which FA nanoparticle incorporation into COL-GEL composite scaffolds is a novel approach that improves the surface COL-GEL composite scaffolds for tissue engineering application in vitro.

Osteogenic differentiation of pre-conditioned bone marrow mesenchymal stem cells with Nisin on modified poly-L-lactic-acid nanofibers.

Introduction: Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are undifferentiated cells with self-renewing ability and multi-lineage differentiation beneficial for regenerative medicine. Nano scaffolds are novel materials employed in bone repair and regeneration. Nisin is a prebiotic that can increase stem cells' lifespan and proliferation. This study attempted to provide a proper strategy for bone marrow mesenchymal stem cells differentiation into the Osteocytes on a Poly-L-lactic-acid (PLLA) scaffold after pretreating with Nisin. Methods: MSC osteogenic differentiation was evaluated by measuring Calcium, Alkaline phosphatase, and quantitative tests such as Real-Time PCR, Acridine Orange, Alizarin Red, Von Kossa, and others. Results: The result of the MTT test showed that the optimal dose of Nisin prebiotic for the MSCs' preconditioning was 200 IU/mL on the 1st, 3rd, and 5th days of culture. Real-time PCR data indicated that the expression rate of ALP, Osteonectin, Osteocalcin, and Collagen I have increased in the presence of Nisin, while the RUNX-2 gene expression has decreased. Furthermore, the results of Alizarin Red and Von Kossa tests, as well as Scanning electron microscopy (SEM), revealed that the cell proliferation in the preconditioned samples with Nisin increased significantly. Conclusions: The study concluded that the cell proliferation and differentiation increased in samples pretreated with Nisin on the PLLA Nano scaffolds.

STAT3-EMT axis in tumors: Modulation of cancer metastasis, stemness and therapy response.

Epithelial-to-mesenchymal transition (EMT) mechanism is responsible for metastasis of tumor cells and their spread to various organs and tissues of body, providing undesirable prognosis. In addition to migration, EMT increases stemness and mediates therapy resistance. Hence, pathways involved in EMT regulation should be highlighted. STAT3 is an oncogenic pathway that can elevate growth rate and migratory ability of cancer cells and induce drug resistance. The inhibition of STAT3 signaling impairs cancer progression and promotes chemotherapy-mediated cell death. Present review focuses on STAT3 and EMT interaction in modulating cancer migration. First of all, STAT3 is an upstream mediator of EMT and is able to induce EMT-mediated metastasis in brain tumors, thoracic cancers and gastrointestinal cancers. Therefore, STAT3 inhibition significantly suppresses cancer metastasis and improves prognosis of patients. EMT regulators such as ZEB1/2 proteins, TGF-ß, Twist, Snail and Slug are affected by STAT3 signaling to stimulate cancer migration and invasion. Different molecular pathways such as miRNAs, lncRNAs and circRNAs modulate STAT3/EMT axis. Furthermore, we discuss how STAT3 and EMT interaction affects therapy response of cancer cells. Finally, we demonstrate targeting STAT3/EMT axis by anti-tumor agents and clinical application of this axis for improving patient prognosis.

Neural Differentiation of Human-Induced Pluripotent Stem Cells (hiPSc) on Surface-Modified Nanofibrous Scaffolds Coated with Platelet-Rich Plasma.

The field of tissue engineering exploits living cells in a variety of ways to restore, maintain, or enhance tissues and organs. Between stem cells, human induced pluripotent stem cells (hiPSCs), are very important due to their wide abilities. Growth factors can support proliferation, differentiation, and migration of hiPSCs. Platelet-rich plasma (PRP) could be used as the source of growth factors for hiPSCs. In the present study, proliferation and neural differentiation of hiPSCs on surface-modified nanofibrous Poly-L-lactic acid (PLLA) coated with platelet-rich plasma was investigated. The results of in vitro analysis showed that on the surface, which was modified nanofibrous scaffolds coated with platelet-rich plasma, significantly enhanced hiPSCs proliferation and neural differentiation were observed. Whereas the MTT ([3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide]) results showed biocompatibility of surface-modified nanofibrous scaffolds coated with platelet-rich plasma and the usage of these modified nanoscaffolds in neural tissue engineering in vivo is promising for the future.

Synthesis of two novel bio-based hydrogels using sodium alginate and chitosan and their proficiency in physical immobilization of enzymes.

Herein, four novel and bio-based hydrogel samples using sodium alginate (SA) and chitosan (CH) grafted with acrylamide (AAm) and glycidyl methacrylate (GMA) and their reinforced nanocomposites with graphene oxide (GO) were synthesized and coded as SA-g-(AAm-co-GMA), CH-g-(AAm-co-GMA), GO/SA-g-(AAm-co-GMA), and GO/CH-g-(AAm-co-GMA), respectively. The morphology, net charge, and water absorption capacity of samples were entirely changed by switching the biopolymer from SA to CH and adding a nano-filler. The proficiencies of hydrogels were compared in the immobilization of a model metagenomic-derived xylanase (PersiXyn9). The best performance was observed for GO/SA-g-poly(AAm-co-GMA) sample indicating better stabilizing electrostatic attractions between PersiXyn9 and reinforced SA-based hydrogel. Compared to the free enzyme, the immobilized PersiXyn9 on reinforced SA-based hydrogel showed a 110.1% increase in the released reducing sugar and almost double relative activity after 180 min storage. While immobilized enzyme on SA-based hydrogel displayed 58.7% activity after twelve reuse cycles, the enzyme on CH-based carrier just retained 8.5% activity after similar runs.

Osteogenic differentiation of human induced pluripotent stem cell in the presence of testosterone and 17 ß-estradiol in vitro.

Recently, numerous scientific approaches have been explored to treat various diseases using stem cells. In 2006, induced pluripotent stem cell (iPSC) were introduced by Takahashi and Yamanaka and showed the potential of self-renewing and differentiation into all types of targeted cells in vitro. In this investigation, we studied the effect of testosterone (T) individually or in the presence of 17 ß-estradiol (E2) on osteogenic differentiation of human iPSC (hiPSC) during 2 wk. The optimal concentrations of sex steroid hormones were examined by MTT assay and acridine orange (AO) staining. The impact of E2 and T either individually or together as a combination was examined by ALP activity; the content of total mineral calcium, by von Kossa and alizarin red staining. Additionally, the expression rate of osteogenic specific markers was studied via real-time RT-PCR and immunocytochemistry analyses at day 14 of differentiation. The obtained results illustrated that the differentiation medium supplemented with T-E2 increased not only the ALP enzyme activity and the content of calcium but also the osteogenic-related gene and protein expressions on the 14th day. Furthermore, the results were confirmed by mineralized matrix staining. In conclusion, these data suggest that T could be used as an effective factor for osteogenic induction of hiPSCs combined with the E2 in bone regeneration.

Anti-hyperglycemic effects of Eryngium billardierei F. Delaroche extract on insulin-resistance HepG2 cells in vitro.

BACKGROUND: Insulin resistance as a major problem is associated with type 2 diabetes mellitus. This study investigated the effect of Eryngium billardierei on insulin-resistance induced HepG2 cells. METHODS AND RESULTS: MTT method was used to evaluate the viability of HepG2 cells treated with various doses of E. billardierei extract. An insulin-resistance model was established in HepG2 cells. Next, MTT assay and Acridine orange staining were performed to investigate the viability of cells in the vicinity of different concentrations of insulin, pioglitazone, and E. billardierei extract in an insulin-resistance media. The glucose uptake test was performed to select the optimal insulin concentration. Expression levels of IR, G6Pase, and PEPCK genes were assessed by real-time RT-PCR. According to obtained data, E. billardierei at concentrations of 0.5 and 1 mg/mL show no toxicity on cells. Furthermore, based on MTT assay and glucose uptake test 10-5 mol/L insulin was chosen as the model group to induce insulin-resistance in HepG2 cells for gene expression analysis. Finally, 1 mg/mL E. billardierei not only induced no cytotoxicity but also showed an increase in the expression of IR as well as a reduction in G6Pase and PEPCK level compared to the control and model groups. CONCLUSIONS: The obtained data indicated that 1 mg/mL E. billardierei might have an anti-insulin resistance effect on insulin-resistance HepG2 cells in vitro and could be a promising candidate with anti-hyperglycemic properties for diabetes treatments.

Enhanced neural differentiation of human-induced pluripotent stem cells on aligned laminin-functionalized polyethersulfone nanofibers; a comparison between aligned and random fibers on neurogenesis.

Despite the numerous attempts in nerve tissue engineering, no ideal strategy has been translated into effective therapy for neuronal regeneration yet. Here, we designed a novel nerve regeneration scaffold combining aligned laminin-immobilized polyethersulfone (PES) nanofibers and human-induced pluripotent stem cells (hiPSCs) for transplantation strategies. Aligned and random PES nanofibers were fabricated by electrospinning method with a diameter of 95-500 nm and were then modified with covalent laminin bounding subsequent to O2 plasma treatment. PES-functionalized fibers found to induce a remarkable higher rate of neuronal genes expression as compared to nontreated group. In addition, hiPSCs cultured on aligned pure fibers exhibited the extension of neurites along with fibers direction and an exponentially elevated expression of neuron specific enolase (early neuroectoderm marker), Tuj-1 (axonal marker), and microtubule-associated protein 2 (dendritic marker) in comparison with random pure fibers. The concomitant of increased hydrophilicity and biocompatibility along with exploiting topographical cues and directional guidance make aligned PES-plasma-laminin a versatile scaffold for adhesion, proliferation, spreading, and differentiation of hiPSCs into nerve cells.

Blockade of TGF-ßR improves the efficacy of doxorubicin by modulating the tumor cell motility and affecting the immune cells in a melanoma model.

TGF-ß contributes to drug resistance and the invasiveness of tumor cells and weakens the anti-tumor immune responses. The present study aimed at examining the efficacy of the combination of SB431542, as a specific inhibitor of TGF-ßR, and doxorubicin in controlling the melanoma tumor in mice. The impact of the combination of the doxorubicin and SB431542 on the cell growth, apoptosis, migration, and invasiveness of B16-F10 cells was examined. Besides, the B16-F10 tumor was induced in C57BL/6 mice, and the effects of the mentioned treatment on the tumor volume, survival, and the exhaustion state of T cells were evaluated. Although the combination of doxorubicin and SB431542 did not exhibit synergism in the inhibition of cell growth and apoptosis induction, it efficiently prohibited the migration and the epithelial to mesenchymal transition of B16-F10 cells, and the combination of doxorubicin and SB431542 caused an increase in mRNA levels of E-cadherin and, on the other hand, led to a decline in the expression of Vimentin. Tumor volume and the survival of tumor-bearing mice were efficiently controlled by the combination therapy. This treatment also eventuated in a decrease in the percentage of PD-L1+, TCD4+, and TCD8+ cells as indicators of exhausted T cells within the spleens of tumor-bearing mice. Blockade of TGF-ßR also propelled the RAW 264.7 cells towards an anti-tumor M1 macrophage phenotype. The inhibition of TGF-ßR demonstrated a potential to increase the efficacy of doxorubicin chemotherapy by the means of affecting cellular motility and restoring the anti-tumor immune responses.

Prospects for Manipulation of Mesenchymal Stem Cells in Tumor Therapy: Anti-Angiogenesis Property on the Spotlight.

The interactions between the tumor microenvironment and the tumor cells confers a condition that accelerate or decelerate the development of tumor. Of these cells, mesenchymal stem cells (MSCs) have the potential to modulate the tumor cells. MSCs have been established with double functions, whereby contribute to a tumorigenic or anti-tumor setting. Clinical studies have indicated the potential of MSCs to be used as tool in treating the human cancer cells. One of the advantageous features of MSCs that make them as a well-suited tool for cancer therapy is the natural tumor-trophic migration potential. A key specification of the tumor development has been stablished to be angiogenesis. As a result, manipulation of angiogenesis has become an attractive approach for cancer therapy. This review article will seek to clarify the anti-angiogenesis strategy in modulating the MSCs to treat the tumor cells.

Donepezil hydrochloride as a novel inducer for osteogenic differentiation of mesenchymal stem cells on PLLA scaffolds in vitro.

Over the past decades, bone defects caused by illness or trauma have been the most common traumatic injuries in humans and treatment of orthopedic infections has always been a serious challenge to experts in the world. In this project, poly L-lactic acid (PLLA) nanofibrous scaffolds were synthesized as a nontoxic, eco-friendly, and cost-effective scaffold by the electrospinning technique. Then, the impact of PLLA on the cell proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) was assayed in the presence and absence of donepezil hydrochloride (DH) which was prescribed in patients with Alzheimer's disease. Also, hMSCs were seeded on PLLA scaffold in the presence (PLLA-DH) and absence of 1 µg mL-1 of DH under osteogenic induction media. Osteogenic differentiation of hMSCs was assessed by specific bone-related tests including alkaline phosphatase (ALP) activity, Alizarin red and von Kossa staining, calcium content assay. Also, Osteocalcin and osteopontin were evaluated as osteogenic proteins as well as ALP, osteonectin, osteocalcin, collagen type I (Col-I) and Runx2 as osteogenic genes via immunocytochemistry (ICC) and Real-time PCR analyses. The obtained data showed the higher ALP enzyme activity and biomineralization, more intensity during von Kossa staining as well as the increase in the expression rate of osteogenic related gene and protein markers in differentiated hMSCs on PLLA-DH. In conclusion, the present study revealed that the combination of PLLA scaffold with DH provides a scope to develop a suitable matrix in bone tissue engineering applications.

Nisin and non-essential amino acids: new perspective in differentiation of neural progenitors from human-induced pluripotent stem cells in vitro.

Over the past decades, stem cell therapy has been investigated as a promising approach towards various diseases, including neurodegenerative disorders. Stem cells show the capability to differentiate into neuronal progenitor cells in vitro. In the present study, the differentiation potential of human-induced pluripotent stem cells (hiPSCs) into neural lineages was examined under the efficient induction media containing forskolin and 3-isobutyl-1-methyl-xanthine (IBMX) in the presence of nisin (Ni), non-essential amino acids (NEAA) and combination of those (NEAA-Ni) in vitro. The optimum concentrations of these factors were obtained by MTT assay and acridine orange (AO) staining. The effect of Ni and NEAA on the expression rate of neural-specific markers including NSE, MAP2, and ß-tubulin III was studied via immunocytochemistry (ICC) and real-time RT-PCR analyses. Our results indicated that the induction medium containing Ni or NEAA increased the gene and protein expression of NSE, MAP2, and ß-tubulin III on the 14th differentiation day. On the other hand, NEAA-Ni showed a less-differentiated hiPSCs compared to Ni and NEAA alone. In conclusion, the obtained results illustrated that Ni and NEAA could be applied as effective factors for neural differentiation of hiPSCs in the future.

The effect of female sex steroid hormones on osteogenic differentiation of endometrial stem cells.

Bone regeneration is a significant and crucial health issue worldwide. Tissue bioengineering has shown itself to be the best substitute for common clinical treatment of bone loss. The suitable cell source is human endometrial stem cells (hEnSCs) which have several suitable characteristics for this approach. Since sex steroid hormones are involved in expansion and conservation of the skeleton, the effect of two sex steroid hormones known as estrogen (17-ß estradiol) and progesterone on osteogenic differentiation of hEnSCs were examined. For this purpose, hEnSCs were treated with 17-ß estradiol and progesterone separately (1 × 10-6 M) and simultaneously (1 × 10-7 M). Osteogenic differentiation tests including measurement of total mineral calcium content, Alizarin Red staining, the quantitative expression levels of some osteogenic markers by Real-time RT-PCR, and immunofluorescence staining were performed at 7 and 14 days of differentiation. To exhibit the morphology of the cells in osteogenic and culture medium, the hEnSCs were stained with Acridine Orange (AO) solution. In this research, MTT assay and AO staining revealed progesterone and 17-ß estradiol increase the proliferation of hEnSCs in a dose-dependent manner. Furthermore, the results of calcium content analysis, Real-time RT-PCR assay, and all tests of differentiation staining have shown that 17-ß estradiol and progesterone cannot induce hEnSCs' osteogenic differentiation. In conclusion, it is indicated that 17-ß estradiol and progesterone do not have positive effects on hEnSCs' osteogenic differentiation in vitro.

A comparative study of hBM-MSCs' differentiation toward osteogenic lineage in the presence of progesterone and estrogen hormones separately and concurrently in vitro.

Promising cell sources for tissue engineering comprise bone marrow derived-mesenchymal stem cells (BM-MSCs) that have multiple differentiation potentials. Also, sex hormones act as important elements in bone development and maintenance, and the roles of two female sex steroid hormones known as estrogen (17-ß estradiol) and progesterone in osteogenic differentiation of human BM-MSCs (hBM-MSCs) are studied. For this purpose, hBM-MSCs were treated with a 1 × 10-6 M concentration of 17-ß estradiol and progesterone separately and simultaneously while the optimum concentrations were obtained by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Osteogenic differentiation tests including measurement of alkaline phosphatase (ALP) enzyme activity, the content of total mineral calcium, mineralized matrix staining by Alizarin Red and Von Kossa solutions, real-time reverse transcription polymerase chain reaction (RT-PCR), and immunofluorescence staining were carried out on Days 7 and 14 of differentiation. To exhibit the morphology of the cells, the BM-MSCs were stained with acridine orange (AO) solution. In this study, the results of ALP activity assay, calcium content and real-time RT-PCR assay and also all tests of differentiation staining have shown that 17-ß estradiol has been recognized as an enhancing factor of osteogenic differentiation. Furthermore, MTT assay and AO staining revealed progesterone as a factor that seriously improved the proliferation of hBM-MSCs. Generally, the 17-ß estradiol individually or in the presence of progesterone has more effects on BM-MSCs' osteogenic differentiation compared to progesterone alone. In this study, it is indicated that the effect of the 17-ß estradiol and progesterone concurrently was the same as individual 17-ß estradiol on the differentiation of hBM-MSCs.

Osteogenic differentiation of hMSCs on semi-interpenetrating polymer networks of polyurethane/poly(2­hydroxyethyl methacrylate)/cellulose nanowhisker scaffolds.

Poly (2­hydroxyethyl methacrylate) (PHEMA) was crosslinked in the presence of biocompatible and biodegradable poly(caprolactone) (PCL) based polyurethanes (PUs) and cellulose nanowhiskers (CNWs). The CNWs were obtained from wastepaper. In order to crosslink PHEMA (10 wt%), a novel acrylic-urethane cross-linker was produced by a condensation reaction of PHEMA and hexamethylene diisocyanate (HDI). The PU-PHEMA-CNWs scaffolds were prepared by solvent casting/particulate leaching method in different weight percentages of CNWs (i.e., 0, 0.1, 0.5, and 1 wt%). The structural, mechanical, and in vitro biological properties of bio-nanocomposites were evaluated via FTIR, SEM, tensile, and MTT assay. The tensile strength of PU-PHEMA-0, PU-PHEMA-0.1, PU-PHEMA-0.5, and PU-PHEMA-1 were 76.2, 95.8, 98.1, and 89.8 kPa, respectively. Incorporation of CNWs also resulted in improved cell proliferation on PU-PHEMA-CNWs scaffolds. The bone marrow derived human mesenchymal stem cells (hMSCs) were seeded on the prepared porous scaffolds and incubated in osteogenic medium. Based on the results including calcium content assay, alkaline phosphatase assay, and mineralization staining, PU-PHEMA-CNW scaffolds were introduced as a suitable election for imitating the behavior of cellular niche. Bone mineralization and osteogenesis differentiation of hMSCs on PU-PHEMA-CNW scaffolds were significantly more than control after 14 days.

A new nanocomposite scaffold based on polyurethane and clay nanoplates for osteogenic differentiation of human mesenchymal stem cells in vitro.

Bone tissue engineering as an alternative strategy, provides a great opportunity for regeneration of large bone tissue lesions. The use of biodegradable porous scaffolds along with stem cells, cytokines and growth factors improves cell survival, adhesion, proliferation and differentiation. In the present study, clay nanoplates (CNPs) were surface-modified (MCNPs) using phosphoric acid and calcium hydroxide, then porous polyurethane (PU) scaffolds and PU-MCNPs nanocomposite scaffolds were synthesized using solvent evaporation-dissolution technique. Physicochemical and morphological properties of scaffolds and MCNPs were studied by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and scanning electron microscopy (SEM). Moreover, thermal behavior of scaffolds was assessed by differential scanning calorimetry (DSC). Degradability, water uptake and mechanical behaviors of scaffolds were evaluated and hydrophilicity properties of them were obtained by contact angle technique. MTT assay and Acridine Orange/Ethidium Bromide (AO/EB) staining were used to assess the biocompatibility of MCNPs and PU scaffolds regarding cell attachment and proliferation support. Osteogenic differentiation of cultured human adipose derived mesenchymal stem cells (hADSCs) on MCNPs, PU and PU-MCNPs scaffolds was evaluated using common osteogenic markers such as alkaline phosphatase (ALP) activity, calcium content assay, Alizarin Red staining, immunocytochemical analysis (ICC) and quantitative real-time PCR (qPCR). According to the results, the surface modification of CNPs and their presence into the PU scaffolds significantly enhanced proliferation and osteogenic differentiation of hADSCs. These results were obtained by higher ALP enzyme activity, biomineralization and expression of osteogenic related proteins and genes in differentiated hADSCs on PU-MCNPs scaffolds. In conclusion, our results revealed that these biocompatible nanocomposites porous scaffolds with proper cell adhesion and proliferation as well as effective osteogenic differentiation and which are able to provide a new and useful matrix for bone tissue engineering purposes.

Composite Nanoscaffolds Modified with Bio-ceramic Nanoparticles (Zn2SiO4) Prompted Osteogenic Differentiation of Human Induced Pluripotent Stem Cells.

Nanofiber scaffolds and bio-ceramic nanoparticles have been widely used in bone tissue engineering. The use of human- induced pluripotent stem cells (hiPSCs) on this scaffold can be considered as a new approach in the differentiation of bone tissue. In the present study, a polyaniline-gelatin-polycaprolactone (PANi-GEL-PCL) composite nanoscaffold was made by electrospinning and modified superficially by plasma method. The synthesized nanoscaffold was then coated with willemite's bio-ceramic nanoparticles (Zn2SiO4). The nanoscaffold's properties were studied by scanning electron microscopy (SEM). Also, nanoparticles characterization was carried out with SEM and dynamic light scattering. The growth and proliferation rate of cells on the synthesized nanoscaffold was examined by MTT assay. Subsequently, hiPSCs were cultured on murine fibroblast cells, incubated in embryoid bodies for 3 days, and placed on the nanoscaffolds. The differentiation potential of hiPSCs was investigated by the examination of common bone markers (e.g. alkaline phosphatase, calcium salt precipitation, and alizarin red test) using bone differentiation factors for 14 days. SEM showed the proper structure of electrospinned nanoscaffolds and coating of nanoparticles on the nanoscaffold surface. The results of MTT assay confirmed the growth and proliferation of cells and the biocompatibility of nanofibers. The results of bone indices also showed that differentiation on the composite nanoscaffold coated with willemite's bio-ceramic nanoparticles dramatically increased in comparison with other groups. Overall, this study demonstrated that PANi-GEL-PCL composite nanoscaffold with willemite's bio-ceramic nanoparticles is a suitable substrate for in vitro growth, proliferation, and differentiation of hiPSCs cells into osteoblasts.