Enhanced growth and myogenic differentiation of spheroid-derived C2C12 cells.

Among many factors of controlling stem cell differentiation, the key transcription factor upregulation via physical force is a good strategy on the lineage-specific differentiation of stem cells. The study aimed to compare growth and myogenic potentials between the parental cells (PCs) and the 1-day-old C2C12 spheroid-derived cells (SDCs) in two-dimensional (2D) and three-dimensional (3D) culture conditions through examination of the cell proliferation and the expression of myogenic genes. The data showed that 1-day-old spheroids had more intense expression of MyoD gene with respect to the PCs. The proliferation of the SDCs is significantly higher than the PCs in a time-dependent manner. The SDCs had also significantly higher myogenic potential than the PCs in 2D and 3D culture conditions. The results suggest that MyoD gene upregulation through cell-cell contacts is the good approach for preparation of seed cells in muscle tissue engineering.

Dynamic cell culture on porous biopolymer microcarriers in a spinner flask for bone tissue engineering: a feasibility study.

Porous microspherical carriers have great promise for cell culture and tissue engineering. Dynamic cultures enable more uniform cell population and effective differentiation than static cultures. Here we applied dynamic spinner flask culture for the loading and multiplication of cells onto porous biopolymer microcarriers. The abilities of the microcarriers to populate cells and to induce osteogenic differentiation were examined and the feasibility of in vivo delivery of the constructs was addressed. Over time, the porous microcarriers enabled cell adhesion and expansion under proper dynamic culture conditions. Osteogenic markers were substantially expressed by the dynamic cell cultures. The cell-cultured microcarriers implanted in the mouse subcutaneous tissue for 4 weeks showed excellent tissue compatibility, with minimal inflammatory signs and significant induction of bone tissues. This first report on dynamic culture of porous biopolymer microcarriers providing an effective tool for bone tissue engineering.

Development of an Injectable Biphasic Hyaluronic Acid-Based Hydrogel With Stress Relaxation Properties for Cartilage Regeneration.

Biomimetic stress-relaxing hydrogels with reversible crosslinks attract significant attention for stem cell tissue regeneration compared with elastic hydrogels. However, stress-relaxing hyaluronic acid (HA)-based hydrogels fabricated using conventional technologies lack stability, biocompatibility, and mechanical tunability. Here, it is aimed to address these challenges by incorporating calcium or phosphate components into the HA backbone, which allows reversible crosslinking of HA with alginate to form interpenetrating networks, offering stability and mechanical tunability for mimicking cartilage. Diverse stress-relaxing hydrogels (τ1/2; SR50, 60-2000 s) are successfully prepared at ≈3 kPa stiffness with self-healing and shear-thinning abilities, favoring hydrogel injection. In vitro cell experiments with RNA sequencing analysis demonstrate that hydrogels tune chondrogenesis in a biphasic manner (hyaline or calcified) depending on the stress-relaxation properties and phosphate components. In vivo studies confirm the potential for biphasic chondrogenesis. These results indicate that the proposed stress-relaxing HA-based hydrogel with biphasic chondrogenesis (hyaline or calcified) is a promising material for cartilage regeneration.

Cooperation between osteoblastic cells and endothelial cells enhances their phenotypic responses and improves osteoblast function.

Osteogenesis requires close co-operation with angiogenesis to create vascularized bone tissue. In this study, an indirect co-culture model using osteoblasts (OBs), primary endothelial cells (ECs) and Matrigel interlayer was established to understand the impact of each cell type on the other. ECs synergistically enhanced osteoblastic gene expression by OBs, while OBs were capable of supporting tubule-like structures formed by ECs on Matrigel, enhancing mean tubule length from 146.5 ± 23.5 µm in ECs alone to 192 ± 28.6 µm in co-culture (p < 0.05). Similar improvements were noted in terms of tubule number. An applicability study of the co-culture model to bone tissue engineering, performed on a biopolymer fibrous membrane, showed substantially enhanced deposition of calcified nodules. These results demonstrate the efficacy of co-culture with ECs to improve osteogenesis for bone tissue engineering.

Novel porous scaffolds of poly(lactic acid) produced by phase-separation using room temperature ionic liquid and the assessments of biocompatibility.

Here we prepared three-dimensional (3D) porous-structured biodegradable polymer scaffolds for tissue regeneration using room temperature ionic liquids (RTILs) as a novel porogen, and addressed their biological properties, including in vitro cell growth and differentiation and in vivo tissue compatibility. RTIL based on 1-butyl-3-methylimidazolium ([bmim]) bearing hydrophilic anion Cl was introduced within the polymer structure to provide a pore network. A mixture of poly(lactic acid) (PLA) with RTIL dissolved in an organic solvent formed a bi-continuous network during the drying process. Selective dissolution of the RTIL phase was facilitated in ethanol, which resulted in a porous network of the polymer phase with complete removal of the RTIL. The RTILs-assisted porous scaffolds showed a typical open-channeled network with pore sizes over 100 µm and porosities of about 86-94%. For the biocompatibility assessments of the scaffolds, mesenchymal stem cells (MSCs) derived from rat bone marrow were seeded onto the PLA scaffold, and the cell proliferation and osteoblastic differentiation behaviors were examined. Results showed a typical on-going increase in the cell population with a level comparable to that observed on the tissue culture plastic control, indicating good cell compatibility. When cultured in an osteogenic medium, the alkaline phosphatase (ALP) activity of the cells on the PLA scaffolds was stimulated to increase with time from 7 to 14 days, in a similar manner to that on the control. Moreover, the expression of genes related to osteoblasts, including collagen type I, osteocalcin and bone sialoprotein, was stimulated on the 3D PLA scaffold during culture for up to 14 days, with levels higher than those on the control, suggesting the developed scaffold provided a 3D matrix condition for osteogenesis. An in vivo pilot study conducted subcutaneously in rat for 4 weeks revealed good tissue compatibility of the scaffold, with the ingrowth of cells and formation of collageneous tissue around and deep within the pores of the scaffold and no significant inflammatory reaction. Taken together, this novel method of using RTILs as a pore generator is considered to be useful in the development of biocompatible porous polymer scaffolds for tissue regeneration.

Performance of evacuated calcium phosphate microcarriers loaded with mesenchymal stem cells within a rat calvarium defect.

Tissue engineering of stem cells in concert with 3-dimensional (3D) scaffolds is a promising approach for regeneration of bone tissues. Bioactive ceramic microspheres are considered effective 3D stem cell carriers for bone tissue engineering. Here we used evacuated calcium phosphate (CaP) microspheres as the carrier of mesenchymal stem cells (MSCs) derived from rat bone marrow. The performance of the CaP-MSCs construct in bone formation within a rat calvarium defect was evaluated. MSCs were first cultured in combination with the evacuated microcarriers for 7 days in an osteogenic medium, which was then implanted in the 6 mm-diameter calvarium defect for 12 weeks. For comparison purposes, a control defect and cell-free CaP microspheres were also evaluated. The osteogenic differentiation of MSCs cultivated in the evacuated CaP microcarriers was confirmed by alkaline phosphatase staining and real time polymerase chain reaction. The in vivo results confirmed the highest bone formation was attained in the CaP microcarriers combined with MSCs, based on microcomputed tomography and histological assays. The results suggest that evacuated CaP microspheres have the potential to be useful as stem cell carriers for bone tissue engineering.

Effect of carbon nanotube coating of aligned nanofibrous polymer scaffolds on the neurite outgrowth of PC-12 cells.

Neurite outgrowth from endogenous or transplanted cells is important for neural regeneration following nerve tissue injury. Modified substrates often provide better environments for cell adhesion and neurite outgrowth. This study was conducted to determine if MWCNT (multiwalled carbon nanotube)-coated electrospun PLCL [poly (l-lactic acid-co-3-caprolactone)] nanofibres improved the neurite outgrowth of PC-12 cells. To accomplish this, two groups, PC-12 cells in either uncoated PLCL scaffolds or MWCNT-coated PLCL scaffolds were cultured for 9 days. MWCNT-coated PLCL scaffolds showed improved adhesion, proliferation and neurite outgrowth of PC-12 cells. These findings suggest that MWCNT-coated nanofibrous scaffolds may be an attractive platform for cell transplantation application in neural tissue engineering.

Research Models of the Nanoparticle-Mediated Drug Delivery across the Blood-Brain Barrier.

Brain diseases and damages come in many forms such as neurodegenerative diseases, tumors, and stroke. Millions of people currently suffer from neurological diseases worldwide. While Challenges of current diagnosis and treatment for neurological diseases are the drug delivery to the central nervous system. The Blood-Brain Barrier (BBB) limits the drug from reaching the targeted site thus showing poor effects. Nanoparticles that have advantage of the assembly at the nanoscale of available biomaterials can provide a delivery platform with potential to raising brain levels of either imaging therapeutic drugs or imaging. Therefore, successful modeling of the BBB is another crucial factor for the development of nanodrugs. In this review, we analyze the in vitro and in vivo findings achieved in various models, and outlook future development of nanodrugs for the successful treatment of brain diseases and damages.

Therapeutic tissue regenerative nanohybrids self-assembled from bioactive inorganic core / chitosan shell nanounits.

Natural inorganic/organic nanohybrids are a fascinating model in biomaterials design due to their ultra-microstructure and extraordinary properties. Here, we report unique-structured nanohybrids through self-assembly of biomedical inorganic/organic nanounits, composed of bioactive inorganic nanoparticle core (hydroxyapatite, bioactive glass, or mesoporous silica) and chitosan shell - namely Chit@IOC. The inorganic core thin-shelled with chitosan could constitute as high as 90%, strikingly contrasted with the conventional composites. The Chit@IOC nanohybrids were highly resilient under cyclic load and resisted external stress almost an order of magnitude effectively than the conventional composites. The nanohybrids, with the nano-roughened surface topography, could accelerate the cellular responses through stimulated integrin-mediated focal adhesions. The nanohybrids were also able to load multiple therapeutic molecules in the core and shell compartment and then release sequentially, demonstrating controlled delivery systems. The nanohybrids compartmentally-loaded with therapeutic molecules (dexamethasone, fibroblast growth factor 2, and phenamil) were shown to stimulate the anti-inflammatory, pro-angiogenic and osteogenic events of relevant cells. When implanted in the in vivo calvarium defect model with 3D-printed scaffold forms, the therapeutic nanohybrids were proven to accelerate new bone formation. Overall, the nanohybrids self-assembled from Chit@IOC nanounits, with their unique properties (ultrahigh inorganic content, nano-topography, high resilience, multiple-therapeutics delivery, and cellular activation), can be considered as promising 3D tissue regenerative platforms.

Preventive role of PD-1 on MPTP-induced dopamine depletion in mice.

Many current studies of Parkinson's disease (PD) suggest that inflammation is involved in the neurodegenerative process. PD-1, a traditional Korean medicine, used to treat various brain diseases in Korea. This study was designed to investigate the effect of PD-1 extract in the Parkinson's model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesioned mice. The MPTP administration caused the dopamine neuron loss in the striatum and substantia nigra pars compacta (SNpc), which was demonstrated by a depletion of tyrosine hydroxylase (TH). In addition, a reduction of bcl-2 expression with elevation of bax expression, caspase-3 activation, and release of cytochrome c into cytosol in dopaminergic neurons of SNpc were noted. Oral administration of PD-1 extract (50 and 100 mg kg(-1)) attenuated the MPTP-induced depletion of TH proteins in the striatum and SNpc and prevented the apoptotic effects. These results indicate that PD-1 extract is able to protect dopaminergic neurons from MPTP-induced neuronal death, with important implications for the treatment of PD.

Current Nanoparticle-Based Technologies for Osteoarthritis Therapy.

Osteoarthritis (OA) is a common chronic joint disease that is characterized by joint pain and stiffness, and limitation of motion and the major cause of disability, which reduces life quality of patients and brings a large economic burden to the family and society. Current clinical treatment is mostly limited to symptomatic treatment aimed at pain alleviation and functional improvement, rather than suppressing the progression of OA. Nanotechnology is a promising strategy for the treatment of OA. In this review, we summarize the current experimental progress that focuses on technologies such as liposomes, micelles, dendrimers, polymeric nanoparticles (PNPs), exosomes, and inorganic nanoparticles (NPs) for their potential treatment of OA.

Targeting with nanoparticles for the therapeutic treatment of brain diseases.

Brain diseases including neurodegenerative disorders and tumours are among the most serious health problems, degrading the quality of life and causing massive economic cost. Nanoparticles that load and deliver drugs and genes have been intensively studied for the treatment of brain diseases, and have demonstrated some biological effects in various animal models. Among other efforts taken in the nanoparticle development, targeting of blood brain barrier, specific cell type or local intra-/extra-cellular space is an important strategy to enhance the therapeutic efficacy of the nanoparticle delivery systems. This review underlies the targeting issue in the nanoparticle development for the treatment of brain diseases, taking key exemplar studies carried out in various in vivo models.

3D culture technologies of cancer stem cells: promising ex vivo tumor models.

Cancer stem cells have been shown to be important in tumorigenesis processes, such as tumor growth, metastasis, and recurrence. As such, many three-dimensional models have been developed to establish an ex vivo microenvironment that cancer stem cells experience under in vivo conditions. Cancer stem cells propagating in three-dimensional culture systems show physiologically related signaling pathway profiles, gene expression, cell-matrix and cell-cell interactions, and drug resistance that reflect at least some of the tumor properties seen in vivo. Herein, we discussed the presently available Cancer stem cell three-dimensional culture models that use biomaterials and engineering tools and the biological implications of these models compared to the conventional ones.

Nano-graphene oxide/polyurethane nanofibers: mechanically flexible and myogenic stimulating matrix for skeletal tissue engineering.

For skeletal muscle engineering, scaffolds that can stimulate myogenic differentiation of cells while possessing suitable mechanical properties (e.g. flexibility) are required. In particular, the elastic property of scaffolds is of importance which helps to resist and support the dynamic conditions of muscle tissue environment. Here, we developed highly flexible nanocomposite nanofibrous scaffolds made of polycarbonate diol and isosorbide-based polyurethane and hydrophilic nano-graphene oxide added at concentrations up to 8%. The nano-graphene oxide incorporation increased the hydrophilicity, elasticity, and stress relaxation capacity of the polyurethane-derived nanofibrous scaffolds. When cultured with C2C12 cells, the polyurethane-nano-graphene oxide nanofibers enhanced the initial adhesion and spreading of cells and further the proliferation. Furthermore, the polyurethane-nano-graphene oxide scaffolds significantly up-regulated the myogenic mRNA levels and myosin heavy chain expression. Of note, the cells on the flexible polyurethane-nano-graphene oxide nanofibrous scaffolds could be mechanically stretched to experience dynamic tensional force. Under the dynamic force condition, the cells expressed significantly higher myogenic differentiation markers at both gene and protein levels and exhibited more aligned myotubular formation. The currently developed polyurethane-nano-graphene oxide nanofibrous scaffolds, due to their nanofibrous morphology and high mechanical flexibility, along with the stimulating capacity for myogenic differentiation, are considered to be a potential matrix for future skeletal muscle engineering.

Neuroprotective effect of methanol extract of Phellodendri Cortex against 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis in PC-12 cells.

Phellodendri Cortex (PC) is a traditional herbal medicine, widely used in Korea and China. The effects of the methanol extract of Phellodendri Cortex (PC extract) on 1-methyl-4-phenylpyridinium (MPP(+))-induced neuronal apoptosis in PC-12 cells have been investigated. MPP(+)-induced apoptosis in PC-12 cells was accompanied by an increased bax/bcl-2 ratio, release of cytochrome c to the cytosol and activation of caspase-3. PC extract inhibited the downregulation of bcl-2 and the upregulation of bax, as well as the release of mitochondrial cytochrome c into the cytosol. In addition, PC extract attenuated caspase-3 activation and cleavage of poly (ADP-ribose) polymerase (PARP). These results suggest that the PC extract has protective effects against MPP(+)-induced neuronal apoptosis in PC-12 cells.

Intrastriatal grafts of mesenchymal stem cells in adult intact rats can elevate tyrosine hydroxylase expression and dopamine levels.

MSCs (mesenchymal stem cells) derived from the bone marrow have shown to be a promising source of stem cells in a therapeutic strategy of neurodegenerative disorder. Also, MSCs can enhance the TH (tyrosine hydroxylase) expression and DA (dopamine) content in catecholaminergic cells by in vitro co-culture system. In the present study, we investigated the effect of intrastriatal grafts of MSCs on TH protein and gene levels and DA content in adult intact rats. When MSCs were transplanted into the striatum of normal rats, the grafted striatum not only had significantly higher TH protein and mRNA levels, but also significantly higher DA content than the untransplanted striatum. Meanwhile, the grafted MSCs differentiated into neurons, astrocytes and oligodendrocytes; however, TH-positive cells could not be detected in our study. These experimental results offer further evidence that MSCs are a promising candidate for treating neurodegenerative diseases such as Parkinson's disease.

Differential chondro- and osteo-stimulation in three-dimensional porous scaffolds with different topological surfaces provides a design strategy for biphasic osteochondral engineering.

Bone/cartilage interfacial tissue engineering needs to satisfy the differential properties and architectures of the osteochondral region. Therefore, biphasic or multiphasic scaffolds that aim to mimic the gradient hierarchy are widely used. Here, we find that two differently structured (topographically) three-dimensional scaffolds, namely, "dense" and "nanofibrous" surfaces, show differential stimulation in osteo- and chondro-responses of cells. While the nanofibrous scaffolds accelerate the osteogenesis of mesenchymal stem cells, the dense scaffolds are better in preserving the phenotypes of chondrocytes. Two types of porous scaffolds, generated by a salt-leaching method combined with a phase-separation process using the poly(lactic acid) composition, had a similar level of porosity (~90%) and pore size (~150 µm). The major difference in the surface nanostructure led to substantial changes in the surface area and water hydrophilicity (nanofibrous ≫ dense); as a result, the nanofibrous scaffolds increased the cell-to-matrix adhesion of mesenchymal stem cells significantly while decreasing the cell-to-cell contracts. Importantly, the chondrocytes, when cultured on nanofibrous scaffolds, were prone to lose their phenotype, including reduced chondrogenic expressions (SOX-9, collagen type II, and Aggrecan) and glycosaminoglycan content, which was ascribed to the enhanced cell-matrix adhesion with reduced cell-cell contacts. On the contrary, the osteogenesis of mesenchymal stem cells was significantly accelerated by the improved cell-to-matrix adhesion, as evidenced in the enhanced osteogenic expressions (RUNX2, bone sialoprotein, and osteopontin) and cellular mineralization. Based on these findings, we consider that the dense scaffold is preferentially used for the chondral-part, whereas the nanofibrous structure is suitable for osteo-part, to provide an optimal biphasic matrix environment for osteochondral tissue engineering.

Rat mesenchymal stem cells increase tyrosine hydroxylase expression and dopamine content in ventral mesencephalic cells in vitro.

Mesenchymal stem cells (MSCs) are pluripotent adult stem cells. It has been shown that MSCs secrete neurotrophic factors involving nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Also, these neurotrophic factors can upregulate tyrosine hydroxylase (TH) gene expression in PC12 cells and neural stem cells. Here, we investigated the effect of co-culturing rat E13.5 ventral mesencephalic cells (VMCs) with MSCs from rat bone marrow on TH expression and dopamine (DA) content. The study consisted of 3 groups: MSC, VMC and a combined MSC+VMC group. All groups were cultured in serum-free neuro-basal medium for 3 days. Thereafter, each group was analyzed by RT-PCR, western blotting, and HPLC. The co-culture group showed a higher expression at TH and DA than the VMC group. However, TH and DA were not present in the MSC group. These observations suggest that MSCs could be an alternative source for treating neurodegenerative diseases such as Parkinson's disease (PD).

Effect of the co-administration of vitamin C and vitamin E on tyrosine hydroxylase and Nurr1 expression in the prenatal rat ventral mesencephalon.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme of dopamine (DA) biosynthesis, which is up-regulated by vitamin C administration. Nurr1 gene is highly expressed in brain and important for midbrain DAergic cell development and survival. But, the role of vitamin C and/or vitamin E during Nurr1 expression is yet to be known. Further, the synergistic effect of vitamins C and E on TH expression has not yet been explored clinically. Therefore, we studied the effects of single or co- administration of vitamin C (0.5 mM) and vitamin E (1 mM) for 72 hr, on both TH and Nurr1 expression in in vitro primary cultured gestational days (GD) 13.5 rat ventral mesencephalon (VM) by Western blot and immunocytochemistry. Our study revealed highest expressions of both TH and Nurr1 in the vitamin C + vitamin E treated group. TH expression was also increased in the vitamin C treated group than that of the control group, but the vitamin E treated group did not show any statistically significant effect. However, both the vitamin C and the vitamin E treated groups revealed increased expression of Nurr1 protein as compared with the control group. The present experimental data suggest that vitamin C can up-regulate the protein expression of TH, but Nurr1 level was elevated either by vitamin C or by vitamin E administration. Further, vitamin E acts synergistically with vitamin C to elevate TH and Nurr1 expression, which is the most novel finding of our study and for the first time; we reported this result, since there have been no published data on the synergistic effect of both the antioxidant vitamins on TH and Nurr1 expression in VM. As the motor function defect due to the progressive loss of DAergic neuron is the major reason of Parkinsonism, therefore, the results of our study finally suggest the effective role of vitamin C and vitamin E during early treatment of Parkinsonism.

Generation of neuronal-like cells from umbilical cord blood-derived mesenchymal stem cells of a RFP-transgenic cloned cat.

Umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) are multipotent adult stem cells, which can differentiation into cells of connective tissue and neural lineages. This study investigated the potential for neuronal differentiation of red fluorescent protein (RFP)-transgenic cat UCB-derived MSCs. The cells were cultured in pre-induction medium for 24 hr and in neuronal-induction medium for 72 hr. Immunofluorescent staining showed that 6.85% of the total cells were beta III-tubulin-positive, 3.37% were neurofilament light (NF-L)-positive and 7.04% were neurofilament medium (NF-M)-positive. A beta III-tubulin band was detected by western blot analysis. Our results demonstrate that RFP-transgenic UCB-derived MSCs can be differentiated into neuronal cells in vitro. Thus, RFP-transgenic MSCs could provide alternative tracing material for stem cell transplantation.