Promotion of Bone Formation in a Rat Osteoporotic Vertebral Body Defect Model via Suppression of Osteoclastogenesis by Ectopic Embryonic Calvaria Derived Mesenchymal Stem Cells.

Osteoporotic vertebral compression fractures (OVCFs) are the most prevalent fractures among patients with osteoporosis, leading to severe pain, deformities, and even death. This study explored the use of ectopic embryonic calvaria derived mesenchymal stem cells (EE-cMSCs), which are known for their superior differentiation and proliferation capabilities, as a potential treatment for bone regeneration in OVCFs. We evaluated the impact of EE-cMSCs on osteoclastogenesis in a RAW264.7 cell environment, which was induced by the receptor activator of nuclear factor kappa-beta ligand (RANKL), using cytochemical staining and quantitative real-time PCR. The osteogenic potential of EE-cMSCs was evaluated under various hydrogel conditions. An osteoporotic vertebral body bone defect model was established by inducing osteoporosis in rats through bilateral ovariectomy and creating defects in their coccygeal vertebral bodies. The effects of EE-cMSCs were examined using micro-computed tomography (µCT) and histology, including immunohistochemical analyses. In vitro, EE-cMSCs inhibited osteoclast differentiation and promoted osteogenesis in a 3D cell culture environment using fibrin hydrogel. Moreover, µCT and histological staining demonstrated increased new bone formation in the group treated with EE-cMSCs and fibrin. Immunostaining showed reduced osteoclast activity and bone resorption, alongside increased angiogenesis. Thus, EE-cMSCs can effectively promote bone regeneration and may represent a promising therapeutic approach for treating OVCFs.

Safety and Feasibility of Intradiscal Administration of Matrilin-3-Primed Adipose-Derived Mesenchymal Stromal Cell Spheroids for Chronic Discogenic Low Back Pain: Phase 1 Clinical Trial.

Functionally enhanced mesenchymal stromal cells participate in the repair of intervertebral disc. This study aimed to assess the safety and tolerability of intradiscal administration of matrilin-3-primed adipose-derived stromal cell (ASC) spheroids with hyaluronic acid (HA) in patients with chronic discogenic low back pain (LBP). In this single-arm, open-label phase I clinical trial, eight patients with chronic discogenic LBP were observed over 6 months. Each patient underwent a one-time intradiscal injection of 1 mL of 6.0 × 106 cells/disc combined with HA under real-time fluoroscopic guidance. Safety and feasibility were gauged using Visual Analogue Scale (VAS) pain and Oswestry Disability Index (ODI) scores and magnetic resonance imaging. All participants remained in the trial, with no reported adverse events linked to the procedure or stem cells. A successful outcome-marked by a minimum 2-point improvement in the VAS pain score and a 10-point improvement in ODI score from the start were observed in six participants. Although the modified Pfirrmann grade remained consistent across all participants, radiological improvements were evident in four patients. Specifically, two patients exhibited reduced high-intensity zones while another two demonstrated decreased disc protrusion. In conclusion, the intradiscal application of matrilin-3-primed ASC spheroids with HA is a safe and feasible treatment option for chronic discogenic LBP.

Molecular Detection of Parvovirus in Manchurian Chipmunks (Tamias sibiricus asiaticus) Captured in Korea.

Cross-species transmission of viral diseases alarms our global community for its potential of novel pandemic events. Of various viral pathogens noted recently, parvoviruses have posed public health threats not only to humans but also to wild animals. To investigate the prevalence of parvoviruses in wild Manchurian chipmunks, here we detected genetic fragments of the nonstructural protein of parvovirus by polymerase chain reaction in wild Manchurian chipmunk specimens captured in the central and southern regions of South Korea and compared their sequence homology with references. Of a total of 348 specimens examined, chipmunk parvovirus (ChpPV)-specific gene fragments were detected with a 31.32% rate (109 chipmunks of 348) in their kidney, liver, lung, and spleen samples, and the chipmunks captured in Gangwon Province exhibited the highest positive rate (45.37%), followed by Gyeongsang (35.29%), Gyeonggi (31.03%), Chungcheong (20.00%), and Jeolla (19.70%). When compared with the reference sequences, a partial ChpPV sequence showed 97.70% identity to the previously reported Korean strain at the nucleic acid level. In the phylogenetic analysis, ChpPV exhibited closer relationship to primate parvoviruses, erythroviruses, and bovine parvovirus than to adeno-associated viruses. Despite limited sample size and genetic sequences examined in this study, our results underline the prevalence of ChpPV in Korea and emphasize the need of close surveillance of parvoviruses in wild animals.

A Synthetic Cell-Penetrating Heparin-Binding Peptide Derived from BMP4 with Anti-Inflammatory and Chondrogenic Functions for the Treatment of Arthritis.

We report dual therapeutic effects of a synthetic heparin-binding peptide (HBP) corresponding to residues 15-24 of the heparin binding site in BMP4 in a collagen-induced rheumatic arthritis model (CIA) for the first time. The cell penetrating capacity of HBP led to improved cartilage recovery and anti-inflammatory effects via down-regulation of the iNOS-IFNγ-IL6 signaling pathway in inflamed RAW264.7 cells. Both arthritis and paw swelling scores were significantly improved following HBP injection into CIA model mice. Anti-rheumatic effects were accelerated upon combined treatment with Enbrel® and HBP. Serum IFNγ and IL6 concentrations were markedly reduced following intraperitoneal HBP injection in CIA mice. The anti-rheumatic effects of HBP in mice were similar to those of Enbrel®. Furthermore, the combination of Enbrel® and HBP induced similar anti-rheumatic and anti-inflammatory effects as Enbrel®. We further investigated the effect of HBP on damaged chondrocytes in CIA mice. Regenerative capacity of HBP was confirmed based on increased expression of chondrocyte biomarker genes, including aggrecan, collagen type II and TNFα, in adult human knee chondrocytes. These findings collectively support the utility of our cell-permeable bifunctional HBP with anti-inflammatory and chondrogenic properties as a potential source of therapeutic agents for degenerative inflammatory diseases.

A national cross-sectional study for poliovirus seroprevalence in the Republic of Korea in 2012: implication for deficiency in immunity to polio among middle-aged people.

BACKGROUND: A worldwide poliomyelitis eradication program was initiated in 1988; however, strains of wild poliovirus (WPV) are still endemic in some countries. Until WPV transmission is eradicated globally, importation and outbreaks of WPV are alarming possibilities. This study is the first report to document the polio immunity after 2004, when an inactivated polio vaccine (IPV) was introduced in the Republic of Korea. METHODS: A total of 745 serum samples from randomly selected patients ranging from 6 to 84 years of age were used for neutralization tests, performed in the World Health Organization polio national reference laboratory. RESULTS: Among the 745 tested sera, 439 (58.9%) were seropositive and 19 (2.6%) were seronegative to all PV serotypes. In all age groups, PV3 showed the lowest level of seroprevalence, at 509 cases (68.3%), compared to 616 (82.7%) for PV1 and 685 (91.9%) for PV2. In the 6-10-year age group, which included IPV-immunized children, the highest seropositive rate was observed and the difference in seroprevalence between PV3 and other serotypes was the lowest compared to the other age groups immunized with oral PV vaccines (OPV). In addition, the seronegative rates of all three PV types in children aged 6-10 in this study were found to be lower than those in OPV-immunized children reported in a previous study from the Republic of Korea. Meanwhile, middle-aged subjects (41-60 years) had the lowest seroprevalence and geometric mean titer. CONCLUSIONS: This study indicates a deficiency in immunity to PV in middle-aged individuals, and low seroprevalence to PV3 in all age groups. In addition, due to the ongoing risk of importing PV, middle-aged people should consider PV vaccination before visiting a PV-endemic country. Our findings provide data to assist those involved in deciding future national polio vaccination strategies for the maintenance of a polio-free status in Korea.

Engineered synthetic cell penetrating peptide with intracellular anti-inflammatory bioactivity: An in vitro and in vivo study.

Various biomaterials have been used for bone and cartilage regeneration, and inflammation associated with biomaterial implantation is also increased. A 15-mer synthetic anti-inflammatory peptide (SAP15) was designed from human ß-defensin 3 to penetrate cells and induce intracellular downregulation of inflammation. The downregulation of inflammation was achieved by the binding of SAP15 to intracellular histone deacetylase (HDAC5). SAP15-mediated inhibition of inflammation was examined in vitro and in vivo using murine macrophages, human articular chondrocytes, and a collagen-induced arthritis (CIA) rat model. Surface plasmon resonance and immunoprecipitation assays indicated that SAP15 binds to HDAC5. SAP15 inhibited the lipopolysaccharide (LPS)-induced phosphorylation of intracellular HDAC5 and NF-κB p65 in murine macrophages. SAP15 treatment increased aggrecan and type II collagen expression and decreased osteocalcin expression in LPS-induced chondrocytes. Subcutaneous injection of SAP15-loaded sodium hyaluronic acid (HA) solution significantly decreased hind paw swelling, joint inflammation, and serum cytokine levels in CIA rats compared with the effects of sodium HA solution alone. The SAP15-loaded HA group exhibited preservation of cartilage and bone structure in CIA rat joints. Moreover, a more robust anti-inflammatory effect of the SAP15 loaded HA was observed than that of etanercept (an anti-tumor necrosis factor-alpha [TNF-α] antibody)-loaded HA. These findings suggest that SAP15 has an anti-inflammatory effect that is not controlled by sodium HA and is mediated by inhibiting HDAC5, unlike the anti-inflammatory mechanism of etanercept. These results demonstrate that SAP15 is useful as an inflammatory regulator of biomaterials and can be developed as a therapeutic for the treatment of inflammation.

Late endothelial progenitor cell-capture stents with CD146 antibody and nanostructure reduce in-stent restenosis and thrombosis.

The restoration of damaged endothelium is promising to reduce side effects, including restenosis and thrombosis, in the stent treatment for vascular diseases. Current technologies based on drug delivery for these complications still do not satisfy patients due to invariant recurrence rate. Recently, even if one approach was applied to clinical trial to develop the firstly commercialized stent employing circulating endothelial progenitor cells (EPCs) in blood vessels, it resulted in failure in clinical trial. Based on instruction of the failed case, we designed an advanced EPC-capture stent covered with anti-CD146 antibody (Ab) immobilized silicone nanofilament (SiNf) for the highly efficient and specific capture of not early but late stage of EPCs. In vitro cell capture test demonstrates enhanced capture efficiency and adhesion morphology of late EPCs on the modified substrate. The modified substrates could capture 8 times more late EPCs and even 3 times more mesenchymal stem cells (MSCs) as compared to unmodified one. A porcine model with high similarity to human reproduced in vivo results ideally translated from in vitro cell capture results. As restenosis indicators, lumen area, neointimal rate and stenosis area for modified stents were reduced at the range of 30-60% as compared to those for bare metal stent (BMS). Fibrin score indicating thrombosis was lowered less than half as comparing to that on BMS. These inspiring results are attributed to ~2-fold increased endothelial coverage, determined by immuno-histological staining. Taken together, the CD146 Ab-armed nanofilamentous stent could show great performance in the reduction of thrombosis and restenosis through re-endothelialization due to highly efficient specific cell capture. STATEMENT OF SIGNIFICANCE: Stents have been developed from simple metal stents to functionalized stents for past decades. However, they have still risks to relapse the occlusion in stented arteries. In this paper, we describe the fabrication and optimization of cell capturing stents to maximize the effective re-endothelialization through the serial coating of silicone nanofilaments and anti-CD146 antibody. The nanofilaments increase the amount of coated antibodies and provide the anchoring points of circulating angiogenic cells for strong focal adhesion. We demonstrate high immobilizing ability of circulating angiogenic cells (endotheliali progenitor cells and mesenchymal stem cells) in vitro under similar shear stress to coronary arteries (15 dyne/cm2). Also, we show accelerating re-endothelialization and the efficient prevention of restenosis in porcine coronary arteries in vivo.

Identification of cell-penetrating osteogenic peptide from copine-7 protein and its delivery system for enhanced bone formation.

Peptide and proteins are recognized as highly selective and therapeutically active biomaterials, as well as relatively safe in clinical application. A calcium phospholipid-binding protein, copine 7 (CPNE7), has been recently identified to induce hard tissue regeneration, including bone and dentin by internalizing into the cells. However, the clinical application of the full length of CPNE7 has limited due to its large size with short half-life. Herein, as an alternative to CPNE7, six bioactive synthetic peptides are designed from CPNE7 (CPNE7-derived peptides, CDP1-CDP6) and investigated their osteogenic potential. Among the CDPs, CDP4 have the highest level of cell-penetrating activity as well as osteogenic efficiency in dental pulp stem cells (DPSCs). CDP4 increased the expression of osteogenesis-related genes and proteins, which was comparable to that by BMP-2. The cell penetration capacity of CDP4 may synergistically induce the osteogenic potential of DPSCs. Moreover, the implantation of the mixture of CDP4 with injectable collagen gel increased bone formation with recovery in the mouse calvarial defect model, comparable to full-length CPNE7 and even BMP-2. In conclusion, these results suggest that our synthetic peptide, CDP4, can be applied in combination with biomaterial to provide high osteogenic efficacy in the field of bone tissue engineering.

Versatile effects of magnesium hydroxide nanoparticles in PLGA scaffold-mediated chondrogenesis.

Artificial scaffolds made up of various synthetic biodegradable polymers have been reported to have many advantages including cheap manufacturing, easy scale up, high mechanical strength, convenient manipulation, and molding into an unlimited variety of shapes. However, the synthetic biodegradable polymers still have the insufficiency for cartilage regeneration owing to their acidic degradation products. To reduce acidification by degradation of synthetic polymers, we incorporated magnesium hydroxide (MH) nanoparticles into porous polymer scaffold not only to effectively neutralize the acidic hydrolysate but also to minimize the structural disturbance of scaffolds. The neutralization effect of poly(D,L-lactic-co-glycolic acid; PLGA)/MH scaffold was confirmed with the maintenance of neutral pH, contrary to a PLGA scaffold with low pH. Further, the scaffolds were applied to evaluate the chondrogenic differentiation of the human bone marrow mesenchymal stem cells. In in vitro study, the PLGA/MH scaffold enhanced the chondrogenesis markers and reduced the calcification, compared to the PLGA scaffold. Additionally, the PLGA/MH scaffold reduced the release of inflammatory cytokines, compared to the PLGA scaffold, as the cell death decreased. Moreover, the addition of MH reduced necrotic cell death at the early stage of chondrogenic differentiation. Further, the necrotic cell death by the PLGA scaffold was mediated by cleavage of caspase-1, the so-called interleukin 1-converting enzyme, and MH alleviated it as well as nuclear factor kappa B expression. Furthermore, the PLGA/MH scaffold highly supported chondrogenic healing of rat osteochondral defect sites in in vivo study. Therefore, it was suggested that a synthetic polymer scaffold containing MH could be a novel healing tool to support cartilage regeneration and further treatment of orthopedic patients. STATEMENT OF SIGNIFICANCE: Synthetic polymer scaffolds have been widely utilized for tissue regeneration. However, they have a disadvantage of releasing acidic products through degradation. This paper demonstrated a novel type of synthetic polymer scaffold with pH-neutralizing ceramic nanoparticles composed of magnesium hydroxide for cartilage regeneration. This polymer showed pH-neutralization property during polymer degradation and significant enhancement of chondrogenic differentiation of mesenchymal stem cells. It reduced not only chondrogenic calcification but also release of proinflammatory cytokines. Moreover, it has an inhibitory effect on necrotic cell death, particularly caspase-1-mediated necrotic cell death (pyroptosis). In in vivo study, it showed higher healing rate of the damaged cartilage in a rat osteochondral defect model. We expected that this novel type of scaffold can be effectively applied to support cartilage regeneration and further treatment of orthopedic patients.

Modified Magnesium Hydroxide Nanoparticles Inhibit the Inflammatory Response to Biodegradable Poly(lactide- co-glycolide) Implants.

Biodegradable polymers have been extensively used in biomedical applications, ranging from regenerative medicine to medical devices. However, the acidic byproducts resulting from degradation can generate vigorous inflammatory reactions, often leading to clinical failure. We present an approach to prevent acid-induced inflammatory responses associated with biodegradable polymers, here poly(lactide- co-glycolide), by using oligo(lactide)-grafted magnesium hydroxide (Mg(OH)2) nanoparticles, which neutralize the acidic environment. In particular, we demonstrated that incorporating the modified Mg(OH)2 nanoparticles within degradable coatings on drug-eluting arterial stents efficiently attenuates the inflammatory response and in-stent intimal thickening by more than 97 and 60%, respectively, in the porcine coronary artery, compared with that of drug-eluting stent control. We also observed that decreased inflammation allows better reconstruction of mouse renal glomeruli in a kidney tissue regeneration model. Such modified Mg(OH)2 nanoparticles may be useful to extend the applicability and improve clinical success of biodegradable devices used in various biomedical fields.

The Effect of Fetal Bovine Serum (FBS) on Efficacy of Cellular Reprogramming for Induced Pluripotent Stem Cell (iPSC) Generation.

Induced pluripotent stem cells (iPSCs) are pivotal to the advancement of regenerative medicine. However, the low efficacy of iPSC generation and insufficient knowledge about the reprogramming mechanisms involved in somatic cell/adult stem cell reversion to a pluripotent phenotype remain critical hurdles to the therapeutic application of iPSCs. The present study investigated whether the concentration of fetal bovine serum (FBS), a widely employed cell culture additive, can influence the cellular reprogramming efficacy (RE) of human adipose-derived stem cells (hADSCs) to generate iPSCs. Compared with the typically employed concentration of FBS (10%), high concentrations (20% and 30%) increased the RE of hADSCs by approximately twofold, whereas a low concentration (5%) decreased the RE by the same extent. Furthermore, cell counting kit-8 (CCK-8), bromodeoxyuridine (BrdU) incorporation, and fluorescence-activated cell sorting (FACS) assays showed that hADSC proliferation during reprogramming was significantly enhanced by FBS at 20% and 30%, whereas quantitative polymerase chain reaction (qPCR) and Western blotting assays revealed a concomitant decrease in p53, p51, and p21 expression. In addition, the efficacy of retrovirus-mediated transduction into hADSCs was increased by approximately 10% at high concentrations of FBS. It was confirmed that platelet-derived growth factor in the FBS enhanced proliferation and reprogramming efficacy. Finally, the generated iPSCs showed a normal karyotype, the same fingerprinting pattern as parental hADSCs, a genome-wide transcriptome pattern similar to that of human embryonic stem cells (hESCs), and in vivo pluripotency. In conclusion, the current investigation demonstrated that high concentrations of FBS can modulate molecular and cellular mechanisms underlying the reprogramming process in hADSCs, thereby augmenting the cellular RE for iPSC generation.

Stiffness of Hydrogels Regulates Cellular Reprogramming Efficiency Through Mesenchymal-to-Epithelial Transition and Stemness Markers.

The stiffness of hydrogels has been reported to direct cell fate. Here, we found that the stiffness of hydrogels promotes the reprogramming of mouse embryonic fibroblasts into induced pluripotent stem cells (iPSCs). We prepared cell culture substrates of various stiffnesses (0.1, 1, 4, 10, and 20 kPa) using a polyacrylamide hydrogel. We found that culture on a soft hydrogel plays an important role in inducing cellular reprogramming into iPSCs via activation of mesenchymal-to-epithelial transition and enhancement of stemness marker expression. These results suggest that physical signals at the interface between cell and substrate can be used as a potent regulator to promote cell fate changes associated with reprogramming into iPSCs, which may lead to effective and reproducible iPSC-production.

Fabrication and characteristics of dual functionalized vascular stent by spatio-temporal coating.

UNLABELLED: Stent implantation with balloon angioplasty is a widely used treatment for coronary artery diseases. Stents have been developed from bare metal stent (BMS) to advanced forms such as drug-eluting stent (DES). However, modern DES still causes thrombosis and/or in-stent restenosis as long-term outcomes. For effective prevention of these problems, we fabricated a dual functionalized stent using spatio-temporal coating, which has two different surfaces, as a novel type of DES. Hyaluronic acid conjugated with dopamine (HA-DA) was applied to a bare cobalt-chromium (CC) stent prior to abluminal coating of sirolimus (SRL)-in-polymer such as poly(d,l-lactide). The SRL-in-polymer (P+S) coated on the abluminal surface of the HA-DA modified stent showed highly stable coating layer and prevented the crack formation after ballooning. In the blood- and cyto-compatibility tests, HA-DA coating displayed suppressive effects on adhesion and activation of platelets and maintained the cell viability and proliferation of human coronary artery endothelial cells even under the existence of SRL. In in vivo study using porcine restenosis model, the neointimal area and inflammation score of the dual functionalized stent with HA-DA and P+S were significantly reduced than those of BMS. It is expected that this novel type of DES can be effectively applied to utilize diverse anti-proliferative drugs and bioactive polymers. STATEMENT OF SIGNIFICANCE: Stents have been developed from bare metal stent to advanced forms such as drug-eluting stents (DESs). However, even DESs can still cause in-stent restenosis as long-term outcomes. This paper demonstrated a novel DES using spatio-temporal coating by dopamine-mediated hyaluronic acid coating (HA-DA) before asymmetric coating of sirolimus-in-poly(d,l-lactide) (P+S). It showed stable coating surface and prevented crack formation after ballooning. HA-DA coating also had an inhibitive effect on adhesion of platelets and maintained cell viability of endothelial cells even under the existence of sirolimus. Additionally, in vivo neointima area and inflammation score of HA-DA/P+S stent significantly decreased than those of BMS. We expected that this novel type of DES can be effectively applied to introduce diverse anti-proliferative drugs and bioactive molecules.

In vivo characterization of the integrin beta3 as a receptor for Hantaan virus cellular entry.

Binding of viruses to cell surface molecules is an essential step in viral infection. In vitro studies suggested that the alpha(v)beta(3) integrin receptor is the epithelial cell receptor for Hantaan virus (HTNV). Whether beta(3) is in vivo the only or central cellular receptor for HTNV infection is not known. To investigate the role of beta(3) integrin for cellular entry of HTNV, we established an HTNV infection model in newborn murine pups. Infected pups died at an average age of 14.2 +/- 1.1 days with high levels of viral antigen detected in their brain, lung, and kidney. Pre-injection of blocking monoclonal antibodies (mAb) specific for either beta(3) or av prolonged survival significantly to a maximal average survival of 19.7 +/- 1.5 days (P <0.01) and 18.4 +/- 0.9 days (P < 0.01), respectively. XT-199, a chemical blocker of the alpha(v)beta(3) receptor also prolonged survival to 19.5 +/- 1.3 days (P < 0.01). In contrast to these receptor blockades, anti-HTNV antibody was not only able to prolong survival, but 20% of infected pups achieved long-term survival. An anti-murine beta(1) antibody comparatively prolonged survival (19.0 +/- 1.2 days), suggesting that HTNV infection is partly mediated through integrin beta(1) receptors as well as through beta(3) receptors in vivo. Our data demonstrate that the beta(3) receptor is important for HTNV infection in vivo, but also suggest that HTNV may utilize additional receptors beyond beta(3) for cellular entry within an organism.

Genomic organization and expression of parkin in Drosophila melanogaster.

We report here the isolation, characterization on genomic structure and expression of the D. melanogaster homolog of human parkin. The 2,122 bp parkin gene sequence contains six exons that form a 1,449 bp transcript encoding a protein of 482 amino acids. 151 bp of 5' and 112 bp of 3' untranslated regions were identified by a combination of 5'-RACE/primer extension and 3'-RACE, respectively. The 5' UTR contains three transcription initiation sites. Neither a classical TATA nor a CAAT box was found in the putative promoter sequence. However, binding sites for AhR-Arnt, AP4, NF1 and GATA transcription factors were identified. Transient transfection analysis of the 5' UTR confirmed its promoter activity in HEK 293 cells and SH-SY5Y neuronal cells using a dual luciferase reporting system. The amino acid sequence of D. melanogaster Parkin exhibits 42%, 43% and 43% identity to that of human, mouse and rat, respectively, representing a 54 kDa protein band via western blot analysis. It shows a high degree of conservation in the Ubiquitin-like domain at the N-terminus (34%), the In-Between RING finger domains (IBR, 65-69%), and the RING finger domains at the C-terminus (56-57%). The expression pattern of D. melanogaster parkin varies during the developmental stages, with the highest expression in the adult stage as measured by competitive RT-PCR. From immunostainings of the embryo, D. melanogaster parkin was expressed slightly higher in the central nervous system (brain and nerve cord) during the late embryonic stage.

Poly-L-lysine increases the ex vivo expansion and erythroid differentiation of human hematopoietic stem cells, as well as erythroid enucleation efficacy.

Hematopoietic stem cells (HSCs) are continuously stimulated by physical interactions with bone marrow or umbilical cord niches as well as by chemical factors found within these niches. The niche can be mimicked by modification of the cytokine composition, elasticity, topography, and/or charge. This work employed cell culture plates coated with several concentrations of poly-L-lysine (PLL), a positively charged synthetic amino-acid chain. Culture substrates that employed relatively high initial coating concentrations of PLL significantly increased the total number of HSCs during ex vivo expansion of CD34(+) cells, as well as erythroid differentiation. Furthermore, the 0.01% PLL substrate stimulated enucleation of erythroid cells, leaving behind a number of extruded nuclei at the bottom of the culture plate, followed by an increase in the number of erythrocytes. Thus, PLL will likely prove useful to enhance the expansion of HSCs and erythroid cells, in addition to the generation of red blood cells.

Mass-producible nano-featured polystyrene surfaces for regulating the differentiation of human adipose-derived stem cells.

In this study, we report an efficient and cost-effective method of fabricating polystyrene (PS) nano-featured substrates containing nanopore (NPo) and nanopillar (NPi) arrays based on hot embossing using nickel nano-stamps. We investigate the behavior of adipose-derived stem cells (ASCs), including adhesion, morphology, proliferation and differentiation, on the replicated PS surfaces. Compared to a flat substrate, NPo- and NPi-featured substrates do not alter the morphology of stem cells. However, both NPo- and NPi-featured substrates induce different integrin expression and lower formation of focal adhesion complexes. In addition, ASCs on the NPo-featured substrate exhibit greater adipogenic differentiation, while the NPi-featured substrate induces higher osteogenic differentiation.

The effect of conjugating RGD into 3D alginate hydrogels on adipogenic differentiation of human adipose-derived stromal cells.

The effects of RGD peptide conjugation to alginate hydrogel on the adipogenic differentiation of ASCs was investigated. After 3 d of culture, RGD-modified alginate hydrogels significantly stimulated FAK and integrin α1 gene expressions and vinculin expression in ASCs. In addition, RGD-modified alginate hydrogels significantly enhanced the adipogenic differentiation of human ASCs to exhibit higher expression levels of oil red O staining and adipogenic genes compared to those of the control group (unmodified gels). These results suggest potential applications of RGD-modified alginate gels for adipose tissue regeneration.

Effect of nucleus pulposus cells having different phenotypes on chondrogenic differentiation of adipose-derived stromal cells in a coculture system using porous membranes.

In this study, adipose-derived stem cells (ASCs) were cocultured with nucleus pulposus (NP) cells using a porous membrane to investigate the effect of NP cell phenotype on ASC chondrogenic differentiation. Human NP cells were collected from 14 patients and classified into two groups (normal vs. degenerative) depending on the level of type II collagen, aggrecan (AGG), type I collagen, and bax gene expression. Human ASCs were then cocultured with each group of NP cells on porous membranes in the absence of chondrogenic supplements. After 2 weeks, real-time-polymerase chain reaction results showed that ASCs cocultured with normal NP cells had much higher type II collagen and AGG gene expression than ASCs cocultured with degenerative NP cells. The production of AGG was also observed only in the group cocultured with normal NP cells. Additionally, coculture of ASC pellets with normal NP cells promoted the production of AGG as compared to coculture of ASC monolayer with normal NP cells. These data demonstrate that a coculture system using porous membranes can induce ASC differentiation into NP cells without chondrogenic supplements. Further, the phenotype of cocultured NP cells significantly influences the extent of ASC differentiation.

Surface modification with fibrin/hyaluronic acid hydrogel on solid-free form-based scaffolds followed by BMP-2 loading to enhance bone regeneration.

Bone tissue engineering often requires a well-defined scaffold that is highly porous. The multi-head deposition system (MHDS), a form of solid freeform fabrication, has raised great interest as a method for fabricating scaffolds, since it yields a highly porous inter-connective structure without the use of cytotoxic solvents, and permits the diffusion of nutrients and oxygen. However, this method is not suitable for introducing proteins, as it includes a heating process. Hydrogels incorporated with protein coating of the scaffold surface could overcome this MHDS limitation. In the present study, the surface of a scaffold fabricated using MHDS was coated with a mixture of fibrin and hyaluronic acid (HA) and used as a vehicle for delivery of both bone morphogenetic protein-2 (BMP-2) and adipose-derived stromal cells (ASCs). Fibrin/HA coating of the scaffold significantly enhanced initial cell attachment. Furthermore, the in vitro release of BMP-2 from fibrin/HA-coated scaffolds was sustained for 3 days and it stimulated the alkaline phosphatase activity of ASCs seeded on the scaffold for 10 days more actively and continuously than did the soluble BMP-2 that was added to the culture media, not the scaffold itself. Importantly, the transplantation of undifferentiated ASCs inoculated on BMP-2-loaded, fibrin/HA-coated scaffolds resulted in more improved bone formation and mineralization than did the transplantation of undifferentiated ASCs seeded on uncoated scaffolds or on fibrin/HA-coated scaffolds without BMP-2, but containing BMP-2 in the cell suspension medium. These results show that BMP-2-loaded, fibrin/HA-coated scaffolds fabricated using MHDS may be useful in stimulating bone regeneration from undifferentiated ASCs in vivo.