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Vascular Endothelial Growth Factor Enhances Axonal Outgrowth in Organotypic Spinal Cord Slices via Vascular Endothelial Growth Factor Receptor 1 and 2.

Hwan Woo PARK ; Hyo Jin JEON ; Mi Sook CHANG

Tissue Engineering and Regenerative Medicine.2016;13(5):601-609. doi:10.1007/s13770-016-0051-9

Enhancing adult nerve regeneration is a potential therapeutic strategy for treating spinal cord injury. Vascular endothelial growth factor (VEGF) is a major contributor to angiogenesis, which can reduce the spinal cord injury by inhibiting the inflammation and improve recovery after spinal cord injury. We have previously demonstrated that exogenous VEGF has neurotrophic effects on injured spinal nerves in organotypic spinal cord slice cultures. However, the mechanisms underlying the neurite growth by exogenous VEGF remain to be explored in spinal cord. In this study, we found out that exogenous VEGF mediated axonal outgrowth through VEGF receptor 1 (VEGFR1) and VEGFR2, both of which were expressed on organotypic spinal cord slices. Although VEGFR1 and VEGFR2 were constitutively expressed in some cells of control spinal cord slices, VEGF treatment upregulated expression of VEGFR1 and VEGFR2. Both VEGFR1 and VEGFR2 were expressed in neuronal cells as well as glial cells of organotypic spinal cord slices. We also observed that VEGF-induced axonal outgrowth was attenuated by a specific mitogen-activated protein kinase (MAPK) inhibitor PD98059 and a specific phosphoinositide 3-kinase (PI3K) inhibitor wortmannin. Thus, these findings suggest that these MAPK and PI3K pathways have important roles in regulating VEGF-induced axonal outgrowth in the postnatal spinal cord.
Adult ; Axons* ; Humans ; Inflammation ; Nerve Regeneration ; Neurites ; Neuroglia ; Neurons ; Protein Kinases ; Receptors, Vascular Endothelial Growth Factor* ; Spinal Cord Injuries ; Spinal Cord* ; Spinal Nerves ; Vascular Endothelial Growth Factor A* ; Vascular Endothelial Growth Factor Receptor-1*

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Alginate-Hyaluronic Acid-Collagen Composite Hydrogel Favorable for the Culture of Chondrocytes and Their Phenotype Maintenance.

Chinmaya MAHAPATRA ; Guang Zhen JIN ; Hae Won KIM

Tissue Engineering and Regenerative Medicine.2016;13(5):538-546. doi:10.1007/s13770-016-0059-1

Articular cartilage has limited regeneration capacity, thus significant challenge has been made to restore the functions. The development of hydrogels that can encapsulate and multiply cells, and then effectively maintain the chondrocyte phenotype is a meaningful strategy to this cartilage repair. In this study, we prepared alginate-hyaluronic acid based hydrogel with type I collagen being incorporated, namely Alg-HA-Col composite hydrogel. The incorporation of Col enhanced the chemical interaction of molecules, and the thermal stability and dynamic mechanical properties of the resultant hydrogels. The primary chondrocytes isolated from rat cartilage were cultured within the composite hydrogel and the cell viability recorded revealed active proliferation over a period of 21 days. The mRNA levels of chondrocyte phenotypes, including SOX9, collagen type II, and aggrecan, were significantly up-regulated when the cells were cultured within the Alg-HA-Col gel than those cultured within the Alg-HA. Furthermore, the secretion of sulphated glycosaminoglycan, a cartilage-specific matrix molecule, was recorded higher in the collagen-added composite hydrogel. Although more in-depth studies are required such as the in vivo functions, the currently-prepared Alg-HA-Col composite hydrogel is considered to provide favorable 3-dimensional matrix conditions for the cultivation of chondrocytes. Moreover, the cell-cultured constructs may be useful for the cartilage repair and tissue engineering.
Aggrecans ; Animals ; Cartilage ; Cartilage, Articular ; Cell Survival ; Chondrocytes* ; Collagen Type I ; Collagen Type II ; Hyaluronic Acid ; Hydrogel* ; Hydrogels ; Phenotype* ; Rats ; Regeneration ; RNA, Messenger ; Tissue Engineering

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Prefabrication of Axially Vascularized Bone by Combining β-Tricalciumphosphate, Arteriovenous Loop, and Cell Sheet Technique.

Dongyang MA ; Liling REN ; Zhen CAO ; Jianxue LI ; Jian CAO ; Wenyan TIAN ; Hong YAO

Tissue Engineering and Regenerative Medicine.2016;13(5):579-584. doi:10.1007/s13770-016-9095-0

The repair of bone defects poses a great challenge for reconstructive surgeons. Although the development of tissue engineering has exhibited promise in replacing damaged bone, the fabrication of large constructs with functional blood vessels remains an obstacle. From the orthopedic surgeon's point of view, the generation of axially vascularized bone, which can anastomose with the recipient vessel, might be a solution to this medical problem. In this study, we aimed to prefabricate an axially vascularized bone by combining a β-TCP scaffold, arteriovenous loop (AVL), and cell sheet in a bioreactor in vivo. Twelve rabbits were randomly allocated into two groups: the experimental group (presence of AVL) and the control group (absence of AVL). The constructs were explanted at 8 weeks postoperatively. The histomorphometric results showed 42.8±5.9% of the bone area in the AVL group and 26.6±3.5% in the control group. Similarly, vessel analysis revealed the average vessel density in the AVL group (12.5±3.3) was significantly more than that in the control group (6.1±1.5, p<0.05). Our research indicated that the combination of a β-TCP scaffold, AVL and cell sheet might engineer vascularized bone. This prefabrication strategy might facilitate clinical translation of bone tissue engineering in reconstructing large bone defects.
Bioreactors ; Blood Vessels ; Bone and Bones ; Orthopedics ; Rabbits ; Surgeons ; Tissue Engineering

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Platelet-Derived Growth Factor-BB-Immobilized Asymmetrically Porous Membrane for Enhanced Rotator Cuff Tendon Healing.

Hyun Ki MIN ; Oh Soo KWON ; Se Heang OH ; Jin Ho LEE

Tissue Engineering and Regenerative Medicine.2016;13(5):568-578. doi:10.1007/s13770-016-9120-3

Rotator cuff tear is a common musculoskeletal disease that often requires surgical repair. Despite of recent advances in surgical techniques, the re-tear rate of the rotator cuff tendon is very high. In this study, a platelet-derived growth factor-BB (PDGF-BB)-immobilized asymmetrically porous membrane was fabricated to investigate the feasibility for enhancing rotator cuff tendon regeneration through the membrane. PDGF-BB is recognized to promote tendon regeneration. The asymmetrically porous membrane was fabricated by polycaprolactone and Pluronic F127 using an immersion precipitation technique, which can allow selective permeability (preventing scar tissue invasion into defect region but allowing permeation of oxygen/nutrients) and incorporation of bioactive molecules (e.g., PDGF-BB) via heparin binding. The PDGF-BB immobilized on the membrane was released in a sustained manner over 42 days. In an animal study using Sprague-Dawley rats, the PDGF-BB-immobilized membrane group showed significantly greater regeneration of rotator cuff tendon in histological and biomechanical analyses compared with the groups of control (suturing) and membrane without PDGF-BB immobilization. The enhancing regeneration of rotator cuff tendon of the PDGF-BB-immobilized membrane may be caused from the synergistic effect of the asymmetrically porous membrane with unique properties (selective permeability and hydrophilicity) as a scaffold for guided tendon regeneration and PDGF-BB sustainedly released from the membrane.
Animals ; Cicatrix ; Heparin ; Immersion ; Immobilization ; Membranes* ; Musculoskeletal Diseases ; Permeability ; Poloxamer ; Rats, Sprague-Dawley ; Regeneration ; Rotator Cuff* ; Tears ; Tendons*

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Efficient Induction of Neural Precursor Cells from Fibroblasts Using Stromal Cell-Derived Inducing Activity.

Mi Sun LIM ; Sang Mi KIM ; Eun Hye LEE ; Chang Hwan PARK

Tissue Engineering and Regenerative Medicine.2016;13(5):554-559. doi:10.1007/s13770-016-0012-3

The direct lineage conversion of fibroblasts into neuronal or neural precursor cells (NPCs) has become a hot issue in recent years as an attractive approach in the field of stem cell regenerative medicine. In this study, we adopted the stromal feeder co-culture method during the early conversion period to enhance conversion efficiency. Stromal cells are often used in directed differentiation of dopaminergic (DA) neurons from pluripotent stem cells. We co-cultured rat embryonic fibroblasts (REFs) on γ-irradiated sonic hedgehog-overexpressing MS5 stromal (MS5-SHH) cells after transduction with Brn2, Ascl1, Myt1L, and BclxL-GFP (BAMXGFP) transcription factors to REFs. One week after co-culture, transduced cells (GFP+ cells) that proliferated on MS5-SHH cells were separated from MS5-SHH cells through a 40 µm cell strainer. Subsequently, the converted cells (GFP+ cells) were expanded on fibronectin-coated culture plates in NPC expansion medium. The induced NPCs (iNPCs) expressed NPC potential (NESTIN+/SOX2+) earlier than seen with non-co-culture methods and were efficiently differentiated into DA neurons by overexpression of Nurr1 and Foxa2 genes, which are specific transcription factors for midbrain DA neuron development. These observations indicated that direct conversion to NPCs using an MS5 stromal cells co-culture method is a suitable technique for efficient generation of iNPC/DA neurons from fibroblasts.
Animals ; Coculture Techniques ; Dopaminergic Neurons ; Fibroblasts* ; Mesencephalon ; Methods ; Neurons ; Pluripotent Stem Cells ; Rats ; Regenerative Medicine ; Stem Cells ; Stromal Cells ; Transcription Factors

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Pigmentation Effect of Electromagnetic Fields at Various Intensities to Melanocytes.

Sang Eun CHO ; Yu Mi KIM ; Kye Hong KANG ; Soo Chan KIM ; Jung Keug PARK ; Young Kwon SEO

Tissue Engineering and Regenerative Medicine.2016;13(5):560-567. doi:10.1007/s13770-016-0090-2

Melanogenesis is the biological process that results in the synthesis of skin pigment of melanin and it has various functions in living systems and is synthesized by the melanosome within the melanocytes. A variety of physical treatments are used to promote melanin production in the melanocytes for pigmentation control. The purpose of this study was to evaluate the intensity-dependent effect of extremely low-frequency electromagnetic fields (ELF-EMFs) on melanogenesis by melanocytes in vitro. Melanocytes were exposed to ELF-EMFs at a frequency of 50 Hz and at intensities in the range of 0.5–20 G over 4 days. The results of lactate dehydrogenase assay showed that there were no significant differences between cells exposed to 0.5 G or 2 G groups and the controls. The melanin contents increased 1.2–1.5-fold in cells exposed to ELF-EMFs and tyrosinase activity increased 1.3-fold in cells exposed to ELF-EMFs, relative to the controls. Also, exposure to ELF-EMFs was associated with activation in cyclic-AMP response element binding protein and microphthalmia-associated transcription factor (MITF) was up-regulated. Up-regulation of MITF induces the expression of melanogenesis-related markers, such as tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2. In conclusion, the present study showed that the exposure to ELF-EMFs at low intensities can stimulate melanogenesis in melanocyte, and these results may be used to a therapeutic devices for inducing repigmentation in vitiligo patients.
Biological Processes ; Carrier Proteins ; Electromagnetic Fields* ; Humans ; In Vitro Techniques ; L-Lactate Dehydrogenase ; Magnets* ; Melanins ; Melanocytes* ; Melanosomes ; Microphthalmia-Associated Transcription Factor ; Monophenol Monooxygenase ; Pigmentation* ; Response Elements ; Skin ; Up-Regulation ; Vitiligo

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Hypoxia Enhances Osteogenic Differentiation in Retinoic Acid-Treated Murine-Induced Pluripotent Stem Cells.

Jeeranan MANOKAWINCHOKE ; Thanaphum OSATHANON ; Hiroshi EGUSA ; Prasit PAVASANT

Tissue Engineering and Regenerative Medicine.2016;13(5):547-553. doi:10.1007/s13770-016-9127-9

Hypoxic condition influences biological responses in various cell types. However, a hypoxic regulating osteogenic differentiation remains controversy. Here, an influence of short-term culture in hypoxic condition on osteogenic marker gene expression by retinoic acid-treated murine gingival fibroblast-derived induced pluripotent stem cells (RA-miPS) was investigated. Results demonstrated that hypoxic condition significantly upregulated Vegf, Runx2, Osx, and Ocn mRNA expression by RA-miPS in normal culture medium at day 3. Further, desferrioxamine significantly downregulated pluripotent marker (Nanog and Oct4) and enhanced osteogenic marker (Runx2, Osx, Dlx5, and Ocn) gene expression as well as promoted in vitro mineral deposition. However, the effect of cobalt chloride on osteogenic differentiation of RA-miPS was not robust. In summary, the results imply that hypoxic condition may be useful in the enhancement of osteogenic differentiation in RA-miPS.
Anoxia* ; Cobalt ; Deferoxamine ; Gene Expression ; In Vitro Techniques ; Induced Pluripotent Stem Cells ; Miners ; Pluripotent Stem Cells* ; RNA, Messenger ; Vascular Endothelial Growth Factor A

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Differentiation of Rat Dermal Mesenchymal Cells and Calcification in Three-Dimensional Cultures.

Taiki SUYAMA ; Mitsutoki HATTA ; Shozaburo HATA ; Hiroyuki ISHIKAWA ; Jun YAMAZAKI

Tissue Engineering and Regenerative Medicine.2016;13(5):527-537. doi:10.1007/s13770-016-9124-z

Three-dimensional (3D) cultures are known to promote cell differentiation. Previously, we investigated the differentiation of rat dermal fibroblasts to α-smooth muscle actin (α-SMA)-positive myofibroblasts through transforming growth factor (TGF)-β production using a 3D culture model. Here, we investigated the phenotypic change from dermal mesenchymal cells (mostly fibroblasts) to osteoblast-like cells, being inspired by the roles of smooth muscle cells or fibroblasts during vascular calcification. Spindle-shaped cells that grew in heterologous populations out of dermal explants from 2-day-old Wistar rats were cultured within a collagen matrix. α-SMA and alkaline phosphatase (ALP) messenger RNA (mRNA) levels initially increased, followed by a rise in Runx2 and osteocalcin (OCN) mRNA levels without calcification. Calcium deposits were produced in the presence of a high concentration of inorganic phosphate (2.1 mM) or β-glycerophosphate (βGP, 10 mM) after 2 weeks of culture, and both were sensitive to an inhibitor of type III phosphate transporters. An ALP inhibitor decreased only βGP-induced calcification. Inhibition of TGF-β type-I receptors attenuated ALP mRNA levels and βGP-induced calcification, suggesting that endogenous TGF-β stimulates ALP activity and then βGP breakdown. An increase in the number of cells embedded in the collagen gel enhanced the mRNA levels of Runx2 and OCN, but not of ALP. Collectively, several factors are likely to promote the differentiation of dermal mesenchymal cells into osteoblast-like cells and ectopic calcification in a 3D collagen matrix, implying the utility of these cells as a potential autologous cell source for tissue engineering.
Actins ; Alkaline Phosphatase ; Animals ; Calcium ; Cell Differentiation ; Collagen ; Dermis ; Fibroblasts ; Myocytes, Smooth Muscle ; Myofibroblasts ; Osteocalcin ; Phosphate Transport Proteins ; Rats* ; Rats, Wistar ; RNA, Messenger ; Tissue Engineering ; Transforming Growth Factors ; Vascular Calcification

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Electrospun Fibrous Silk Fibroin/Poly(L-Lactic Acid) Scaffold for Cartilage Tissue Engineering.

Weiwei LIU ; Zhengqiang LI ; Lu ZHENG ; Xiaoyan ZHANG ; Peng LIU ; Ting YANG ; Bing HAN

Tissue Engineering and Regenerative Medicine.2016;13(5):516-526. doi:10.1007/s13770-016-9099-9

For successful tissue engineering of articular cartilage, a scaffold with mechanical properties that match those of natural cartilage as closely as possible is needed. In the present study, we prepared a fibrous silk fibroin (SF)/poly(L-lactic acid) (PLLA) scaffold via electrospinning and investigated the morphological, mechanical, and degradation properties of the scaffolds fabricated using different electrospinning conditions, including collection distance, working voltage, and the SF:PLLA mass ratio. In addition, in vitro cell-scaffold interactions were evaluated in terms of chondrocyte adhesion to the scaffolds as well as the cytotoxicity and cytocompatibility of the scaffolds. The optimum electrospinning conditions for generating a fibrous SF/PLLA scaffold with the best surface morphology (ordered alignment and suitable diameter) and tensile strength (~1.5 MPa) were a collection distance of 20 cm, a working voltage of 15 kV, and a SF:PLLA mass ratio of S50P50. The degradation rate of the SF/PLLA scaffolds was found to be determined by the SF:PLLA mass ratio, and it could be increased by reducing the PLLA proportion. Furthermore, chondrocytes spread well on the fibrous SF/PLLA scaffolds and secreted extracellular matrix, indicating good adhesion to the scaffold. The cytotoxicity of SF/PLLA scaffold extract to chondrocytes over 24 and 48 h in culture was low, indicating that the SF/PLLA scaffolds are biocompatible. Chondrocytes grew well on the SF/PLLA scaffold after 1, 3, 5, and 7 days of direct contact, indicating the good cytocompatibility of the scaffold. These results demonstrate that the fibrous SF/PLLA scaffold represents a promising composite material for use in cartilage tissue engineering.
Cartilage* ; Cartilage, Articular ; Chondrocytes ; Extracellular Matrix ; Fibroins ; In Vitro Techniques ; Silk* ; Tensile Strength ; Tissue Engineering*

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Bioreactor Conditioning of Valve Scaffolds Seeded Internally with Adult Stem Cells.

Allison KENNAMER ; Leslie SIERAD ; Richard PASCAL ; Nicholas RIERSON ; Christopher ALBERS ; Marius HARPA ; Ovidiu COTOI ; Lucian HARCEAGA ; Peter OLAH ; Preda TEREZIA ; Agneta SIMIONESCU ; Dan SIMIONESCU

Tissue Engineering and Regenerative Medicine.2016;13(5):507-515. doi:10.1007/s13770-016-9114-1

The goal of this study was to test the hypothesis that stem cells, as a response to valve-specific extracellular matrix “niches” and mechanical stimuli, would differentiate into valvular interstitial cells (VICs). Porcine aortic root scaffolds were prepared by decellularization. After verifying that roots exhibited adequate hemodynamics in vitro, we seeded human adipose-derived stem cells (hADSCs) within the interstitium of the cusps and subjected the valves to in vitro pulsatile bioreactor testing in pulmonary pressures and flow conditions. As controls we incubated cell-seeded valves in a rotator device which allowed fluid to flow through the valves ensuring gas and nutrient exchange without subjecting the cusps to significant stress. After 24 days of conditioning, valves were analyzed for cell phenotype using immunohistochemistry for vimentin, alpha-smooth muscle cell actin (SMA) and prolyl-hydroxylase (PHA). Fresh native valves were used as immunohistochemistry controls. Analysis of bioreactor-conditioned valves showed that almost all seeded cells had died and large islands of cell debris were found within each cusp. Remnants of cells were positive for vimentin. Cell seeded controls, which were only rotated slowly to ensure gas and nutrient exchange, maintained about 50% of cells alive; these cells were positive for vimentin and negative for alpha-SMA and PHA, similar to native VICs. These results highlight for the first time the extreme vulnerability of hADSCs to valve-specific mechanical forces and also suggest that careful, progressive mechanical adaptation to valve-specific forces might encourage stem cell differentiation towards the VIC phenotype.
Actins ; Adult Stem Cells* ; Adult* ; Bioreactors* ; Extracellular Matrix ; Heart Valves ; Hemodynamics ; Humans ; Immunohistochemistry ; In Vitro Techniques ; Islands ; Muscle Cells ; Phenotype ; Stem Cells ; Vimentin

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