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BACKGROUND@#To prevent unsolved problems of medical devices, we hypothesized that combinatorial effects of zwitterionic functional group and anti-bacterial metal ions can reduce effectively the thrombosis and bacterial infection of polymeric biomaterials. In this research, we designed a novel series of zwitterionic polyurethane (zPU) additives to impart anti-thrombotic properties to a polyvinyl chloride (PVC) matrix. @*METHODS@#We have synthesized zPUs by combination of various components and zPUs complexed with metal ions. Zwitterion group was prepared by reaction with 1,3-propane sultone and Nmethyldiethanolamine and metal ions were incorporated into sulfobetaine chains via molecular complexation. These zPU additives were characterized using FT-IR, 1H-NMR, elemental analysis, and thermal analysis. The PVC film blended with zPU additives were prepared by utilizing a solvent casting and hot melting process. @*RESULTS@#Water contact angle demonstrated that the introduction of zwitterion group has improved hydrophilicity of polyurethanes dramatically. Protein adsorption test resulted in improved anti-fouling effects dependent on additive concentration and decreases in their effects by metal complexation. Platelet adhesion test revealed anti-fouling effects by additive blending but not significant as compared to protein resistance results. @*CONCLUSION@#With further studies, the synthesized zPUs and zPUs complexed with metal ions are expected to be used as good biomaterials in biomedical fields. Based on our results, we can carefully estimate that the enhanced anti-fouling effect contributed to reduced platelet adhesion.
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BACKGROUND@#Immunoglobulin A (IgA) nephropathy (IgAN) is one of an important cause of progressive kidney disease and occurs when IgA settles in the kidney resulted in disrupts kidney’s ability to filter waste and excess water.Hydrogels are promising material for medical applications owing to their excellent adaptability and filling ability. Herein, we proposed a hyaluronic acid/gelatin (CHO-HA/Gel-NH2 ) bioactive hydrogel as a cell carrier for therapeutic kidney regeneration in IgAN. @*METHODS@#CHO-HA/Gel-NH2 hydrogel was fabricated by Schiff-base reaction without any additional crosslinking agents. The hydrogel concentrations and ratios were evaluated to enhance adequate mechanical properties and biocompatibility for further in vivo study. High serum IgA ddY mice kidneys were treated with human urine-derived renal progenitor cells encapsulated in the hydrogel to investigate the improvement of IgA nephropathy and kidney regeneration. @*RESULTS@#The stiffness of the hydrogel was significantly enhanced and could be modulated by altering the concentrations and ratios of hydrogel. CHO-HA/Gel-NH2 at a ratio of 3/7 provided a promising milieu for cells viability and cells proliferation. From week four onwards, there was a significant reduction in blood urea nitrogen and serum creatinine level in Cell/Gel group, as well as well-organized glomeruli and tubules. Moreover, the expression of pro-inflammatory and profibrotic molecules significantly decreased in the Gel/Cell group, whereas anti-inflammatory gene expression was elevated compared to the Cell group. @*CONCLUSION@#Based on in vivo studies, the renal regenerative ability of the progenitor cells could be further increased by this hydrogel system.
ABSTRACT
BACKGROUND@#Immunoglobulin A (IgA) nephropathy (IgAN) is one of an important cause of progressive kidney disease and occurs when IgA settles in the kidney resulted in disrupts kidney’s ability to filter waste and excess water.Hydrogels are promising material for medical applications owing to their excellent adaptability and filling ability. Herein, we proposed a hyaluronic acid/gelatin (CHO-HA/Gel-NH2 ) bioactive hydrogel as a cell carrier for therapeutic kidney regeneration in IgAN. @*METHODS@#CHO-HA/Gel-NH2 hydrogel was fabricated by Schiff-base reaction without any additional crosslinking agents. The hydrogel concentrations and ratios were evaluated to enhance adequate mechanical properties and biocompatibility for further in vivo study. High serum IgA ddY mice kidneys were treated with human urine-derived renal progenitor cells encapsulated in the hydrogel to investigate the improvement of IgA nephropathy and kidney regeneration. @*RESULTS@#The stiffness of the hydrogel was significantly enhanced and could be modulated by altering the concentrations and ratios of hydrogel. CHO-HA/Gel-NH2 at a ratio of 3/7 provided a promising milieu for cells viability and cells proliferation. From week four onwards, there was a significant reduction in blood urea nitrogen and serum creatinine level in Cell/Gel group, as well as well-organized glomeruli and tubules. Moreover, the expression of pro-inflammatory and profibrotic molecules significantly decreased in the Gel/Cell group, whereas anti-inflammatory gene expression was elevated compared to the Cell group. @*CONCLUSION@#Based on in vivo studies, the renal regenerative ability of the progenitor cells could be further increased by this hydrogel system.
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BACKGROUND@#Autologous fat grafting is one of the most common procedures used in plastic surgery to correct soft tissue deficiency or depression deformity. However, its clinical outcomes are often suboptimal, and lack of metabolic and architectural support at recipient sites affect fat survival leading to complications such as cyst formation, calcification.Extracellular matrix-based scaffolds, such as allograft adipose matrix (AAM) and poly(lactic-co-glycolic) acid (PLGA), have shown exceptional clinical promise as regenerative scaffolds. Magnesium hydroxide (MH), an alkaline ceramic, has attracted attention as a potential additive to improve biocompatibility. We attempted to combine fat graft with regenerative scaffolds and analyzed the changes and viability of injected fat graft in relation to the effects of injectable natural, and synthetic (PLGA/MH microsphere) biomaterials. @*METHODS@#In vitro cell cytotoxicity, angiogenesis of the scaffolds, and wound healing were evaluated using human dermal fibroblast cells. Subcutaneous soft-tissue integration of harvested fat tissue was investigated in vivo in nude mouse with random fat transfer protocol Fat integrity and angiogenesis were identified by qRT-PCR and immunohistochemistry. @*RESULTS@#In vitro cell cytotoxicity was not observed both in AAM and PLGA/MH with human dermal fibroblast.PLGA/MH and AAM showed excellent wound healing effect. in vivo, the AAM and PLGA/MH retained volume compared to that in the only fat group. And the PLGA/MH showed the highest angiogenesis and anti-inflammation. @*CONCLUSION@#In this study, a comparison of the volume retention effect and angiogenic ability between autologous fat grafting, injectable natural, and synthetic biomaterials will provide a reasonable basis for fat grafting.
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BACKGROUND@#In order to produce and isolate the exosome derived from the cell of interests, a serum free environment (starvation) has been essential for excluding the unknown effect from serum-derived exosomes. Recently, serum-free culture media have been developed as a substitute for serum supplemented media so that MSC proliferates with maintaining the original characteristics of the cells in a serum free condition. Due to the different properties of the exosomes representing the states and characteristics of the origin cells, a study is needed to compare the properties of the cell-derived exosomes according to the cell culture media. @*METHODS@#To compare the cell culture condition on exosomes, human umbilical cord mesenchymal stem cells (UCMSCs) were cultured with two different media, serum containing media, 10% FBS supplemented DMEM (NM) and serum-free chemically defined media, CellCor TM CD MSC (CDM). To remove FBS-derived exosomes from UCMSC cultured with NM, the medium was replaced with FBS-free DMEM for starvation during exosome isolation. The production yield and expression levels of angiogenic and pro-inflammatory factors were compared. And, the subpopulations of exosome were classified depending on the surface properties and loaded cytokines. Finally, the wound healing and angiogenic effects have been evaluated using in vitro assays. @*RESULTS@#The UCMSC-derived exosomes under two different cell culture media could be classified into subpopulations according to the surface composition and loaded cytokines. Especially, exosome derived from UCMSC cultured with CDM showed higher expression levels of cytokines related to regenerative bioactivities which resulted in enhanced wound healing and angiogenesis. @*CONCLUSION@#CDM has the advantages to maintain cell proliferation even during the period of exosome isolations and eliminate unknown side effects caused by serumderived exosomes. Additionally, exosomes derived from UCMSC cultured with CDM show better wound healing and angiogenic effects due to a lot of regeneration-related cytokines and less pro-inflammatory cytokines compared to with NM.
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BACKGROUND@#Poly(lactic-co-glycolic acid) (PLGA) microspheres have been actively used in various pharmaceutical formulations because they can sustain active pharmaceutical ingredient release and are easy to administer into the body using a syringe. However, the acidic byproducts produced by the decomposition of PLGA cause inflammatory reactions in surrounding tissues, limiting biocompatibility. Magnesium hydroxide (MH), an alkaline ceramic, has attracted attention as a potential additive because it has an acid-neutralizing effect. @*METHODS@#To improve the encapsulation efficiency of hydrophilic MH, the MH particles were capped with hydrophobic ricinoleic acid (RA-MH). PLGA microspheres encapsulated with RA-MH particles were manufactured by the O/W method. To assess the in vitro cytotoxicity of the degradation products of PLGA, MH/PLGA, and RA-MH/PLGA microspheres, CCK-8 and Live/Dead assays were performed with NIH-3T3 cells treated with different concentrations of their degradation products. in vitro anti-inflammatory effect of RA-MH/PLGA microspheres was evaluated with quantitative measurement of pro-inflammatory cytokines. @*RESULTS@#The synthesized RA-MH was encapsulated in PLGA microspheres and displayed more than four times higher loading content than pristine MH. The PLGA microspheres encapsulated with RA-MH had an acid-neutralizing effect better than that of the control group. In an in vitro cell experiment, the degradation products obtained from RA-MH/PLGA microspheres exhibited higher biocompatibility than the degradation products obtained from PLGA microspheres. Additionally, the RA-MH/PLGA microsphere group showed an excellent anti-inflammatory effect. @*CONCLUSION@#Our results proved that RA-MH-encapsulated PLGA microspheres showed excellent biocompatibility with an anti-inflammatory effect. This technology can be applied to drug delivery and tissue engineering to treat various incurable diseases in the future.
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BACKGROUND@#In order to produce and isolate the exosome derived from the cell of interests, a serum free environment (starvation) has been essential for excluding the unknown effect from serum-derived exosomes. Recently, serum-free culture media have been developed as a substitute for serum supplemented media so that MSC proliferates with maintaining the original characteristics of the cells in a serum free condition. Due to the different properties of the exosomes representing the states and characteristics of the origin cells, a study is needed to compare the properties of the cell-derived exosomes according to the cell culture media. @*METHODS@#To compare the cell culture condition on exosomes, human umbilical cord mesenchymal stem cells (UCMSCs) were cultured with two different media, serum containing media, 10% FBS supplemented DMEM (NM) and serum-free chemically defined media, CellCor TM CD MSC (CDM). To remove FBS-derived exosomes from UCMSC cultured with NM, the medium was replaced with FBS-free DMEM for starvation during exosome isolation. The production yield and expression levels of angiogenic and pro-inflammatory factors were compared. And, the subpopulations of exosome were classified depending on the surface properties and loaded cytokines. Finally, the wound healing and angiogenic effects have been evaluated using in vitro assays. @*RESULTS@#The UCMSC-derived exosomes under two different cell culture media could be classified into subpopulations according to the surface composition and loaded cytokines. Especially, exosome derived from UCMSC cultured with CDM showed higher expression levels of cytokines related to regenerative bioactivities which resulted in enhanced wound healing and angiogenesis. @*CONCLUSION@#CDM has the advantages to maintain cell proliferation even during the period of exosome isolations and eliminate unknown side effects caused by serumderived exosomes. Additionally, exosomes derived from UCMSC cultured with CDM show better wound healing and angiogenic effects due to a lot of regeneration-related cytokines and less pro-inflammatory cytokines compared to with NM.
ABSTRACT
BACKGROUND@#Poly(lactic-co-glycolic acid) (PLGA) microspheres have been actively used in various pharmaceutical formulations because they can sustain active pharmaceutical ingredient release and are easy to administer into the body using a syringe. However, the acidic byproducts produced by the decomposition of PLGA cause inflammatory reactions in surrounding tissues, limiting biocompatibility. Magnesium hydroxide (MH), an alkaline ceramic, has attracted attention as a potential additive because it has an acid-neutralizing effect. @*METHODS@#To improve the encapsulation efficiency of hydrophilic MH, the MH particles were capped with hydrophobic ricinoleic acid (RA-MH). PLGA microspheres encapsulated with RA-MH particles were manufactured by the O/W method. To assess the in vitro cytotoxicity of the degradation products of PLGA, MH/PLGA, and RA-MH/PLGA microspheres, CCK-8 and Live/Dead assays were performed with NIH-3T3 cells treated with different concentrations of their degradation products. in vitro anti-inflammatory effect of RA-MH/PLGA microspheres was evaluated with quantitative measurement of pro-inflammatory cytokines. @*RESULTS@#The synthesized RA-MH was encapsulated in PLGA microspheres and displayed more than four times higher loading content than pristine MH. The PLGA microspheres encapsulated with RA-MH had an acid-neutralizing effect better than that of the control group. In an in vitro cell experiment, the degradation products obtained from RA-MH/PLGA microspheres exhibited higher biocompatibility than the degradation products obtained from PLGA microspheres. Additionally, the RA-MH/PLGA microsphere group showed an excellent anti-inflammatory effect. @*CONCLUSION@#Our results proved that RA-MH-encapsulated PLGA microspheres showed excellent biocompatibility with an anti-inflammatory effect. This technology can be applied to drug delivery and tissue engineering to treat various incurable diseases in the future.
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Background@#Inflammation induces dysfunction of endothelial cells via inflammatory cell adhesion, and this phenomenon and reactive oxygen species accumulation are pivotal triggers for atherosclerosis-related vascular disease. Although exosomes are excellent candidate as an inhibitor in the inflammation pathway, it is necessary to develop exosome-mimetic nanovesicles (NVs) due to limitations of extremely low release rate and difficult isolation of natural exosomes. NVs are produced in much larger quantities than natural exosomes, but due to the low flexibility of the cell membranes, the high loss caused by hanging on the filter membranes during extrusion remains a challenge to overcome. Therefore, by making cell membranes more flexible, more efficient production of NVs can be expected. @*Methods@#To increase the flexibility of the cell membranes, the suspension of umbilical cord-mesenchymal stem cells (UC-MSCs) was subjected to 5 freeze and thaw cycles (FT) before serial extrusion. After serial extrusion through membranes with three different pore sizes, FT/NVs were isolated using a tangential flow filtration (TFF) system. NVs or FT/NVs were pretreated to the human coronary artery endothelial cells (HCAECs), and then inflammation was induced using tumor necrosis factor-α (TNF-α). @*Results@#With the freeze and thaw process, the production yield of exosome-mimetic nanovesicles (FT/NVs) was about 3 times higher than the conventional production method. The FT/NVs have similar biological properties as NVs for attenuating TNF-α induced inflammation. @*Conclusion@#We proposed the efficient protocol for the production of NVs with UC-MSCs using the combination of freeze and thaw process with a TFF system. The FT/NVs successfully attenuated the TNF-α induced inflammation in HCAECs.
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BACKGROUND: Despite major progress in stem cell therapy, our knowledge of the characteristics and tissue regeneration potency of long-term transported cells is insufficient. In a previous in vitro study, we established the optimal cell transport conditions for amniotic fluid stem cells (AFSCs). In the present study, the target tissue regeneration of long-term transported cells was validated in vivo. METHODS: For renal regeneration, transported AFSCs were seeded on a poly(lactide-co-glycolide) scaffold and implanted in a partially resected kidney. The target tissue regeneration of the transported cells was compared with that of freshly harvested cells in terms of morphological reconstruction, histological microstructure reformation, immune cell infiltration, presence of induced cells, migration into remote organs, expression of inflammation/fibrosis/renal differentiation-related factors, and functional recovery. RESULTS: The kidney implanted with transported cells showed recovery of total kidney volume, regeneration of glomerular/renal tubules, low CD4/CD8 infiltration, and no occurrence of cancer during 40 weeks of observation. The AFSCs gradually disappeared and did not migrate into the liver, lung, or spleen. We observed low expression levels of proinflammatory cytokines and fibrotic factors; enhanced expression of the genes Wnt4, Pax2, Wt1, and Emx2; and significantly reduced blood urea nitrogen and creatinine values. There were no statistical differences between the performance of freshly harvested cells and that of the transported cells. CONCLUSION: This study demonstrates that long-term transported cells under optimized conditions can be used for cell therapy without adverse effects on stem cell characteristics, in vivo safety, and tissue regeneration potency.
Subject(s)
Female , Amniotic Fluid , Blood Urea Nitrogen , Cell- and Tissue-Based Therapy , Creatinine , Cytokines , In Vitro Techniques , Kidney , Liver , Lung , Polyglactin 910 , Regeneration , Spleen , Stem CellsABSTRACT
BACKGROUND: We fabricated anti-inflammatory scaffold using Mg(OH)2-incorporated polylactic acid-polyglycolic acid copolymer (MH-PLGA). To demonstrate the anti-inflammatory effects of the MH-PLGA scaffold, an animal model should be sensitive to inflammatory responses. The interleukin-10 knockout (IL-10 KO) mouse is a widely used bowel disease model for evaluating inflammatory responses, however, few studies have evaluated this mouse for the anti-inflammatory scaffold. METHODS: To compare the sensitivity of the inflammatory reaction, the PLGA scaffold was implanted into IL-10 KO and C57BL/6 mouse kidneys. Morphology, histology, immunohistochemistry, and gene expression analyses were carried out at weeks 1, 4, 8, and 12. The anti-inflammatory effect and renal regeneration potency of the MH-PLGA scaffold was also compared to those of PLGA in IL-10 KO mice. RESULTS: The PLGA scaffold-implanted IL-10 KO mice showed kidneys relatively shrunken by fibrosis, significantly increased inflammatory cell infiltration, high levels of acidic debris residue, more frequent CD8-, C-reactive protein-, and ectodysplasin A-positive cells, and higher expression of pro-inflammatory and fibrotic factors compared to the control group. The MH-PLGA scaffold group showed lower expression of pro-inflammatory and fibrotic factors, low immune cell infiltration, and significantly higher expression of anti-inflammatory factors and renal differentiation related genes compared to the PLGA scaffold group. CONCLUSION: These results indicate that the MH-PLGA scaffold had anti-inflammatory effects and high renal regeneration potency. Therefore, IL-10 KO mice are a suitable animal model for in vivo validation of novel anti-inflammatory scaffolds.
Subject(s)
Animals , Mice , Ectodysplasins , Fibrosis , Gene Expression , Immunohistochemistry , Interleukin-10 , Kidney , Mice, Knockout , Models, Animal , RegenerationABSTRACT
BACKGROUND: Kidney ischemia-reperfusion (IR) via laparotomy is a conventional method for kidney surgery in a mouse model. However, IR, an invasive procedure, can cause serious acute and chronic complications through apoptotic and inflammatory pathways. To avoid these adverse responses, a Non-IR and dorsal slit approach was designed for kidney surgery. METHODS: Animals were divided into three groups, 1) sham-operated control; 2) IR, Kidney IR via laparotomy; and 3) Non-IR, Non-IR and dorsal slit. The effects of Non-IR method on renal surgery outcomes were verified with respect to animal viability, renal function, apoptosis, inflammation, fibrosis, renal regeneration, and systemic response using histology, immunohistochemistry, real-time polymerase chain reaction, serum chemistry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and Masson's trichrome staining. RESULTS: The Non-IR group showed 100% viability with mild elevation of serum blood urea nitrogen and creatinine values at day 1 after surgery, whereas the IR group showed 20% viability and lethal functional abnormality. Histologically, renal tubule epithelial cell injury was evident on day 1 in the IR group, and cellular apoptosis enhanced TUNEL-positive cell number and Fas/caspase-3 and KIM-1/NGAL expression. Inflammation and fibrosis were high in the IR group, with enhanced CD4/CD8-positive T cell infiltration, inflammatory cytokine secretion, and Masson's trichrome stain-positive cell numbers. The Non-IR group showed a suitable microenvironment for renal regeneration with enhanced host cell migration, reduced immune cell influx, and increased expression of renal differentiation-related genes and anti-inflammatory cytokines. The local renal IR influenced distal organ apoptosis and inflammation by releasing circulating pro-inflammatory cytokines. CONCLUSION: The Non-IR and dorsal slit method for kidney surgery in a mouse model can be an alternative surgical approach for researchers without adverse reactions such as apoptosis, inflammation, fibrosis, functional impairment, and systemic reactions.
Subject(s)
Animals , Mice , Apoptosis , Blood Urea Nitrogen , Cell Count , Cell Movement , Chemistry , Creatinine , Cytokines , DNA Nucleotidylexotransferase , Epithelial Cells , Fibrosis , Immunohistochemistry , Inflammation , Kidney , Laparotomy , Methods , Nephrectomy , Real-Time Polymerase Chain Reaction , RegenerationABSTRACT
PURPOSE: In neurogenic bladder, both smooth muscle contraction and nerve regeneration are very important for functional improvement. Glycine-isoleucine-lysine-valine-alanine-valine (GIKVAV) is a peptide that can induce nerve regeneration in vivo. In this study, we evaluated bladder function after injection of muscle-derived stem cells (MDSCs) and GIKVAV into the cryo-injured bladder of nude mice. MATERIALS AND METHODS: Human muscle samples were obtained from the rectus abdominis muscle of 12 patients who underwent laparotomy. The purpose and entire method of the study were explained to the patients, and all subjects who participated in this study provided written informed consent. The MDSCs were isolated by a modified preplate technique, and only CD34+ human MDSC were extracted by use of Mini-MACS kits. The nude mice were subdivided into 5 groups (n=40): normal group (N, n=8), saline injection group after cryo-injury (S, n=8), GIKVAV injection group after cryo-injury (G, n=8), human MDSC injection group after cryo-injury (M, n=8), and GIKVAV and human MDSC injection group after cryo-injury (GM, n=8). At 2 weeks after injection, we compared the contractility of a bladder muscle strip of each group by organ bath and polygraph by using electronic field stimulation (EFS). Nerve regeneration was evaluated by choline acetyl transferase (ChAT) immunostaining. RESULTS: The contractile powers of the N, S, G, M, and GM groups were 3.58+/-0.27, 1.54+/-0.25, 1.54+/-0.31, 2.49+/-0.36, and 2.44+/-0.34 mN/mg, respectively, by EFS. The contractility of the bladder muscle strip in the S and G groups was lower than that in the N group. The contractile powers of the M and GM groups were lower than those of the N group but greater than those of the S and G groups. In ChAT immunohistochemical staining, nerve regeneration was increased in the G and GM groups compared with the S and M groups. CONCLUSIONS: Nerve regeneration was induced by GIKVAV injection regardless of human MDSC injection. There was no direct effect of GIKVAV on bladder muscle contractility.
Subject(s)
Animals , Humans , Mice , Baths , Choline , Contracts , Electronics , Electrons , Informed Consent , Laparotomy , Mice, Nude , Muscle, Smooth , Muscles , Nerve Regeneration , Rectus Abdominis , Stem Cells , Transferases , Urinary Bladder , Urinary Bladder, NeurogenicABSTRACT
Skeletal muscle contains several precursor cells that generate muscle, bone, cartilage and blood cells. Although there are reports that skeletal muscle-derived cells can trans-differentiate into neural-lineage cells, methods for isolating precursor cells, and procedures for successful neural induction have not been fully established. Here, we show that the preplate cell isolation method, which separates cells based on their adhesion characteristics, permits separation of cells possessing neural precursor characteristics from other cells of skeletal muscle tissues. We term these isolated cells skeletal muscle-derived neural precursor cells (SMNPs). The isolated SMNPs constitutively expressed neural stem cell markers. In addition, we describe effective neural induction materials permitting the neuron-like cell differentiation of SMNPs. Treatment with retinoic acid or forskolin facilitated morphological changes in SMNPs; they differentiated into neuron-like cells that possessed specific neuronal markers. These results suggest that the preplate isolation method, and treatment with retinoic acid or forskolin, may provide vital assistance in the use of SMNPs in cell-based therapy of neuronal disease.
Subject(s)
Animals , Mice , Antigens, Differentiation/metabolism , Cell Adhesion , Cell Differentiation , Cell Lineage , Cell Separation , Cells, Cultured , Colforsin/pharmacology , Mice, Inbred ICR , Muscle, Skeletal/cytology , Neurons/cytology , Stem Cells/cytology , Tretinoin/pharmacologyABSTRACT
High-density micromass culture was needed to take three dimensions culture with ASCs(adipose derived stromal cells) and chondrogenesis. However, the synthetic polymer has hydrophobic character and low affinity to cells and other biomolecules. Therefore, the surface modification without changes of physical and chemical properties is necessary for more suitable condition to cells and biomolecules. This study was performed to investigate the effect of surface modification of poly (lactic-co-glycolic acid)(PLGA) scaffold by plasma treatment (P(+)) on the adhesion, proliferation and chondrogenesis of ASCs, and not plasma treatment (P(-)). ASCs were isolated from human subcutaneous adipose tissue obtained by lipectomy and liposuction. At 1 hour 30 minutes and 3days after cell seeding onto the P(-) group and the P(+) group, total DNA amount of attached and proliferated ASCs markedly increased in the P(+) group (p < 0.05). The changes of the actin under confocal microscope were done for evaluation of cellular affinity, at 1 hour 30 minutes, the shape of the cells was spherical form in all group. At 3rd day, the shape of the cells was fiber network form and finely arranged in P(+) group rather than in P(-) group. RT-PCR analysis of cartilage-specific type II collagen and link protein were expressed in 1, 2 weeks of induction. Amount of Glycoaminoglycan (GAG) markedly increased in P(+) group(p < 0.05). In a week, extracellular matrix was not observed in the Alcian blue and Safranin O staining. However in 2 weeks, it was observed that sulfated proteoglycan increased in P(+) group rather than in P(-) group. In conclusion, we recognized that plasma treatment of PLGA scaffold could increase the hydrophilic property of cells, and provide suitable environment for high-density micromass culture to chondrogenesis.
Subject(s)
Humans , Actins , Alcian Blue , Chondrogenesis , Collagen Type II , DNA , Extracellular Matrix , Lipectomy , Plasma , Polymers , Proteoglycans , Stromal Cells , Subcutaneous Fat , Surface PropertiesABSTRACT
Cadmium is known to exert toxic effects on multiple organs, including the testes. To determine if alpha-tocopherol, an antioxidant, could protect testicular tissues and spermatogenesis from the toxic effects of cadmium, six-week old male Sprague-Dawley rats were randomized to receive cadmium at doses of 0 (control), 1, 2, 4 or 8 mg/kg by the intraperitoneal route (Group A) or alpha-tocopherol for 5 days before being challenged with cadmium (Group B) in an identical dose-dependent manner. When both groups received cadmium at 1 mg/kg, there were no changes in testicular histology relative to controls. When Group A received cadmium at 2 mg/kg, undifferentiated spermatids and dead Sertoli cells increased in the seminiferous tubules while interstitial cells decreased and inflammatory cells increased in the interstitial tissues. On flow cytometric analysis, the numbers of elongated spermatids (M1) and round spermatids (M2) decreased while 2c stage cells (M3, diploid) increased. In contrast, when Group B received cadmium at 2 mg/kg, the histological insults were reduced and the distribution of the germ cell population remained comparable to controls. However, alpha-tocopherol had no protective effects with higher cadmium doses of 4 and 8 mg/kg. These findings indicate that alpha-tocopherol treatment can protect testicular tissue and preserve spermatogenesis from the detrimental effects of cadmium but its effectiveness is dependent on the dose of cadmium exposed.
Subject(s)
Rats , Male , Animals , alpha-Tocopherol/pharmacology , Testis/drug effects , Spermatogenesis/drug effects , Rats, Sprague-Dawley , Inflammation , Flow Cytometry , Dose-Response Relationship, Drug , Cadmium Poisoning/pathology , Cadmium/metabolism , Antioxidants/pharmacologyABSTRACT
PURPOSE: To investigate the feasibility of using a poly (epsilon-caprolactone) (PCL) sheet seeded with autologous muscle-derived stem cells as a bladder substitute. MATERIALS AND METHODS: Muscle-derived stem cells were isolated from the gastrocnemius muscle of 9 female Sprague-Dawley rats using a preplate technique, and cultured on a 5x5mm PCL sheet. The sheets were implanted into the mesentery of the rats in an autologous manner. Three rats were sacrificed 2, 4 and 8 weeks after implantation, and the morphological changes were assessed by H&E and immunofluorescence staining including DAPI, myosin heavy chain (MHC) and choline acetyl transferase (CAT). RESULTS: All the rats survived for the scheduled time. A mild inflammatory reaction was observed around the PCL sheet in the postoperative 2-week specimen but this receded with time. Muscle cells on the sheet were observed over the experimental period. The 8-week specimen showed a moderate amount of muscle cells on the sheet, and MHC and CAT immunofluorescence staining showed a positive reaction. The muscle layer was not well organized. Angiogenesis was quite noticable between the sheet and the muscle cells on the 8-week specimen. CONCLUSIONS: A PCL sheet seeded with autologous muscle-derived stem cells showed skeletal muscle differentiation on the sheets 8 weeks after mesenteric implantation in an autologous manner. This suggests the feasibility of using a PCL sheet seeded with autologous muscle-derived stem cell as a bladder substitute.
Subject(s)
Animals , Cats , Female , Humans , Rats , Atrophy , Choline , Fluorescent Antibody Technique , Mesentery , Muscle Cells , Muscle, Skeletal , Myosin Heavy Chains , Rats, Sprague-Dawley , Regeneration , Stem Cells , Transferases , Urinary BladderABSTRACT
The advantages of wielding synthetic polymer are that the pore size, physical strength and chemical composition can be easily controlled. However, the synthetic polymer has hydrophobic character and low affinity to cells and other bio molecules. This study was performed to investigate the effect of plasma glow's discharge on the surface modification of poly(lactic-co-glycolic acid)(PLGA) sponge on the adhesion and bio-activity of chondrocytes in vitro culture. PLGA sponges(lactic acid: glycolic acid = 85 : 15, pore size=200 - 300 mrcro m, dimension=15 x 2 mm) were prepared. The experimental group(n=8) was treated with a radiofrequency plasma glow-discharge(acrylic acid and oxygen: 50 W, 0.2 torr for 30 seconds) but the control group (n=8) was treated with nothing. 1 x 10(6)ml/20 microliter of P3 chondrocytes from rabbit ears were used for seeding. Eight hours after cell seeding, the total DNA amount of chondrocytes attached to the PLGA and the changes of actin were evaluated under a confocal microscope. Type I and II collagen expression were detected by RT-PCR three weeks after seeding for an evaluation of phenotypic maintenance. The total DNA amount of attached chondrocytes was remarkably increased in the experimental group(p < 0.05). After scrutinizing with a confocal microscope, the actin of cells was more spread out and finely arranged in the experimental group than in the control group. Both types of collagen expression were significantly increased in their assigned groups. Plasma treatment of the PLGA sponge could increase the adhesion property of chondrocytes, and provide suitable environment for maintaining the phenotype of chondrocytes.
Subject(s)
Actins , Chondrocytes , Collagen , DNA , Ear , Oxygen , Phenotype , Plasma , Polymers , PoriferaABSTRACT
In many tissue engineering application, highly open porous scaffolds are required for efficient cell seeding and culture. Synthetic biodegradable polymers such as poly (L-lactic acid)(PLLA) and its copolymers with D-lactic and glycolic acids(PLGA) are widely used as a porous scaffold. The suitable biodegradability and dimensional stability of porous scaffolds during in vivo implantation play an important role in tissue engineering application. In this study, we investigated in vivo biodegradation and dimensional stability of acellular porous polymer scaffolds prepared by using a gas foaming technique with non-toxic effervescent mixture. In addition, we have engineered cartilage tissue 3D cultured on PLGA scaffolds in nude mouse in order to compare with degradation and deformation on acellular porous polymer scaffolds and to form tissue-engineered cartilage tissue. Sodium bicarbonate and citric acid crystals were used as an effervescent mixture. These particles were milled and sieved to yield various range of sizes(50 - 100, 100 - 300, and > 300 micrometer). After polymer scaffolds fabricated, biodegradation test was performed in subcutaneous tissue of male rats during 12 weeks. Degradability of polymer scaffolds were evaluated by weight difference, gel permeation chromatography(GPC), and SEM as each period. Tissue-engineered cartilage by transplanting 3D cultured chondrocytes onto PLGA 85:15 scaffolds in nude mouse was also made and compared with acellular scaffolds. In conclusion, highly open porous biodegradable scaffolds are prepared by gas foaming method using sodium bicarbonate and citric acid as a non-toxic effervescent mixture. Furthermore, tissue-engineered cartilage formation by in vivo 3D culture onto modified PLGA scaffolds in nude mouse was significantly improved as compared to controls.
Subject(s)
Animals , Humans , Male , Mice , Rats , Cartilage , Chondrocytes , Citric Acid , Mice, Nude , Polymers , Porifera , Sodium Bicarbonate , Subcutaneous Tissue , Tissue EngineeringABSTRACT
For tissue-engineered neocartilage formation in vivo, most studies have cultured chondrocytes within a biodegradable polymer in vitro before implantation of cell-polymer complex into an animal. The present study was performed to investigate the necessity of in vitro culture (preconditioning). Cell-polymer complex was made of chondrocytes obtained from a rabbit ear and a 85:15 poly(DL-lactic-co- glycolic acid) (PLGA) sponge. The complex was implanted into a nude mouse either after or without in vitro preconditioning. For control groups, PLGA sponge without chondrocytes was used. One, 2, and 4 weeks after the implantation, each group was examined by measurement of weight and volume of sponges as well as histologic study with Safranin-O staining. The control groups showed loss of weight as time passed. The non-preconditioned group, on the other hand, showed weight loss for the first week, but increased in weight afterwards. The preconditioned group also had weight loss in the first week after the implantation with no noticeable weight changes thereafter. Neither weight nor volume of PLGA sponges in preconditioned group was significantly different from those in non-preconditioned group until the 2nd week. In the 4th week, volume of sponges in non-preconditioned group was significantly larger than that in preconditioned group. On histological observation, chondrocytes seeded into a PLGA sponges proliferated and differentiated into cartilage tissues both in preconditioned and non-preconditioned groups, but non-preconditioned group formed cartilage tissue more extensively than the preconditioned group. Based on the above results, it is suggested that new cartilage tissues can be formed successfully following the implantation of cell- polymer complex into a living body without any prior conditioning in a separate culture system.