RESUMO
BACKGROUND: This study aims to investigate the effects of a conditioned medium (CM) from human umbilical cord mesenchymal stem cells (HuMSCs) cultivated in gelatin sponge (GS-HuMSCs-CM) on hair growth in a mouse model. METHODS: CM was collected from the HuMSCs cultivated in a monolayer or in a gelatin sponge. Vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF) levels in CMs were measured by enzyme-linked immunosorbent assays (ELISAs). A hair loss model by a C57 BL/6J mouse was prepared. The effects of GS-HuMSCs-CM and HuMSCs on hair regrowth in mice were investigated by intradermal injection in the depilated back skin with normal saline (NS) as the control. The time for hair regrowth and full covering in depilated areas was observed, and the hair growth was evaluated histologically and by grossly measuring hair length and diameter. RESULTS: Compared with monolayer cultured cells, the three-dimensional (3D) culture of HuMSCs in gelatin sponge drastically increased VEGF, IGF-1, KGF, and HGF production. GS-HuMSCs-CM and HuMSCs injection both promoted hair regeneration in mice, while GS-HuMSCs-CM presented more enhanced effects in hair length, hair diameter, and growth rate. GS-HuMSCs-CM significantly promoted angiogenesis in injected skin areas, which might also contribute to faster hair regrowth. CONCLUSION: GS-HuMSCs-CM exerted significant effects on inducing hair growth and promoted skin angiogenesis in C57BL/6J mice.
Assuntos
Cabelo , Fator de Crescimento Insulin-Like I , Células-Tronco Mesenquimais , Cordão Umbilical , Animais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Humanos , Meios de Cultivo Condicionados/farmacologia , Camundongos , Cordão Umbilical/citologia , Cabelo/crescimento & desenvolvimento , Cabelo/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Gelatina/química , Alicerces Teciduais/química , Camundongos Endogâmicos C57BL , Células Cultivadas , Fator 7 de Crescimento de Fibroblastos/metabolismoRESUMO
BACKGROUND: In the domain of plastic surgery, nasal cartilage regeneration is of significant importance. The extracellular matrix (ECM) from porcine nasal septum cartilage has shown potential for promoting human cartilage regeneration. Nonetheless, the specific biological inductive factors and their pathways in cartilage tissue engineering remain undefined. METHODS: The decellularized matrix derived from porcine nasal septum cartilage (PN-DCM) was prepared using a grinding method. Human umbilical cord mesenchymal stem cells (HuMSCs) were cultured on these PN-DCM scaffolds for 4 weeks without exogenous growth factors to evaluate their chondroinductive potential. Subsequently, proteomic analysis was employed to identify potential biological inductive factors within the PN-DCM scaffolds. RESULTS: Compared to the TGF-ß3-cultured pellet model serving as a positive control, the PN-DCM scaffolds promoted significant deposition of a Safranin-O positive matrix and Type II collagen by HuMSCs. Gene expression profiling revealed upregulation of ACAN, COL2A1, and SOX9. Proteomic analysis identified potential chondroinductive factors in the PN-DCM scaffolds, including CYTL1, CTGF, MGP, ITGB1, BMP7, and GDF5, which influence HuMSC differentiation. CONCLUSION: Our findings have demonstrated that the PN-DCM scaffolds promoted HuMSC differentiation towards a nasal chondrocyte phenotype without the supplementation of exogenous growth factors. This outcome is associated with the chondroinductive factors present within the PN-DCM scaffolds.
Assuntos
Diferenciação Celular , Condrogênese , Células-Tronco Mesenquimais , Septo Nasal , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Septo Nasal/citologia , Septo Nasal/química , Animais , Suínos , Células Cultivadas , Alicerces Teciduais/química , Matriz Extracelular Descelularizada/química , Matriz Extracelular Descelularizada/farmacologia , Matriz Extracelular/metabolismo , Matriz Extracelular/química , Engenharia Tecidual , Cordão Umbilical/citologiaRESUMO
OBJECTIVE: To report the efficacy and long-term outcomes of treating the skin defects of aplasia cutis congenita (ACC) with cryopreserved amniotic membrane (AM). METHOD: Human amnion was obtained from the caesarean delivery of a full-term healthy pregnancy and processed in a sterile laminar flow hood, and cryopreserved in liquid nitrogen. The structure of the AM was investigated histologically and the viability of the epithelial cells was assessed after cryopreservation and compared with fresh AM and with AM preserved in phosphate-buffered saline (PBS) at 4°C. The cryopreserved AM was applied onto the lower limb skin defects of a one-month old baby with ACC. Timely AM changes were performed as necessary until the wounds healed. RESULTS: The structure of the cryopreserved AM was intact, with little visible difference compared with fresh AM. The viability of the epithelial cells was partially lost but still much better retained than in those preserved in PBS at 4°C. The limb skin defects were gradually re-epithelialised upon application of the AM and were completely healed after one month. The 4-month and 2-year follow-ups presented good skin texture and colour, without hypertrophic scar formation. CONCLUSION: In this case study, cryopreservation of AM presented a well preserved stromal compartment and viable epithelial layer. It also offered features such as pain relief, good attachment and adhesiveness, improved wound healing and suppressed scar formation in the treatment of ACC skin defects.
Assuntos
Âmnio , Displasia Ectodérmica , Gravidez , Feminino , Humanos , Lactente , Criopreservação , Pele , Células Epiteliais , Displasia Ectodérmica/terapiaRESUMO
BACKGROUND: To maintain and enhance the wound healing effects of mesenchymal stem cells (MSCs), a scaffold for hosting MSCs is needed, which ought to be completely biocompatible, durable, producible, and of human source. OBJECTIVE: To build a cell-extracellular matrix (ECM) complex assembled by human umbilical cord mesenchymal stem cells (HuMSCs) and to investigate its clinical potentials in promoting wound healing. METHOD: HuMSCs were isolated and expanded. When the cells of third passage reached confluency, ascorbic acid was added to stimulate the cells to deposit ECM where the cells grew in. Four weeks later, a cells-loaded ECM sheet was formed. The cell-ECM complex was observed under the scanning electron microscopy (SEM) and subjected to histological studies. The supernatants were collected and the cell-ECM complex was harvested at different time points and processed for enzyme-linked immune sorbent assay (ELISA) and mRNA analysis. The in vivo experiments were performed by means of implanting the cell-ECM complex on the mice back for up to 6 months and the specimens were collected for histological studies. RESULTS: After 4 weeks of cultivation with ascorbic stimulation, a sheet was formed which is mainly composed with HuMSCs, collagen and hyaluronic acid. The cell-ECM complex can sustain to certain tensile force. The mRNA and protein levels of vascular endothelial growth factor-α (VEGF-α), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), and transforming growth factor-ß1 (TGF-ß1) were remarkably increased compared to monolayer-cultured cells. The implanted cell-ECM complex on mice was still noticeable with host cells infiltration and vascularization on 6 months. CONCLUSION: Our studies suggested that HuMSCs can be multi-cultivated through adding ascorbic stimulation and ECM containing collagen and hyaluronic acid were enriched around the cells which self-assembly formed a cell-ECM complex. Cell-ECM complex can improve growth factors secretion remarkably which means it may promote wound healing by paracrine.
Assuntos
Ácido Hialurônico , Fator A de Crescimento do Endotélio Vascular , Camundongos , Humanos , Animais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/fisiologia , Colágeno , Matriz ExtracelularRESUMO
BACKGROUND: Umbilical cord mesenchymal stem cell (HUCMSC)-based therapies were previously utilised for cartilage regeneration because of the chondrogenic potential of MSCs. However, chondrogenic differentiation of HUCMSCs is limited by the administration of growth factors like TGF-ß that may cause cartilage hypertrophy. It has been reported that extracellular vesicles (EVs) could modulate the phenotypic expression of stem cells. However, the role of human chondrogenic-derived EVs (C-EVs) in chondrogenic differentiation of HUCMSCs has not been reported. RESULTS: We successfully isolated C-EVs from human multi-finger cartilage and found that C-EVs efficiently promoted the proliferation and chondrogenic differentiation of HUCMSCs, evidenced by highly expressed aggrecan (ACAN), COL2A, and SOX-9. Moreover, the expression of the fibrotic marker COL1A and hypertrophic marker COL10 was significantly lower than that induced by TGF-ß. In vivo, C-EVs induced HUCMSCs accelerated the repair of the rabbit model of knee cartilage defect. Furthermore, C-EVs led to an increase in autophagosomes during the process of chondrogenic differentiation, indicating that C-EVs promote cartilage regeneration through the activation of autophagy. CONCLUSIONS: C-EVs play an essential role in fostering chondrogenic differentiation and proliferation of HUCMSCs, which may be beneficial for articular cartilage repair.
Assuntos
Autofagia/fisiologia , Cartilagem/metabolismo , Condrócitos/metabolismo , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Cordão Umbilical/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Condrócitos/citologia , Condrogênese , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Coelhos , Cordão Umbilical/citologiaRESUMO
BACKGROUND: Acetaminophen (APAP) overdose is the common cause of acute liver failure (ALF) due to the oxidative damage of multiple cellular components. This study aimed to investigate whether plasma membrane vesicles (PMVs) from human umbilical cord mesenchymal stem cells (hUCMSCs) could be exploited as a novel stem cell therapy for APAP-induced liver injury. METHODS: PMVs from hUCMSCs were prepared with an improved procedure including a chemical enucleation step followed by a mechanical extrusion. PMVs of hUCMSCs were characterized and supplemented to hepatocyte cultures. Rescue of APAP-induced hepatocyte damage was evaluated. RESULTS: The hUCMSCs displayed typical fibroblastic morphology and multipotency when cultivated under adipogenic, osteogenic, or chondrogenic conditions. PMVs of hUCMSCs maintained the stem cell phenotype, including the presence of CD13, CD29, CD44, CD73, and HLA-ABC, but the absence of CD45, CD117, CD31, CD34, and HLA-DR on the plasma membrane surface. RT-PCR and transcriptomic analyses showed that PMVs were similar to hUCMSCs in terms of mRNA profile, including the expression of stemness genes GATA4/5/6, Nanog, and Oct1/2/4. GO term analysis showed that the most prominent reduced transcripts in PMVs belong to integral membrane components, extracellular vesicular exosome, and extracellular matrix. Immunofluorescence labeling/staining and confocal microscopy assays showed that PMVs enclosed cellular organelles, including mitochondria, lysosomes, proteasomes, and endoplasmic reticula. Incorporation of the fusogenic VSV-G viral membrane glycoprotein stimulated the endosomal release of PMV contents into the cytoplasm. Further, the addition of PMVs and a mitochondrial-targeted antioxidant Mito-Tempo into cultures of APAP-treated HepG2 cells resulted in reduced cell death, enhanced viability, and increased mitochondrial membrane potential. Lastly, this study demonstrated that the redox state and activities of aminotransferases were restored in APAP-treated HepG2 cells. CONCLUSIONS: The results suggest that PMVs from hUCMSCs could be used as a novel stem cell therapy for the treatment of APAP-induced liver injury.
Assuntos
Acetaminofen , Células-Tronco Mesenquimais , Acetaminofen/toxicidade , Diferenciação Celular , Membrana Celular , Terapia Baseada em Transplante de Células e Tecidos , Células Hep G2 , Humanos , Cordão UmbilicalRESUMO
BACKGROUND: Nevus flammeus (NF) is a congenital vascular malformation. OBJECTIVES: To investigate the acute effect of a variable pulse width Nd:YAG laser combined with hematoporphyrin monomethyl ether (HMME)-mediated photodynamic therapy (PDT) on a cockscomb model of NF. MATERIAL AND METHODS: Forty-two leghorn roosters were randomly divided into the following 7 groups: group A1 (treated with HMME-mediated PDT; energy density of 75 J/cm2), group A2 (treated with HMME-mediated PDT; 125 J/cm2), group A3 (treated with HMME-mediated PDT; 150 J/cm2), group A4 (treated with HMME-mediated PDT; 175 J/cm2), group B (treated with a variable pulse width Nd:YAG laser), group C (treated with a variable pulse width Nd:YAG laser and HMME-mediated PDT), and group D (the control group). Changes in the cockscomb tissues were observed visually and microscopically on days 1, 3, 7, and 14 after treatment. The capillary reduction and the ratio of collagen type I to type III were examined. RESULTS: The response rate was higher in groups A3 and A4 than in group B. In group A, a higher energy density resulted in a higher response rate and a greater capillary reduction (p < 0.05 for all). However, we concluded that PDT at an energy density of 175 J/cm2 is not suitable for treating NF, as severe tissue damage, markedly lower capillary numbers, and markedly higher collagen type I:III ratios were observed in the cockscombs treated at this energy density; instead, 150 J/cm2 may be a more appropriate energy density. Moreover, HMME-mediated PDT at 150 J/cm2 combined with a variable pulse width Nd:YAG laser achieved better treatment outcomes than PDT or a variable pulse width Nd:YAG laser alone (p < 0.05 for both). CONCLUSIONS: Compared to PDT or a variable pulse width Nd:YAG laser alone, the combination of the 2 therapies achieved a better acute effect in treating a cockscomb model of NF, and 150 J/cm2 may be a suitable energy density for PDT.
Assuntos
Terapia a Laser , Lasers de Estado Sólido , Fotoquimioterapia , Mancha Vinho do Porto , Animais , Galinhas , Hematoporfirinas , Lasers de Estado Sólido/uso terapêutico , Masculino , Mancha Vinho do Porto/tratamento farmacológicoRESUMO
Star-block copolymers PEI-g-PZLL with a branched polyethylenimine (PEI) core and multiple grafted poly(ε-benzyloxycarbonyl-L-lysine) (PZLL) peripheral chains were designed, synthesized, and evaluated as nanocarriers for indomethacin (IND). In an aqueous solution, PEI-g-PZLL self-assembled into spherical nanoparticles capable of encapsulating IND at high loading capacity and loading efficiency. Differential scanning calorimetry and X-ray diffraction measurements indicated that IND was molecularly or amorphously dispersed in the nanoparticles. Fourier transform infrared spectra revealed the presence of multiple intermolecular interactions, including hydrogen bonding, electrostatic forces, π-π stacking and hydrophobic interactions, between the block copolymer and the IND molecules. IND-loaded nanoparticles exhibited fast release under intestinal pH. Compared with raw IND, the utilization of PEI-g-PZLL as a carrier significantly enhanced the oral bioavailability of IND and improved its protective effect on renal ischemia-reperfusion injury, as evidenced by in vivo pharmacokinetic and pharmacodynamic studies. Cytotoxicity assay, histological observation and cellular uptake study suggested that PEI-g-PZLL was fairly biocompatible. All these results indicated that star-block copolymers PEI-g-PZLL could be used as efficient nanocarriers for IND and other poorly water-soluble drugs. STATEMENT OF SIGNIFICANCE: The use of polyethylenimine (PEI) as an oral drug delivery carrier is limited because it is not biodegradable and the use of higher molecular weight PEI leads to improved efficiency but also increased cytotoxicity. The design of functionalized PEIs with low cytotoxicity and high efficiency is crucial for developing a successful oral drug delivery system. In our study, poly(ε-benzyloxycarbonyl-L-lysine) (PZLL)-grafted branched PEI (PEI-g-PZLL) was reported as an oral nanocarrier for indomethacin (IND). The low cytotoxicity and biodegradability, well-defined self-assembled nano-sized polymeric micelles, high loading capacity and loading efficiency, amorphous state of the encapsulated IND, as well as the enhanced oral bioavailability of IND, makes the copolymer PEI-g-PZLL a promising nanocarrier for the oral administration of IND and possibly other poorly water-soluble drugs.
Assuntos
Portadores de Fármacos/química , Indometacina/farmacologia , Nanopartículas/química , Polietilenoimina/química , Polilisina/análogos & derivados , Administração Oral , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Disponibilidade Biológica , Células CACO-2 , Varredura Diferencial de Calorimetria , Morte Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Endocitose/efeitos dos fármacos , Humanos , Indometacina/administração & dosagem , Indometacina/farmacocinética , Interleucina-6/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Nanopartículas/ultraestrutura , Polilisina/química , Ratos Sprague-Dawley , Espectroscopia de Infravermelho com Transformada de Fourier , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Objective: To investigate the feasibility of human amniotic membrane-living skin equivalent (AM-LSE) in repairing the skin defect. Methods: A 5-year-old boy with giant nevus at neck, shoulder, and back was admitted in July 2016. Normal skin tissue of the patient was harvested and keratinocytes and dermal fibroblasts were separated and expanded in vitro. Human AM was donated from a normal delivery and de-epithelialized for constructing an LSE as a matrix. Keratinocytes were seeded on the epithelial side of the AM which was previously seeded with fibroblasts on the stromal side and then the complex was lifted for air-liquid surface cultivation for 10 days and observed under naked eyes and sampled for histological study. The nevus was excised to deep fascia and the skin defect in size of 20 cm×15 cm was covered with artificial skin of collagen sponge for 2 weeks to enhance granulation tissue formation, and then the AM-LSE grafts of stamp size were grafted on. The dressing was changed until the wound healed. Results: After 10 days of air-liquid surface cultivation, the AM-LSE developed a multilayered and differentiated epidermis with the fibroblasts-populated amnion as the dermal matrix. The LSE stamps survived and expanded to cover the whole wound. The grafted area showed normal skin color and soft contexture at 6 months after operation, and histological study showed well developed epidermis with compactly aligned basal cells, stratified and well differentiated squamous, granular layers and stratum corneum and well vascularized dermal compartment without inflammatory cells infiltration. Conclusion: The cultivated AM-LSE with autologous cells can repair skin defect and survive for a long term without rejection.
Assuntos
Âmnio/transplante , Nevo Pigmentado/cirurgia , Pele Artificial , Pré-Escolar , Epiderme , Humanos , Queratinócitos , Masculino , PeleRESUMO
Polysaccharides from Grateloupia livida (Harv.) Yamada (GL) were extracted by a heating circumfluence method. Single-factor experiments were performed for the three parameters: extraction time (X1), extraction temperature (X2) and the ratio of water to raw material (X3) and their test range. From preliminary experimental results, one type of the response surface methodology, the Box-Behnken design was applied for the optimizing polysaccharide extraction conditions. The experimental data obtained were fitted to a second-order polynomial equation. The optimal conditions were extraction time 5 h, extraction temperature 100 °C and ratio of water to raw material 70 mL/g. Under these conditions, the experimental yield was 39.22% ± 0.09%, which well matched the predicted value (39.25%), with 0.9774 coefficient of determination (R²). GL polysaccharides had scavenging activities for DPPH and hydroxyl radicals in vitro. The scavenging rates for both radicals peaked at 20 mg/mL GL concentration. However, the positive standard, VC (ascorbic acid), possessed stronger antioxidant activities than GL polysaccharides. Furthermore, the anticancer activity of GL polysaccharides on HepG2 cell proliferation increased dose- and time-dependently, but the positive standard, 5-fluorouracil (5-fu) showed more significant anticancer activity in this study. Overall, GL polysaccharides may have potential applications in the medical and food industries.