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Titanium has a long history of clinical use, but the naturally forming oxide is not ideal for bacterial resistance. Anodization processes can modify the crystallinity, surface topography, and surface chemistry of titanium oxides. Anatase, rutile, and mixed phase oxides are known to exhibit photocatalytic activity (PCA)-driven bacterial resistance under UVA irradiation. Silver additions are reported to enhance PCA and reduce bacterial attachment. This study investigated the effects of silver-doping additions to three established anodization processes. Silver doping showed no significant influence on oxide crystallinity, surface topography, or surface wettability. Oxides from a sulfuric acid anodization process exhibited significantly enhanced PCA after silver doping, but silver-doped oxides produced from phosphoric-acid-containing electrolytes did not. Staphylococcus aureus attachment was also assessed under dark and UVA-irradiated conditions on each oxide. Each oxide exhibited a photocatalytic antimicrobial effect as indicated by significantly decreased bacterial attachment under UVA irradiation compared to dark conditions. However, only the phosphorus-doped mixed anatase and rutile phase oxide exhibited an additional significant reduction in bacteria attachment under UVA irradiation as a result of silver doping. The antimicrobial success of this oxide was attributed to the combination of the mixed phase oxide and higher silver-doping uptake levels.
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Clinical and basic science applications using adipose-derived stem cells (ADSCs) are gaining popularity. The current adipose tissue harvesting procedures introduce nonphysiological conditions, which may affect the overall performance of the isolated ADSCs. In this study, we elucidate the differences between ADSCs isolated from adipose tissues harvested within the first 5 min of the initial surgical incision (well-vascularized, nonpremedicated condition) versus those isolated from adipose tissues subjected to medications and deprived of blood supply during elective free flap procedures (ischemic condition). ADSCs isolated from well-vascularized and ischemic tissues positively immunostained for several standard stem cell markers. Interestingly, the percent change in the CD36 expression for ADSCs isolated from ischemic versus well-vascularized tissue was significantly lower in males than females (p < 0.05). Upon differentiation and maturation to adipocytes, spheroids formed using ADSCs isolated from ischemic adipose tissue had lower triglyceride content compared to those formed using ADSCs isolated from the well-vascularized tissue (p < 0.05). These results indicate that ADSCs isolated from ischemic tissue either fail to uptake fatty acids or fail to efficiently convert those fatty acids into triglycerides. Therefore, more robust ADSCs suitable to establish in vitro adipose tissue models can be obtained by harvesting well-vascularized and nonpremedicated adipose tissues.
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Using a three-dimensional (3-D) in vitro culture model, we report the dose dependent effect of 17ß-estradiol and testosterone on the adipogenic differentiation and maturation of human adipose derived stem cells (hASCs) obtained from female and male patients. Considering sexual dimorphism, we expected male and female adipocytes to respond differently to the sex steroids. Both male and female hASC spheroids were exposed to 100 nM and 500 nM of 17ß-estradiol and testosterone either at the beginning of the adipogenic maturation (Phase I) to discourage intracellular triglyceride accumulation or exposed after adipogenic maturation (Phase II) to reduce the intracellular triglyceride accumulation. The results show that 17ß-estradiol leads to a dose dependent reduction in intracellular triglyceride accumulation in female hASC spheroids compared to the both untreated and testosterone-treated cells. Affirming our hypothesis, 17ß-estradiol prevented intracellular triglyceride accumulation during Phase I, while it stimulated lipolysis during Phase II. PPAR-γ and adiponectin gene expression also reduced upon 17ß-estradiol treatment in female cells. Interestingly, 17ß-estradiol and testosterone had only a modest effect on the male hASC spheroids. Collectively, our findings suggest that 17ß-estradiol can prevent fat accumulation in adipocytes during early and late stages of maturation in females.
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Adipogenia , Adiponectina , Estradiol , Caracteres Sexuais , Testosterona , Humanos , Adipogenia/efeitos dos fármacos , Masculino , Feminino , Estradiol/farmacologia , Testosterona/farmacologia , Adiponectina/metabolismo , Triglicerídeos/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/citologia , Células Cultivadas , PPAR gama/metabolismo , PPAR gama/genética , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipócitos/citologia , Células-Tronco/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/citologia , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Tecido Adiposo/efeitos dos fármacos , Lipólise/efeitos dos fármacosRESUMO
OBJECTIVES: Guided Tissue Regeneration (GTR) is a popular clinical procedure for periodontal tissue regeneration. However, its key component, the barrier membrane, is largely collagen-based and is still quite expensive, posing a financial burden to the patients as well as healthcare systems and negatively impacting the patient's decision-making. Thus, our aim is to prepare a novel biomimetic GTR membrane utilizing a natural biomaterial, soluble eggshell membrane protein (SEP), which is economical as it comes from an abundant industrial waste from food and poultry industries, unlike collagen. Additive polymer, poly (lactic-co-glycolic acid) (PLGA), and a bioceramic, nano-hydroxyapatite (HAp), were added to improve its mechanical and biological properties. METHODS: For this barrier membrane preparation, we initially screened the significant factors affecting its mechanical properties using Taguchi orthogonal array design and further optimized the significant factors using response surface methodology. Furthermore, this membrane was characterized using SEM, EDAX, and ATR-FTIR, and tested for proliferation activity of human periodontal ligament fibroblasts (HPLFs). RESULTS: Optimization using response surface methodology predicted that the maximal tensile strength of 3.1 MPa and modulus of 39.9 MPa could be obtained at membrane composition of 8.9 wt% PLGA, 7.2 wt% of SEP, and 2 wt% HAp. Optimized PLGA/SEP/HAp membrane specimens that were electrospun on a static collector showed higher proliferation activity of HPLFs compared to tissue culture polystyrene and a commercial collagen membrane. SIGNIFICANCE: From the results observed, we can conclude that SEP-based nanofibrous GTR membrane could be a promising, environment-friendly, and cost-effective alternative for commercial collagen-based GTR membrane products.
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Materiais Biocompatíveis , Regeneração Tecidual Guiada , Animais , Humanos , Materiais Biocompatíveis/farmacologia , Casca de Ovo , Teste de Materiais , Colágeno , DurapatitaRESUMO
Titanium anodization has been shown to produce crystalline oxides exhibiting photocatalytic reactions that form reactive oxygen species (ROS) when exposed to UV light. The ROS subsequently attack bacteria cells, and thus reduce bacteria attachment on titanium implant surfaces. Polyaniline (PANI) is a conductive polymer that has shown antibacterial properties when electropolymerized onto titanium. Our research group hypothesized the addition of PANI to crystalline titanium oxide surfaces would increase the available free electrons and thus increase photocatalytic activity (PCA). This research led to the development of a novel single-step anodization approach for PANI doping crystalline titanium oxide layers. The objective of the present study was to determine the proper aniline electrolyte concentration needed to maximize the PCA and reduce bacterial attachment on the formed oxides. Aniline concentrations up to 1 M were added into a 1 M sulfuric acid electrolyte. The formed oxides exhibited increased PANI surface coverage but decreased anatase and rutile crystalline titanium oxide phase formation with increasing aniline electrolyte concentrations. Despite exhibiting the lowest levels of anatase and rutile formation, the 0.75 M and 1 M aniline oxides with the greatest PANI surface coverage also exhibited the highest PCA levels. 1 M aniline oxides showed significantly higher PCA under UVA irradiation compared to oxides formed from aniline concentrations up to 0.5 M (p < 0.001). 0.75 M aniline oxides exhibited significant reductions in Staphylococcus aureus attachment with or without UVA irradiation compared to control oxides without PANI. MTT and live/dead assays confirmed cytocompatibility and nearly 100% cell viability for the PANI doped oxides.
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Óxidos , Titânio , Titânio/farmacologia , Titânio/química , Espécies Reativas de Oxigênio , Óxidos/química , Compostos de Anilina/farmacologia , Compostos de Anilina/química , Antibacterianos/farmacologiaRESUMO
Critical-sized cranial bone defects fail to re-ossify and require the surgical intervention of cranioplasty. To achieve superior bone healing in such cases, a hydrogel consisting of an interpenetrating network of collagen and elastin-like polypeptide to encapsulate bone morphogenetic protein-2 (BMP-2), doxycycline, and 45S5 Bioglass is developed. This hydrogel has an appropriate elastic modulus of 39 ± 2.2 kPa to allow proper handling during implantation. The hydrogel promotes human adipose-derived stem attachment, proliferation, and differentiation toward the osteogenic lineage, including the deposition of hydroxyapatite particles embedded within a collagenous fibrillar structure after 21 days of in vitro culture. After eight weeks of implantation of the acellular hydrogel in a critical-sized rat cranial defect model, only a small quantity of various pro-inflammatory (< 20 pg mg-1 ) and anti-inflammatory (< 10 pg mg-1 ) factors in the adjacent cranial tissue is noticed, indicating the overall biocompatibility of the hydrogel. Scanning electron microscopy evidenced the presence of new fibrous extracellular matrix and mineral aggregates at the defect site, with calcium/phosphorus ratio of 0.5 and 2.0 by eight and twelve weeks, respectively. Microcomputed tomography (Micro-CT) and histological analyses showed formation of mature mineralized tissue that bridged with the surrounding bone. Taken together, the acellular composite hydrogel shows great promise for superior bone healing after cranioplasty.
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Elastina , Hidrogéis , Ratos , Humanos , Animais , Hidrogéis/farmacologia , Hidrogéis/química , Elastina/farmacologia , Elastina/química , Microtomografia por Raio-X , Regeneração Óssea , Osteogênese , Peptídeos , Colágeno/farmacologia , Colágeno/química , Crânio/diagnóstico por imagem , Proteína Morfogenética Óssea 2/farmacologia , Diferenciação CelularRESUMO
Glioblastoma (GBM) is a primary brain tumor that carries a dismal prognosis, which is primarily attributed to tumor recurrence after surgery and resistance to chemotherapy. Since the tumor recurrence appears near the site of surgical resection, a concept of immediate and local application of chemotherapeutic after initial tumor removal could lead to improved treatment outcome. With the ultimate goal of developing a locally-applied, injectable drug delivery vehicle for GBM treatment, we created elastin-like polypeptide (ELP) hydrogels. The ELP hydrogels can be engineered to release anti-cancer drugs over an extended period. The purpose of this study was to evaluate the biomechanical properties of ELP hydrogels, to characterize their ability to release doxorubicin over time, and to investigate, in vitro, the anti-proliferative effect of Dox-laden ELP hydrogels on GBM. Here, we present microstructural differences, swelling ratio measurements, drug release characteristics, and in vitro effects of different ELP hydrogel compositions. We found that manipulation of the ELP-collagen ratio allows for tunable drug release, that the released drug is taken up by cells, and that incubation with a small volume of ELP-Dox hydrogel drastically reduced survival and proliferation of GBM cells in vitro. These results underscore the potential of ELP hydrogels as a local delivery strategy to improve prognosis for GBM patients after tumor resection.
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The design and development of multifunctional nanoparticles have attracted great interest in biomedical research. This study aims to prepare pH-responsive melanin-like nanoparticles for T1-weighted magnetic resonance imaging (MRI) and photothermal therapy. The new multifunctional nanoparticles (amino-Fe-PDANPs) are synthesized by copolymerization of dopamine and its derivative amino-N-[2-(diethylamino) ethyl]-3,4-dihydroxy-benzenepropanamide (N-Dopa) at room temperature. The size of nanoparticles can be controlled by NaOH concentration. The incorporation of N-Dopa is characterized by NMR and FT-IR. From transmission electron microscopy (TEM), the nanoparticles exhibit excellent dispersion stability in water and are spherical in shape. The MRI measurement has demonstrated that amino-Fe-PDANPs have a significant signal enhancement in responding to the acidic solution. Confirmed by the photothermal study, the nanoparticles exhibit a high photothermal conversion efficiency. The melanin-like multifunctional nanoparticles integrate both diagnosis and therapeutic functionalities, indicating the potential for theranostic application.
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Elastin-like polypeptides (ELP) have been used as a genetically-engineered, biocompatible substitute for elastin. Cell culture coatings prepared using ELP conjugated to low molecular weight polyethyleneimine (PEI) entices cells to form three-dimensional cellular aggregates that mimic their in vivo counterparts. This study seeks to control the deposition of the ELP and ELP-PEI molecules to control the roughness of the final coatings. The two polymers were coated onto three different substrates (glass, polystyrene, tissue-culture polystyrene) and the solution environment was altered by changing the polymer concentration (0.5, 1.0, 1.5 mg/mL) and/or salt concentration (None, 0.2 M phosphate buffered saline) for a total of 36 conditions. Atomic force microscopy (AFM) was used to measure the average roughness (Ra) of the samples and found that ELP coated samples had a higher Ra than their ELP-PEI counterparts. The coatings were tested for stability by performing cell culture media changes every three days for 11 days. AFM showed that the average roughness of the tested samples increased with each media change. To address this, the surfaces were crosslinked using hexamethyl diisocyanate, which minimized the change in surface roughness even when subjected to an intense sonication process. This study provides parameters to achieve elastin-based coatings with controlled roughness that can be used to support stable, long-term in vitro cell culture.
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Materiais Revestidos Biocompatíveis/química , Elastina/química , Peptídeos/química , Técnicas de Cultura de Células , Reagentes de Ligações Cruzadas/química , Microscopia de Força Atômica , Propriedades de SuperfícieRESUMO
As life expectancy and the age of the general population increases so does the need for improved implants. A major contributor to the failure of implants is poor osseointegration, which is typically described as the direct connection between bone and implant. This leads to unnecessary complications and an increased burden on the patient population. Modification of the implant surfaces through novel techniques, such as varying topography and/or applying coatings, has become a popular method to enhance the osseointegration capability of implants. Recent research has shown that particular surface features influence how bone cells interact with a material; however, it is unknown which exact features achieve optimal bone integration. In this review, current methods of modifying surfaces will be highlighted, and the resulting surface characteristics and biological responses are discussed. Review of the current strategies of surface modifications found that many coating types are more advantageous when used in combination; however, finding a surface modification that utilizes the mutual beneficial effects of important surface characteristics while still maintaining commercial viability is where future challenges exist.
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Materiais Revestidos Biocompatíveis , Osseointegração , Próteses e Implantes , Animais , Humanos , Propriedades de SuperfícieRESUMO
Thermosensitive chitosan hydrogels-renewable, biocompatible materials-have many applications as injectable biomaterials for localized drug delivery in the treatment of a variety of diseases. To combat infections such as Staphylococcus aureus osteomyelitis, localized antibiotic delivery would allow for higher doses at the site of infection without the risks associated with traditional antibiotic regimens. Fosfomycin, a small antibiotic in its own class, was loaded into a chitosan hydrogel system with varied beta-glycerol phosphate (ß-GP) and fosfomycin (FOS) concentrations. The purpose of this study was to elucidate the interactions between FOS and chitosan hydrogel. The Kirby Bauer assay revealed an unexpected concentration-dependent inhibition of S. aureus, with reduced efficacy at the high FOS concentration but only at the low ß-GP concentration. No effect of FOS concentration was observed for the planktonic assay. Rheological testing revealed that increasing ß-GP concentration increased the storage modulus while decreasing gelation temperature. NMR showed that FOS was removed from the liquid portion of the hydrogel by reaction over 12 h. SEM and FTIR confirmed gels degraded and released organophosphates over 5 days. This work provides insight into the physicochemical interactions between fosfomycin and chitosan hydrogel systems and informs selection of biomaterial components for improving infection treatment.
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Antibacterianos/administração & dosagem , Quitosana/química , Fosfomicina/administração & dosagem , Glicerofosfatos/química , Antibacterianos/química , Antibacterianos/farmacologia , Sistemas de Liberação de Medicamentos , Fosfomicina/química , Fosfomicina/farmacologia , Hidrogéis , Reologia , Staphylococcus aureus/efeitos dos fármacos , Temperatura , Fatores de TempoRESUMO
This paper evaluates the aggregation behavior of a potential drug and gene delivery system that combines branched polyethyleneimine (PEI), a positively-charged polyelectrolyte, and elastin-like polypeptide (ELP), a recombinant polymer that exhibits lower critical solution temperature (LCST). The LCST behavior of ELP has been extensively studied, but there are no quantitative ways to control the size of aggregates formed after the phase transition. The aggregate size cannot be maintained when the temperature is lowered below the LCST, unless the system exhibits hysteresis and forms irreversible aggregates. This study shows that conjugation of ELP with PEI preserves the aggregation behavior that occurs above the LCST and achieves precise aggregate radii when the solution conditions of pH (3, 7, 10), polymer concentration (0.1, 0.15, 0.3 mg/mL), and salt concentration (none, 0.2, 1 M) are carefully controlled. K-means cluster analyses showed that salt concentration was the most critical factor controlling the hydrodynamic radius and LCST. Conjugating ELP to PEI allowed crosslinking the aggregates and achieved stable particles that maintained their size below LCST, even after removal of the harsh (high salt or pH) conditions used to create them. Taken together, the ability to control aggregate sizes and use of crosslinking to maintain stability holds excellent potential for use in biological delivery systems.
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Sistemas de Liberação de Medicamentos , Elastina/química , Partículas Elementares/uso terapêutico , Aprendizado de Máquina , Temperatura Baixa , Elastina/uso terapêutico , Técnicas de Transferência de Genes , Humanos , Hidrodinâmica , Interações Hidrofóbicas e Hidrofílicas , Peptídeos/química , Transição de Fase , Temperatura , Temperatura de TransiçãoRESUMO
Elastin-like polypeptides (ELP) belong to a family of recombinant polymers that shows great promise as biocompatible drug delivery and tissue engineering materials. ELPs aggregate above a characteristic transition temperature (Tt). We have previously shown that the Tt and size of the resulting aggregates can be controlled by changing the ELP's solution environment (polymer concentration, salt concentration, and pH). When coupled to a synthetic polyelectrolyte, polyethyleneimine (PEI), ELP retains its Tt behavior and gains the ability to be crosslinked into defined particle sizes. This paper explores several machine learning models to predict the Tt and hydrodynamic radius (Rh) of ELP and two ELP-PEI polymers in varying solution conditions. An exhaustive design of experiments matrix consisting of 81 conditions of interest with varying salt concentration (0, 0.2, 1 M NaCl), pH (3, 7, 10), polymer concentration (0.1, 0.17, 0.3 mg/mL), and polymer type (ELP, ELP-PEI800, ELP-PEI10K) was investigated. The five models used in this study were multiple linear regression, elastic-net, support vector regression, multi-layer perceptron, and random forest. A multi-layer perceptron model was found to have the highest accuracy, with an R2 score of 0.97 for both Rh and Tt. This was followed closely by the random forest model, with an R2 of 0.94 for Rh and 0.95 for Tt. Feature importance was determined using the random forest and linear regression models. Both models showed that salt concentration and polymer type were the two most influential factors that determined Rh, while salt concentration was the dominant factor for Tt.
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Hidrodinâmica , Rádio (Anatomia) , Algoritmos , Elastina , Aprendizado de Máquina , Temperatura , Temperatura de TransiçãoRESUMO
This study aimed to probe the effect of formulation of scaffolds prepared using collagen and elastin-like polypeptide (ELP) and their resulting physico-chemical and mechanical properties on the adipogenic differentiation of human adipose derived stem cells (hASCs). Six different ELP-collagen scaffolds were prepared by varying the collagen concentration (2 and 6 mg/mL), ELP addition (6 mg/mL), or crosslinking of the scaffolds. FTIR spectroscopy indicated secondary bonding interactions between collagen and ELP, while scanning electron microscopy revealed a porous structure for all scaffolds. Increased collagen concentration, ELP addition, and presence of crosslinking decreased swelling ratio and increased elastic modulus and compressive strength of the scaffolds. The scaffold characteristics influenced cell morphology, wherein the hASCs seeded in the softer, non-crosslinked scaffolds displayed a spread morphology. We determined that stiffer and/or crosslinked elastin-collagen based scaffolds constricted the spreading of hASCs, leading to a spheroid morphology and yielded an enhanced adipogenic differentiation as indicated by Oil Red O staining. Overall, this study underscored the importance of spheroid morphology in adipogenic differentiation, which will allow researchers to create more physiologically-relevant three-dimensional, in vitro culture models.
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Studies have shown ultraviolet-A (UVA) irradiation of crystalline titanium oxides leads to the production of reactive oxygen species (ROS) via a photocatalytic process. The ROS exhibit antimicrobial properties that may be of benefit in preventing bacterial attachment to implant devices. Recent studies have suggested a potential benefit of mixed anatase and rutile oxides and dopants on the photocatalytic properties of titanium oxides. The goal of this work was to compare the photocatalytic activity of different anodized commercially pure titanium grade 4 (CPTi4) surfaces. CPTi4 specimens were anodized in three mixed-acid electrolytes to create crystalline oxide surfaces that were either primarily anatase, primarily rutile, or a combination of anatase and rutile. Additionally, the primarily anatase and combination oxides incorporated some phosphorous from the phosphoric acid component in the electrolyte. The photocatalytic activity of the anodized specimens was measured using both methylene blue (MB) degradation assay and comparing the attachment of S. aureus under irradiation with UVA light of differing intensities (1 mW/cm2, 8 mW/cm2, and 23 mW/cm2). Primarily rutile oxides exhibited significantly higher levels of MB degradation after exposure to 1 mW/cm2 UVA lights. Primarily rutile specimens also had the largest reduction in bacterial attachment followed by the mixed phase specimens and the primarily anatase specimens at 1 mW/cm2 UVA lights. Phosphorous-doped, mixed phase oxides exhibited an accelerated MB degradation response during exposure to 8 mW/cm2 and 23 mW/cm2 UVA lights. All anodized and unanodized CPTi4 groups revealed similar S. aureus attachment at the two higher UVA intensities. Although MB degradation assay and the bacteria attachment assay both confirmed photocatalytic activity of the oxides formed in this study, the results of the MB degradation assay did not accurately predict the oxides performance against S. aureus.
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Antibacterianos/química , Titânio/química , Antibacterianos/farmacologia , Catálise , Azul de Metileno/química , Oxirredução , Processos Fotoquímicos , Espécies Reativas de Oxigênio/química , Staphylococcus aureus/efeitos dos fármacos , Propriedades de Superfície , Titânio/farmacologia , Raios UltravioletaRESUMO
While three-dimensional spheroids outperform traditional two-dimensional monolayer culture for human adipose-derived stem cells (hASCs), there is not a consensus on the most successful method for enhancing their adipogenic differentiation and minimizing the loss of physiologically relevant, fatty spheroids during culture. To this end, we compared three culture methods, namely, elastin-like polypeptide-polyethyleneimine (ELP-PEI) coated surfaces, ultra-low attachment static culture, and suspension culture for their ability to form and retain productive hASC spheroids. The ELP-PEI coatings used the ELP conjugated to two molecular weights of PEI (800 or 25,000 g/mol). FTIR spectroscopy, atomic force microscopy, and contact angle goniometry revealed that the ELP-PEI coatings had similar chemical structures, surface topography, and hydrophobicity. Time-lapse microscopy showed that increasing the PEI molecular weight resulted in smaller spheroids. Measurement of triglyceride content showed that the three methods induced comparable differentiation of hASCs toward the adipogenic lineage. DNA content and morphometric analysis revealed merging of spheroids to form larger spheroids in the ultra-low attachment static culture and suspension culture methods. In contrast, the retention of hASC spheroid sizes and numbers with a regular spheroid size (~100 µm) were best atop the ELP-PEI800 coatings. Overall, this research shows that the spheroid culture atop the ELP-PEI coatings is a suitable cell culture model for future studies involving long-term, three-dimensional culture of mature adipocytes derived from hASCs.
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Adipócitos/metabolismo , Adipogenia , Tecido Adiposo/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Esferoides Celulares/metabolismo , Adipócitos/citologia , Tecido Adiposo/citologia , Materiais Revestidos Biocompatíveis/química , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Esferoides Celulares/citologiaRESUMO
Previously, we found that elastin-like polypeptide (ELP), when dried above the lower critical solution temperature on top of a hydrophilic fused silica disk, exhibited a dynamic coalescence behavior. The ELP initially wet the silica, but over the next 12 h, dewett the surface and formed aggregates of precise sizes and shapes. Using Fourier-transform infrared (FT-IR) spectroscopy, the present study explores the role of secondary structures present in ELP during this progressive desiccation and their effect on aggregate size. The amide I peak (1600-1700 cm-1) in the ELP's FT-IR spectrum was deconvoluted using the second derivative method into eight subpeaks (1616, 1624, 1635, 1647, 1657, 1666, 1680, 1695 cm-1). These peaks were identified to represent extended strands, ß-turns, 3(10)-helix, polyproline I, and polyproline II using previous studies on ELP and molecules similar in peptide composition. Positive correlations were established between the various subpeaks, water content, and aggregate size to understand the contributions of the secondary structures in particle formation. The positive correlations suggest that type II ß-turns, independent of the water content, contributed to the growth of the aggregates at earlier time points (1-3.5 h). At later time points (6-12 h), the aggregate growth was attributed to the formation of 3(10)-helices that relied on a decrease in water content. Understanding these relationships gives greater control in creating precisely sized aggregates and surface coatings with varying roughness.
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We have developed a multicomponent hydrogel scaffold that can mimic the bone extracellular matrix by incorporating collagen, elastin-like polypeptide (ELP), and Bioglass. We examined the effects of Bioglass addition to collagen-ELP scaffolds on mechanical properties, physical characteristics, and in vitro osteogenic differentiation, by varying the Bioglass amount and particle size. Response surface methodology with a central composite design predicted 5 mg (6.6 mg/mL) Bioglass with a particle size of 142 ± 5 µm as the optimal amount and particle size to be mixed with 6 mg/mL collagen and 18 mg/mL ELP to obtain a combination of maximized compressive properties. Swelling ratio and FTIR spectroscopy indicated lower hydrophilicity and the presence of hydrophobic and secondary interactions between collagen, ELP, and Bioglass. Scanning electron microscopy showed a nanofibrous morphology of intermingled collagen-ELP-Bioglass network. In vitro osteogenic characterization using human adipose-derived stem cells revealed increased cell attachment and proliferation with increased ALP activity, osteocalcin content, and mineralized deposit formation during a three-week culture. Numerous mineralized deposits composed of calcium and phosphorous were shown by energy dispersive spectroscopy. Overall, our results show that the collagen-ELP-Bioglass multicomponent composites have enhanced mechanical properties with adequate physical features and cell culture properties for bone tissue engineering.
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The goals of this study are to evaluate the ability of the multicomponent collagen-elastin-like polypeptide (ELP)-Bioglass scaffolds to support osteogenesis of rat mesenchymal stem cells (rMSCs), demonstrate in vivo biocompatibility by subcutaneous implantation in Sprague-Dawley rats, monitor degradation noninvasively, and finally assess the scaffold's ability in healing critical-sized cranial bone defects. The collagen-ELP-Bioglass scaffold supports the in vitro osteogenic differentiation of rMSCs over a 3 week culture period. The cellular (rMSC-containing) or acellular scaffolds implanted in the subcutaneous pockets of rats do not cause any local or systemic toxic effects or tumors. The real-time monitoring of the fluorescently labeled scaffolds by IVIS reveals that the scaffolds remain at the site of implantation for up to three weeks, during which they degrade gradually. Micro-CT analysis shows that the bilateral cranial critical-sized defects created in rats lead to greater bone regeneration when filled with cellular scaffolds. Bone mineral density and bone microarchitectural parameters are comparable among different scaffold groups, but the histological analysis reveals increased formation of high-quality mature bone in the cellular group, while the acellular group has immature bone and organized connective tissue. These results suggest that the rMSC-seeded collagen-ELP-Bioglass composite scaffolds can aid in better bone healing process.