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Baryte (BaSO4) is a critical raw material with no functional recycling since the used applications are dissipative. Significant quantities of baryte end up in tailings as a side stream from the mining industry. Baryte from a secondary raw material source was used as a filler in plastics for low duty radiation shielding and as an aggregate in radiation shielding geopolymers needed for safely storing low-radioactive waste ash. Mechanical strength in geopolymers remained at a high level with 0-50 % baryte additions. Low-cost plastic composites with baryte additions showed promising attenuation for X-rays and gamma-rays. The results showed improved qualities in the direct use of the secondary baryte material in concrete and plastics in comparison to primary baryte. Baryte from an industrial waste stream was shown to be applicable to be used in radiation shielding in geopolymers for storing low-radioactive waste, and in plastics. Primary baryte can be replaced with secondary baryte to bring environmental, economic, and even functional benefits.
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BACKGROUND: Patient-derived xenografts (PDXs) are considered to better recapitulate the histopathological and molecular heterogeneity of human cancer than other preclinical models. Despite technological advances, PDX models from hormone naïve primary prostate cancer are scarce. We performed a detailed analysis of PDX methodology using a robust subcutaneous model and fresh tissues from patients with primary hormone naïve prostate cancer. METHODS: Clinical prostate tumor specimens (n=26, Gleason score 6-10) were collected from robotic-assisted laparoscopic radical prostatectomies at Turku University Hospital (Turku, Finland), cut into pieces, and implanted subcutaneously into 84 immunodeficient mice. Engraftments and the adjacent material from prostatic surgical specimens were compared using histology, immunohistochemistry and DNA sequencing. RESULTS: The probability of a successful engraftment correlated with the presence of carcinoma in the implanted tissue. Tumor take rate was 41%. Surprisingly, mouse hormone supplementation inhibited tumor take rate, whereas the degree of mouse immunodeficiency did not have an effect. Histologically, the engrafted tumors closely mimicked their parental tumors, and the Gleason grades and copy number variants of the engraftments were similar to those of their primary tumors. Expression levels of androgen receptor, prostate-specific antigen, and keratins were retained in engraftments, and a detailed genomic analysis revealed high fidelity of the engraftments with their corresponding primary tumors. However, in the second or third passage of tumors, the carcinoma areas were almost completely replaced by benign tissue with frequent degenerative or metaplastic changes. CONCLUSIONS: Subcutaneous primary prostate engraftments preserve the phenotypic and genotypic landscape. Thus, they serve a potential model for personalized medicine and preclinical research but their use may be limited to the first passage.
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The Cellulose nanofibrils (CNF), also referred to as nanocellulose, is one of the most studied bio-based material in recent year, which has good potential in the future for packaging applications due to its excellent mechanical strength and oxygen barrier properties. In the future, CNF films may also find new applications for example in printed electronics, if the surface smoothness of CNF films can be improved. One way to improve surface smoothness is to use thin coating solutions with zero porosity, such as molar mass controlled cellulose ester coatings. In this study, we have coated CNF films using molar mass controlled cellulose esters with different side chain lengths forming 3-layer film (ester-CNF-ester). These coatings improved significantly the smoothness of CNF films. The 3-layer films have also good water vapor barrier and mechanical properties and the films are heat-sealable, which enable various new applications in the future.
Assuntos
Celulose/química , Ésteres/química , Nanofibras/química , Interações Hidrofóbicas e Hidrofílicas , Peso Molecular , PorosidadeRESUMO
We investigated the use of polypyrrole (PPy)-coated polymer scaffolds and electrical stimulation (ES) to differentiate adipose stem cells (ASCs) towards smooth muscle cells (SMCs). Since tissue engineering lacks robust and reusable 3D ES devices we developed a device that can deliver ES in a reliable, repeatable, and cost-efficient way in a 3D environment. Long pulse (1 ms) or short pulse (0.25 ms) biphasic electric current at a frequency of 10 Hz was applied to ASCs to study the effects of ES on ASC viability and differentiation towards SMCs on the PPy-coated scaffolds. PPy-coated scaffolds promoted proliferation and induced stronger calponin, myosin heavy chain (MHC) and smooth muscle actin (SMA) expression in ASCs compared to uncoated scaffolds. ES with 1 ms pulse width increased the number of viable cells by day 7 compared to controls and remained at similar levels to controls by day 14, whereas shorter pulses significantly decreased viability compared to the other groups. Both ES protocols supported smooth muscle expression markers. Our results indicate that electrical stimulation on PPy-coated scaffolds applied through the novel 3D ES device is a valid approach for vascular smooth muscle tissue engineering.
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Tecido Adiposo/metabolismo , Materiais Revestidos Biocompatíveis/química , Miócitos de Músculo Liso/metabolismo , Polímeros/química , Pirróis/química , Células-Tronco/metabolismo , Alicerces Teciduais/química , Tecido Adiposo/citologia , Adulto , Estimulação Elétrica , Feminino , Humanos , Pessoa de Meia-Idade , Miócitos de Músculo Liso/citologia , Células-Tronco/citologia , Engenharia Tecidual/métodosRESUMO
Tumorigenesis is a multistep process involving co-operation between several deregulated oncoproteins. In this study, we unravel previously unrecognized interactions and crosstalk between Pim kinases and the Notch signaling pathway, with implications for both breast and prostate cancer. We identify Notch1 and Notch3, but not Notch2, as novel Pim substrates and demonstrate that for Notch1, the serine residue 2152 is phosphorylated by all three Pim family kinases. This target site is located in the second nuclear localization sequence (NLS) of the Notch1 intracellular domain (N1ICD), and is shown to be important for both nuclear localization and transcriptional activity of N1ICD. Phosphorylation-dependent stimulation of Notch1 signaling promotes migration of prostate cancer cells, balances glucose metabolism in breast cancer cells, and supports in vivo growth of both types of cancer cells on chick embryo chorioallantoic membranes. Furthermore, Pim-induced growth of orthotopic prostate xenografts in mice is associated with enhanced nuclear Notch1 activity. Finally, simultaneous inhibition of Pim and Notch abrogates the cellular responses more efficiently than individual treatments, opening up new vistas for combinatorial cancer therapy.
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Neoplasias da Mama/patologia , Carcinogênese/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Animais , Movimento Celular , Embrião de Galinha , Feminino , Humanos , Células MCF-7 , Masculino , Camundongos , Fosforilação , Receptor Notch2/metabolismo , Receptor Notch3/metabolismo , Serina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Research on human enteroviruses has resulted in the identification of more than 100 enterovirus types, which use more than 10 protein receptors and/or attachment factors required in cell binding and initiation of the replication cycle. Many of these "viral" receptors are overexpressed in cancer cells. Receptor binding and the ability to replicate in specific target cells define the tropism and pathogenesis of enterovirus types, because cellular infection often results in cytolytic response, i.e., disruption of the cells. Viral tropism and cytolytic properties thus make native enteroviruses prime candidates for oncolytic virotherapy. Copy DNA cloning and modification of enterovirus genomes have resulted in the generation of enterovirus vectors with properties that are useful in therapy or in vaccine trials where foreign antigenic epitopes are expressed from or on the surface of the vector virus. The small genome size and compact particle structure, however, set limits to enterovirus genome modifications. This review focuses on the therapeutic use of native and recombinant enteroviruses and the methods that have been applied to modify enterovirus genomes for therapy.
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Enterovirus/genética , Enterovirus/fisiologia , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Recombinação Genética , Tropismo Viral , Animais , Humanos , Internalização do VírusRESUMO
Polypyrrole (PPy) has gained interest as an implant material due to its multifunctional properties and its high compatibility with several cell and tissue types. For the first time, the biocompatibility and osteointegration of PPy coating, incorporated with chondroitin sulfate (CS), were studied in vivo by implanting PPy-coated bioabsorbable bone fixation composite screws of poly-(lactide/glycolide) copolymer (PLGA) and ß-tricalcium phosphate (TCP) into New Zealand white rabbits. Uncoated bioabsorbable polymer composite screws and commercially available stainless steel cortical screws were used as reference implants. The rabbits were euthanized 12 and 26 weeks after the implantation. The systemic effects were evaluated from food and water consumption, body weight, body temperature, clinical signs, blood samples, internal organ weights, and histological examination. Local effects were studied from bone tissue and surrounding soft tissue histology. New bone formation was evaluated by micro-computed tomography, tetracycline labeling and torsion tests. Torsion tests were performed in order to capture the peak value of the torsion force during the course of the screw's loosening. The coated screws induced significantly more bone formation than the uncoated screws. In addition, none of the implants induced any systemic or local toxicity. The results suggest that PPy is biocompatible with bone tissue and is a potential coating for enhancing osteointegration in orthopedic implants.
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Parafusos Ósseos , Fosfatos de Cálcio/química , Materiais Revestidos Biocompatíveis/síntese química , Ácido Láctico/química , Osteogênese/fisiologia , Ácido Poliglicólico/química , Polímeros/química , Pirróis/química , Implantes Absorvíveis , Animais , Desenho de Equipamento , Análise de Falha de Equipamento , Teste de Materiais , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , CoelhosRESUMO
Polystyrene (PS, 1), polycaprolactone homopolymers (PCL, 2) and 3-Iodo-2-propynyl n-butylcarbamate (IPBC, 3) were physically mixed in dichloromethane (DCM) and processed into solid microspheres by using emulsion solvent evaporation method. Five different compositions with varying PS/PCL ratio were tested. The phase morphology of the microspheres was studied using Phase imaging atomic force microscopy (AFM) of polished cross-sections. Scanning electron microscopy was utilized to assess the distribution of IPBC in the polymer microspheres. The phase separation of the PS and PCL polymers in solvent cast films was assessed using polarized light optical microscopy of 11 polymer blends (0-100 wt-% PCL in PS). The PS/PCL-IPBC microspheres were incubated in water at RT and the release of IPBC was studied using high performance liquid chromatography (HPLC) at time points 1, 7 and 30 days. The microspheres dispersed in water borne outdoor paint matrix were tested for their antifouling activity against moulds in vitro.
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Incrustação Biológica/prevenção & controle , Carbamatos/administração & dosagem , Desinfetantes/administração & dosagem , Microesferas , Poliésteres/química , Poliestirenos/química , Carbamatos/toxicidade , Desinfetantes/toxicidade , Composição de Medicamentos , Fungos/efeitos dos fármacos , Transição de FaseRESUMO
Polypyrrole (PPy) is a conductive polymer that has aroused interest due to its biocompatibility with several cell types and high tailorability as an electroconductive scaffold coating. This study compares the effect of hyaluronic acid (HA) and chondroitin sulfate (CS) doped PPy films on human adipose stem cells (hASCs) under electrical stimulation. The PPy films were synthetized electrochemically. The surface morphology of PPy-HA and PPy-CS was characterized by an atomic force microscope. A pulsed biphasic electric current (BEC) was applied via PPy films non-stimulated samples acting as controls. Viability, attachment, proliferation and osteogenic differentiation of hASCs were evaluated by live/dead staining, DNA content, Alkaline phosphatase activity and mineralization assays. Human ASCs grew as a homogenous cell sheet on PPy-CS surfaces, whereas on PPy-HA cells clustered into small spherical structures. PPy-CS supported hASC proliferation significantly better than PPy-HA at the 7 day time point. Both substrates equally triggered early osteogenic differentiation of hASCs, although mineralization was significantly induced on PPy-CS compared to PPy-HA under BEC. These differences may be due to different surface morphologies originating from the CS and HA dopants. Our results suggest that PPy-CS in particular is a potential osteogenic scaffold coating for bone tissue engineering.
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Sulfatos de Condroitina , Ácido Hialurônico , Polímeros , Pirróis , Células-Tronco/fisiologia , Alicerces Teciduais , Tecido Adiposo/citologia , Adulto , Idoso , Adesão Celular , Proliferação de Células , Sobrevivência Celular , Estimulação Elétrica , Feminino , Humanos , Pessoa de Meia-Idade , OsteogêneseRESUMO
Genomes of three strains (Parker, USSR, and 275/58) of coxsackievirus A7 (CV-A7) were amplified by the long reverse transcription (RT)-PCR method and sequenced. While the sequences of Parker and USSR were identical, the similarities of 275/58 to the CV-A7 reference sequence, accession no. AY421765, were 82.6% and 96.2% for nucleotides and amino acids, respectively.
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Surface properties and electrical charges are critical factors elucidating cell interactions on biomaterial surfaces. The surface potential distribution and the nanoscopic and microscopic surface elasticity of organic polypyrrole-hyaluronic acid (PPy-HA) were studied by atomic force microscopy (AFM) in a fluid environment in order to explain the observed enhancement in the attachment of human adipose stem cells on positively charged PPy-HA films. The electrostatic force between the AFM tip and a charged PPy-HA surface, the tip-sample adhesion force, and elastic moduli were estimated from the AFM force curves, and the data were fitted to electrostatic double-layer and elastic contact models. The surface potential of the charged and dried PPy-HA films was assessed with Kelvin probe force microscopy (KPFM), and the KPFM data were correlated to the fluid AFM data. The surface charge distribution and elasticity were both found to correlate well with the nodular morphology of PPy-HA and to be sensitive to the electrochemical charging conditions. Furthermore, a significant change in the adhesion was detected when the surface was electrochemically charged positive. The results highlight the potential of positively charged PPy-HA as a coating material to enhance the stem cell response in tissue-engineering scaffolds.
Assuntos
Tecido Adiposo/citologia , Ácido Hialurônico/química , Polímeros/química , Pirróis/química , Células-Tronco/citologia , Adesão Celular , Sobrevivência Celular , Células Cultivadas , Técnicas Eletroquímicas , Humanos , Microscopia de Varredura por Sonda , Propriedades de SuperfícieRESUMO
An electrically conductive polypyrrole (PPy) doped with a bioactive agent is an emerging functional biomaterial for tissue engineering. We therefore used chondroitin sulfate (CS)-doped PPy coating to modify initially electrically insulating polylactide resulting in novel osteogenic scaffolds. In situ chemical oxidative polymerization was used to obtain electrically conductive PPy coating on poly-96L/4D-lactide (PLA) nonwoven scaffolds. The coated scaffolds were characterized and their electrical conductivity was evaluated in hydrolysis. The ability of the coated and conductive scaffolds to enhance proliferation and osteogenic differentiation of human adipose stem cells (hASCs) under electrical stimulation (ES) in three-dimensional (3D) geometry was compared to the noncoated PLA scaffolds. Electrical conductivity of PPy-coated PLA scaffolds (PLA-PPy) was evident at the beginning of hydrolysis, but decreased during the first week of incubation due to de-doping. PLA-PPy scaffolds enhanced hASC proliferation significantly compared to the plain PLA scaffolds at 7 and 14 days. Furthermore, the alkaline phosphatase (ALP) activity of the hASCs was generally higher in PLA-PPy seeded scaffolds, but due to patient variation, no statistical significance could be determined. ES did not have a significant effect on hASCs. This study highlights the potential of novel PPy-coated PLA scaffolds in bone tissue engineering.
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Tecido Adiposo/citologia , Diferenciação Celular/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Poliésteres/farmacologia , Polímeros/farmacologia , Pirróis/farmacologia , Células-Tronco/citologia , Alicerces Teciduais/química , Adulto , Idoso , Fosfatase Alcalina/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/farmacologia , Meios de Cultura/farmacologia , DNA/metabolismo , Espectroscopia Dielétrica , Estimulação Elétrica , Eletrodos , Feminino , Humanos , Hidrólise/efeitos dos fármacos , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Espectrometria de Massas por Ionização por Electrospray , Células-Tronco/efeitos dos fármacos , Células-Tronco/enzimologiaRESUMO
Stabilized bioreceptor layers are of great importance in the design of novel biosensors. In earlier work, chimeric avidins enabled immobilization of biotinylated antibodies onto gold surfaces with greater stability compared to more conventional avidins (wild-type avidin and streptavidin). In the present study, the applicability of chimeric avidins as a general binding scaffold for biotinylated antibodies on spin-coated functionalized polythiophene thin films has been studied by surface plasmon resonance and atomic force microscopy. Novel chimeric avidins showed remarkably increased binding characteristics compared with other avidins, such as wild-type avidin, streptavidin, and bacterial avidin when merely physically adsorbed onto the polythiophene surface. They gave the highest binding capacities, the highest affinity constant, and the highest stability for biotinylated probe immobilization. Introduction of carboxylic acid groups to polythiophene layer further enhanced the binding level of the avidins. Polythiophene layers functionalized with chimeric avidins thus offered a promising generic platform for biosensor applications.
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Avidina/química , Técnicas Biossensoriais/métodos , Polímeros/química , Tiofenos/química , Adsorção , Anticorpos/química , Ouro/química , Imunoglobulina G/química , Microscopia de Força Atômica/métodos , Modelos Químicos , Conformação Molecular , Ligação Proteica , Estreptavidina/química , Ressonância de Plasmônio de SuperfícieRESUMO
Coxsackievirus A7 (CAV7) is a rarely detected and poorly characterized serotype of the Enterovirus species Human enterovirus A (HEV-A) within the Picornaviridae family. The CAV7-USSR strain has caused polio-like epidemics and was originally thought to represent the fourth poliovirus type, but later evidence linked this strain to the CAV7-Parker prototype. Another isolate, CAV7-275/58, was also serologically similar to Parker but was noninfectious in a mouse model. Sequencing of the genomic region encoding the capsid proteins of the USSR and 275/58 strains and subsequent comparison with the corresponding amino acid sequences of the Parker strain revealed that the Parker and USSR strains are nearly identical, while the 275/58 strain is more distant. Using electron cryomicroscopy and three-dimensional image reconstruction, the structures of the CAV7-USSR virion and empty capsid were resolved to 8.2-Å and 6.1-Å resolutions, respectively. This is one of the first detailed structural analyses of the HEV-A species. Using homology modeling, reconstruction segmentation, and flexible fitting, we constructed a pseudoatomic T = 1 (pseudo T = 3) model incorporating the three major capsid proteins (VP1 to VP3), addressed the conformational changes of the capsid and its constituent viral proteins occurring during RNA release, and mapped the capsid proteins' variable regions to the structure. During uncoating, VP4 and RNA are released analogously to poliovirus 1, the interfaces of VP2 and VP3 are rearranged, and VP1 rotates. Variable regions in the capsid proteins were predicted to map mainly to the surface of VP1 and are thus likely to affect the tropism and pathogenicity of CAV7.
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Proteínas do Capsídeo/genética , Proteínas do Capsídeo/ultraestrutura , Enterovirus/genética , Enterovirus/ultraestrutura , Internalização do Vírus , Microscopia Crioeletrônica , Enterovirus/fisiologia , Humanos , Processamento de Imagem Assistida por Computador , Modelos Biológicos , Dados de Sequência Molecular , Conformação Proteica , Análise de Sequência de DNARESUMO
Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material.
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DNA Circular/metabolismo , DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Mutagênese , Técnicas de Amplificação de Ácido Nucleico , DNA Circular/genética , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/genética , Biblioteca Gênica , Mutação/genética , Moldes Genéticos , Uracila/metabolismo , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismoRESUMO
Efficient display of antibody on filamentous phage M13 coat is crucial for successful biopanning selections. We applied a directed evolution strategy to improve the oligovalent display of a poorly behaving Fab fragment fused to phage gene-3 for minor coat protein (g3p). The Fab displaying clones were enriched from a randomly mutated Fab gene library with polyclonal anti-mouse IgG antibodies. Contribution of each mutation to the improved phenotype of one selected mutant was studied. It was found out that two point mutations had significant contribution to the display efficiency of Fab clones superinfected with hyperphage. The most dramatic effect was connected to a start codon mutation, from AUG to GUG, of the PelB signal sequence preceding the heavy chain. The clone carrying this mutation, FabM(GUG), displayed Fab 19-fold better and yielded twofold higher phage titers than the original Fab.
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Bacteriófago M13/genética , Evolução Molecular Direcionada , Fragmentos Fab das Imunoglobulinas/metabolismo , Biblioteca de Peptídeos , Animais , Sequência de Bases , Clonagem Molecular , Códon de Iniciação , Fragmentos Fab das Imunoglobulinas/genética , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Sinais Direcionadores de Proteínas/genéticaRESUMO
Electroactivity of polypyrrole hyaluronic acid, electropolymerized in the presence of oxidized carbon nanotubes (PPyHA-CNT) was studied in situ by electrochemical atomic force microscopy (EC-AFM) in physiological electrolyte solution. In situ Raman spectroscopic and quartz crystal microbalance (QCM) studies were conducted on layers of the polymer grown on AT-cut 5 MHz quartz crystals. Human adipose stem cell (ASC) attachment and viability were studied by Live/Dead staining, and the proliferation was evaluated by WST-1 Cell proliferation assay for polypyrrole samples electropolymerized on titanium. According to cyclic voltammetry, the measured specific capacitance of the material on gold is roughly 20% of the reference polypyrrole dodecylbenzene sulfonate (PPyDBS). Electrochemical-QCM (EC-QCM) analysis of a 210-nm thick film reveals that the material is very soft G' approximately 100 kPa and swells upon reduction. EC-AFM of samples polymerized on microelectrodes show that there are areas of varying electroactivity, especially for samples without a hydrophopic backing PPyDBS layer. AFM line scans show typically 20-25% thickness change during electrochemical reduction. Raman spectroscopic analysis suggests that the material supports noticeable polaron conduction. Biocompatibility study of the PPyHA-CNT on titanium with adipose stem cells showed equal or better cell attachment, viability, and proliferation compared with the reference polylactide.