A new waterborne chitosan-based polyurethane hydrogel as a vehicle to transplant bone marrow mesenchymal cells improved wound healing of ulcers in a diabetic rat model.

Foot ulcers, a common complication of diabetes, can cause physical incapacity and are derived from several factors, including poor wound healing. New therapeutic strategies are needed to minimize this complication for the sake of patients' health. We therefore developed a new chitosan- polyurethane hydrogel membrane (HPUC) and the test results confirmed that HPUC present low cytotoxicity and improved wound healing when used with mononuclear bone marrow fraction cells in the diabetic rat model. The biodegradable hydrogels were produced in block copolymer networks with a combination of chitosan blocks and biodegradable polyurethane. The membranes were characterized by FTIR, 13C-NMR and thermogravimetry. Swelling and hydrolytic degradation were also evaluated. The non-solubility of the membranes in good solvents and the chemical characterization confirmed that the network structure was formed between the PU and the chitosan through urea/urethane bonds. The findings confirm that the HPUC have interesting properties that make them suitable for wound healing applications.

Comparison of 2D and 3D cell culture models for cell growth, gene expression and drug resistance.

In vitro drug screening is widely used in the development of new drugs, because they constitute a cost-effective approach to select compounds with more potential for therapy. They are also an attractive alternative to in vivo testing. However, most of these assays are done in two-dimensional culture models, where cells are grown on a polystyrene or glass flat surface. In order to develop in vitro models that would more closely resemble physiological conditions, three-dimensional models have been developed. Here, we introduce two novel fully synthetic scaffolds produced using the polymer polyhydroxybutyrate (PHB): a Solvent-Casting Particle-Leaching (SCPL) membrane; and an electrospun membrane, to be used for 3D cultures of B16 F10 murine melanoma cells and 4T1 murine breast cancer cells. A 2D cell culture system in regular tissue culture plates and a classical 3D model where cells are grown on a commercially available gel derived from Engelbreth-Holm Swarm (EHS) tumor were used for comparison with the synthetic scaffolds. Cells were also collected from in vivo tumors grown as grafts in syngeneic mice. Morphology, cell viability, response to chemotherapy and gene expression analysis were used to compare all systems. In the electrospun membrane model, cells were grown on nanometer-scale fibers and in the SCPL membrane, which provides a foam-like structure for cell growth, pore sizes varied. Cells grown on all 3D models were able to form aggregates and spheroids, allowing for increased cell-cell contact when compared with the 2D system. Cell morphology was also more similar between 3D systems and cells collected from the in vivo tumors. Cells grown in 3D models showed an increase in resistance to dacarbazine, and cisplatin. Gene expression analysis also revealed similarities among all 3D platforms. The similarities between the two synthetic systems to the classic EHS gel model highlight their potential application as cost effective substitutes in drug screening, in which fully synthetic models could represent a step towards higher reproducibility. We conclude PHB synthetic membranes offer a valuable alternative for 3D cultures.

Effect of standard and neutral-pH peritoneal dialysis solutions upon fibroblasts proliferation.

INTRODUCTION: Continuous exposition of the peritoneal membrane to conventional dialysis solutions is an important risk factor for inducing structural and functional alterations. OBJECTIVE: To compare in vitro mouse fibroblast NIH-3T3 cell viability after exposition to a neutral pH dialysis solution in comparison to cells exposed to a standard solution. METHODS: Experimental study to compare the effects of a conventional standard or a neutral-pH, low-glucose degradation products peritoneal dialysis solution on the viability of exposed fibroblasts in cell culture. Both solutions were tested in all the commercially available glucose concentrations. Cell viability was evaluated with tetrazolium salt colorimetric assay. RESULTS: Fibroblast viability was significantly superior in the neutral pH solution in comparison to control, in all three glucose concentrations (Optical density in nm-means ± SD: 1.5% 0.295 ± 0.047 vs. 0.372 ± 0.042, p < 0.001; 2.3% 0.270 ± 0.036 vs. 0.337 ± 0.051, p < 0.001; 4.25% 0.284 ± 0.037 vs. 0.332 ± 0.032, p < 0.001; control vs. neutral pH respectively, Student t Test). There was no significant difference in cell viability between the three concentrations of glucose when standard solution was used (ANOVA p = 0.218), although cell viability was higher after exposition to neutral pH peritoneal dialysis fluid at 1.5% in comparison to 2.3 and 4.25% glucose concentrations (ANOVA p = 0.008: Bonferroni 1.5% vs. 2.3% p = 0.033, 1.5% vs. 4.25% p = 0.014, 2.3% vs. 4.25% p = 1.00). CONCLUSION: Cell viability was better in neutral pH dialysis solution, especially in the lower glucose concentration. A more physiological pH and lower glucose degradation products may be responsible for such results.

Effect of standard and neutral-pH peritoneal dialysis solutions upon fibroblasts proliferation / Efeito da solução de diálise peritoneal com pH neutro e padrão sobre a proliferação de fibroblastos

Introduction: Continuous exposition of the peritoneal membrane to conventional dialysis solutions is an important risk factor for inducing structural and functional alterations. Objective: To compare in vitro mouse fibroblast NIH-3T3 cell viability after exposition to a neutral pH dialysis solution in comparison to cells exposed to a standard solution. Methods: Experimental study to compare the effects of a conventional standard or a neutral-pH, low-glucose degradation products peritoneal dialysis solution on the viability of exposed fibroblasts in cell culture. Both solutions were tested in all the commercially available glucose concentrations. Cell viability was evaluated with tetrazolium salt colorimetric assay. Results: Fibroblast viability was significantly superior in the neutral pH solution in comparison to control, in all three glucose concentrations (Optical density in nm-means ± SD: 1.5% 0.295 ± 0.047 vs. 0.372 ± 0.042, p < 0.001; 2.3% 0.270 ± 0.036 vs. 0.337 ± 0.051, p < 0.001; 4.25% 0.284 ± 0.037 vs. 0.332 ± 0.032, p < 0.001; control vs. neutral pH respectively, Student t Test). There was no significant difference in cell viability between the three concentrations of glucose when standard solution was used (ANOVA p = 0.218), although cell viability was higher after exposition to neutral pH peritoneal dialysis fluid at 1.5% in comparison to 2.3 and 4.25% glucose concentrations (ANOVA p = 0.008: Bonferroni 1.5% vs. 2.3% p = 0.033, 1.5% vs. 4.25% p = 0.014, 2.3% vs. 4.25% p = 1.00). Conclusion: Cell viability was better in neutral pH dialysis solution, especially in the lower glucose concentration. A more physiological pH and lower glucose degradation products may be responsible for such results. .

Introdução: A exposição contínua da membrana peritoneal a soluções convencionais de diálise é um importante fator de risco para induzir alterações estruturais e funcionais. Objetivo: Comparar a viabilidade in vitro dos fibroblastos NIH-3T3 de camundongo após exposição à solução de diálise com pH neutro com células expostas à solução padrão. Métodos: Estudo experimental; ambas as soluções foram testadas em todas as concentrações de glicose comercialmente disponíveis. A viabilidade celular foi avaliada por ensaio colorimétrico de sal tetrazólio. Resultados: A viabilidade de fibroblastos foi melhor na solução de pH neutro em relação ao controle nas três concentrações de glicose (densidade óptica em nm-médias ± DP: 1,5% 0,295 ± 0,047 vs. 0,372 ± 0,042, p < 0,001; 2,3% 0,270 ± 0,036 vs. 0,337 ± 0,051, p < 0,001; 4,25% 0,284 ± 0,037 vs. 0,332 ± 0,032, p < 0,001; controle vs. pH neutro respectivamente, teste t de Student). Não houve diferença significativa na viabilidade celular entre as três concentrações de glicose quando solução padrão foi utilizada (ANOVA p = 0,218), embora a viabilidade celular tenha sido superior após exposição aos fluidos de diálise peritoneal neutros, pH 1,5% em comparação com 2,3 e 4,25% de concentrações de glicose (ANOVA p = 0,008: Bonferroni 1,5% vs. 2,3% p = 0,033, 1,5% vs. 4,25% p = 0,014, 2,3% vs. 4,25% p = 1,0). Conclusão: A viabilidade celular foi melhor em solução neutra de pH de diálise, especialmente nas menores concentrações de glicose. O pH fisiológico e com menos produtos de degradação de glicose podem ser responsáveis por estes resultados. .

Osteoinduction of human bone marrow cells: an in vitro study / Introdução osteogênica de células da medula óssea humana: um estudo in vitro

Objective: This study evaluated the osteogenic induction of human bone marrow cells by human recombinant bone morphogenetic protein-4 (rhBMP-4) and proteins released by Saos-2 (human osteosarcoma cell line). Study design: Osteoinduction in the presence or absence of Saos-2 and/or rhBMP-4 was evaluated in cultured human bone marrow cells. Morphological aspects and bone protein markers (osteonectin, osteopontin, and osteocalcin) were analyzed on days 1, 2, 5, 8, 11 and 14. Osteonectin expression was evaluated using immunohistochemistry with anti-secreted protein acidic and rich in cystein (anti-SPARC) antibody. mRNA transcripts for osteopontin were determined using RT-PCR with specific primers. Results: Bone marrow cells were adherent since the first day of culture and were positive for osteonectin. mRNA transcripts were detected in all culture conditions since the first day of culture. As human osteosarcoma cells are a source of additional growth they did not affect osteoinduction. rhBMP-4 up regulates osteoinduction during the first days of culture only. Osteoblasts were obtained from human bone marrow cells even in the absence of growth factors and showed a typical morphology. Cells derived from bone marrow can undergo osteoinduction in vitro in the absence of osteoinductive factors such as bone morphogenetic proteins. Conclusions: This study showed that an osteoblastic cell lineage may be obtained from human bone marrow derived from adherent cells, and that the presence of the rhBMP-4 seems to have an effect during the first stages of differentiation only.

Use of calcium phosphate cement scaffolds for bone tissue engineering: in vitro study / A utilização do cimento de fosfato de cálcio como arcabouço para engenharia de tecido ósseo: estudo in vitro

Purpose: To evaluate the ability of macroporous tricalcium phosphate cement (CPC) scaffolds to enable the adhesion, proliferation, and differentiation of mesenchymal stem cells derived from human bone marrow. Methods: Cells from the iliac crest of an adult human donor were processed and cultured on macroporous CPC discs. Paraffin spheres sized between 100 and 250µm were used as porogens. Cells were cultured for 5, 10, and 15 days. Next, we assessed cells' behavior and morphology on the biomaterial by scanning electron microscopy. The expression levels of the BGLA and SSP1 genes and the alkaline phosphatase (ALP) activity were quantified by the quantitative real-time polymerase chain reaction technique (QT-PCR) using the fluorophore SYBR GREEN®. Results: QT-PCR detected the expression of the BGLA and SSP1 genes and the ALP activity in the periods of 10 and 15 days of culture. Thus, we found out that there was cell proliferation and differentiation in osteogenic cells. Conclusion: Macroporous CPC, with pore sized between 100 and 250µm and developed using paraffin spheres, enables adhesion, proliferation, and differentiation of mesenchymal stem cells in osteogenic cells and can be used as a scaffold for bone tissue engineering.

Objetivo: Avaliar a capacidade de suportes tridimensionais macroporosos de cimento de fosfato de cálcio (CFC), de permitir a adesão, proliferação e diferenciação de células-tronco mesenquimais derivadas da medula óssea humana. Métodos: células obtidas da crista ilíaca de um doador humano adulto foram processadas e cultivadas sobre suportes de CFC, macroporosos, que tiveram como corpo gerador de poros, microesferas de parafina, com tamanho entre 100 e 250µm. Os períodos de cultura estabelecidos foram de cinco, 10 e 15 dias. Após estes períodos, o comportamento e a morfologia das células junto ao biomaterial foram avaliados por meio de Microscopia Eletrônica de Varredura. Os níveis de expressão dos genes BGLA e SSP1 bem como a atividade da Fosfatase Alcalina (ALP) foram quantificados pela técnica de PCR em Tempo Real (QT-PCR) utilizando o fluoróforo SYBR Green®. Resultados: O QT-PCR detectou a expressão dos genes BGLA e SSP1 e a atividade da fosfatase alcalina nos períodos de 10 e 15 dias de cultura. No período de cinco dias, não foi observada a expressão de nenhum dos genes investigados. Conclusão: O CFC, macroporoso, com tamanho de poros entre 100 e 250µm, criados por meio da utilização de microesferas de parafina, permite a adesão, proliferação e diferenciação de células-tronco mesenquimais em células osteogênicas, podendo ser utilizado como arcabouço para engenharia de tecido ósseo.

Use of calcium phosphate cement scaffolds for bone tissue engineering: in vitro study.

PURPOSE: To evaluate the ability of macroporous tricalcium phosphate cement (CPC) scaffolds to enable the adhesion, proliferation, and differentiation of mesenchymal stem cells derived from human bone marrow. METHODS: Cells from the iliac crest of an adult human donor were processed and cultured on macroporous CPC discs. Paraffin spheres sized between 100 and 250µm were used as porogens. Cells were cultured for 5, 10, and 15 days. Next, we assessed cells' behavior and morphology on the biomaterial by scanning electron microscopy. The expression levels of the BGLA and SSP1 genes and the alkaline phosphatase (ALP) activity were quantified by the quantitative real-time polymerase chain reaction technique (QT-PCR) using the fluorophore SYBR GREEN(®). RESULTS: QT-PCR detected the expression of the BGLA and SSP1 genes and the ALP activity in the periods of 10 and 15 days of culture. Thus, we found out that there was cell proliferation and differentiation in osteogenic cells. CONCLUSION: Macroporous CPC, with pore sized between 100 and 250µm and developed using paraffin spheres, enables adhesion, proliferation, and differentiation of mesenchymal stem cells in osteogenic cells and can be used as a scaffold for bone tissue engineering.

Cytotoxicity of silver solder employed in orthodontics.

OBJECTIVE: To test the null hypothesis that the silver soldering employed in orthodontics is not cytotoxic for fibroblasts. MATERIALS AND METHODS: This in vitro study was performed using a culture of mice fibroblasts (lineage NIH/3T3), divided into four groups (n = 10 each): control, negative control (stainless steel archwire), positive control (amalgam disks), and test group (silver soldering). After cell culture in complete Dulbecco modified eagle medium and achievement of confluence in 80%, the suspension was added to the plates of 24 wells containing the specimens and incubated in an oven at 37 degrees C for 24 hours. The plates were analyzed on an inverted light microscope, photomicrographs were obtained, and the results were recorded as response rates based on modifications of the parameters of Stanford according to the size of the diffusion halo of the toxic substance and quantity of cell lysis. RESULTS: The results revealed a maximum response rate for the silver soldering group, as well as severe inhibition of cell proliferation and growth, more round cells with mostly darkened and granular aspects, suggesting lysis with cell death. A similar response was seen in the positive control group. CONCLUSION: The hypothesis is rejected. The silver soldering used in orthodontics represents a highly cytotoxic material for the cells analyzed.

Effect of titanium surface roughness on human bone marrow cell proliferation and differentiation: an experimental study.

PURPOSE: To assess the proliferation and differentiation of human bone marrow-derived cells cultured on titanium surfaces with different roughness characteristics. METHODS: Cells obtained from the iliac crest of an adult human donor were routinely processed and cultured on titanium surfaces of varying roughness, according to their preparation method: polishing only (smooth surface) and polishing followed by etching with HF/HNO3 for 15 and 30 minutes (rough surfaces). Surfaces were assessed using scanning electronic microscopy and profilometry. RESULTS: Titanium disks etched with acid for 15 minutes allowed greater cell proliferation in all culture periods. The level of osteopontin and osteocalcin expression was increased in both acid-etched groups, which indicates an advanced stage of differentiation of cells into osteoblasts. CONCLUSIONS: Increased surface roughness accelerates the differentiation of undifferentiated mesenchymal cells into osteogenic lineage cells, but does not necessarily favor cell proliferation. An intermediate surface roughness of 0.5microm (acid etching for 15 minutes) favors both initial and final cell responses.

Effect of titanium surface roughness on human bone marrow cell proliferation and differentiation: an experimental study / Efeito da rugosidade de superfície de discos de titânio sobre a proliferação e diferenciação de células de medula óssea humana: estudo experimental

PURPOSE: To assess the proliferation and differentiation of human bone marrow-derived cells cultured on titanium surfaces with different roughness characteristics. METHODS: Cells obtained from the iliac crest of an adult human donor were routinely processed and cultured on titanium surfaces of varying roughness, according to their preparation method: polishing only (smooth surface) and polishing followed by etching with HF/HNO3 for 15 and 30 minutes (rough surfaces). Surfaces were assessed using scanning electronic microscopy and profilometry. RESULTS: Titanium disks etched with acid for 15 minutes allowed greater cell proliferation in all culture periods. The level of osteopontin and osteocalcin expression was increased in both acid-etched groups, which indicates an advanced stage of differentiation of cells into osteoblasts. CONCLUSIONS: Increased surface roughness accelerates the differentiation of undifferentiated mesenchymal cells into osteogenic lineage cells, but does not necessarily favor cell proliferation. An intermediate surface roughness of 0.5µm (acid etching for 15 minutes) favors both initial and final cell responses.

OBJETIVO: Avaliar a proliferação e diferenciação de células derivadas da medula óssea humana sobre superfícies de titânio com diferentes rugosidades de superfície. MÉTODOS: Células obtidas da crista ilíaca de um doador humano adulto foram rotineiramente processadas e cultivadas sobre superfícies de titânio preparadas através de polimento apenas, ou polimento seguido de condicionamento com HF/HNO3 por 15 e 30 minutos, para produzir superfícies com rugosidades variadas, conforme determinado por Microscopia Eletrônica de Varredura e perfilometria. RESULTADOS: Discos de titânio condicionados com ácido por 15 minutos permitiram maior proliferação celular em todos os períodos de cultura. O nível de expressão das proteínas osteopontina e osteocalcina estava aumentado em ambos os grupos condicionados com ácido, indicando que as células estavam comprometidas com o fenótipo osteogênico. CONCLUSÕES: O aumento na rugosidade de superfície acelera a diferenciação de células mesenquimais indiferenciadas em células de linhagem osteogênica, mas não necessariamente favorece a proliferação celular. Uma superfície com rugosidade intermediária de 0,5µm (condicionada com ácido por 15 minutos) favorece tanto as respostas celulares iniciais quanto as finais.

Effect of low-level laser therapy on inflammatory reactions during wound healing: comparison with meloxicam.

OBJECTIVE: This study evaluated the action of low-level laser therapy (LLLT) on the modulation of inflammatory reactions during wound healing in comparison with meloxicam. BACKGROUND DATA: LLLT has been recommended for the postoperative period because of its ability to speed healing of wounds. However, data in the literature are in disagreement about its anti-inflammatory action. METHODS: Standardized circular wounds were made on the backs of 64 Wistar rats. The animals were divided into four groups according to the selected postoperative therapy: group A-control; group B-administration of meloxicam; and groups C and D-irradiation with red (lambda = 685 nm) and infrared (lambda = 830 nm) laser energy, respectively. The animals were killed at 12, 36, and 72 h and 7 days after the procedure. RESULTS: Microscopic analysis revealed significant vascular activation of irradiated sites in the first 36 h. Only group B showed decreases in the intensity of polymorphonuclear infiltrates and edema. Group D showed a higher degree of organization and maturation of collagen fibers than the other groups at 72 h. The animals in group C showed the best healing pattern at 7 days. The anti-inflammatory action of meloxicam was confirmed by the results obtained in this research. The quantification of interleukin-1beta (IL-1beta) mRNA by real-time polymerase chain reaction (PCR) did not show any reduction in the inflammatory process in the irradiated groups when compared to the other groups. CONCLUSIONS: LLLT improves the quality of histologic repair and is useful during wound healing. However, with the methods used in this study the laser energy did not minimize tissue inflammatory reactions.

Efeitos das células tronco adultas de medula óssea e do plasma rico em plaquetas na regeneração e recuperação funcional nervosa em um modelo de defeito agudo em nervo perfiférico em rato

OBJETIVOS: Foram avaliados os efeitos do uso de células tronco da medula óssea (CTM) e do plasma rico em plaquetas (PRP) na regeneração de nervos periféricos, utilizando um modelo estabelecido de regeneração de nervo ciático em ratos. MÉTODOS: Um defeito nervoso de 10 mm foi reconstruído com a utilização de um tubo de silicone preenchido com CTM, PRP ou ambos. O grupo controle recebeu somente o tubo de silicone. Foi realizado ainda um quinto grupo no qual o intervalo foi reconstruído utilizando o segmento ressecado do nervo. A função motora foi testada seis semanas após a cirurgia utilizando teste de marcha. Após o teste motor, os ratos foram anestesiados, o nervo ciático e o tubo foram ressecados e foi realizada microscopia eletrônica de transmissão. RESULTADOS: A análise quantitativa demonstra uma melhora na recuperação funcional no grupo CTM em comparação com os demais grupos. Regeneração nervosa foi demonstrada no grupo CTM por microscopia eletrônica de trasmissão com uma recuperação praticamente completa da anatomia neural. CONCLUSÃO: Nossos resultados sugerem que o uso de CTM associado com a técnica de tubulização promove uma satisfatória recuperação da função motora e regeneração nervosa.

OBJECTIVES: The effects of the use of bone marrow stem cells (MSC) and platelet-rich plasma (PRP) on peripheral nerves regeneration were assessed by using an established model of sciatic nerve regeneration in rats. METHODS: A 10-mm nervous defect was reconstructed by using a silicone tube filled with MSC, PRP or both. The control group received only the silicone tube. A fifth group was also set, in which the interval was reconstructed by using a dried segment of the nerve. Motor function was tested six weeks after surgery, by means of a gait test. After motor test, the rats were anesthetized, the sciatic nerve and the tube were dried, and the transmission electronic microscopy was performed. RESULTS: The quantitative analysis shows an improved functional recovery in MSC group compared to the other groups. Nervous regeneration was reported for MSC group by means of transmission electronic microscopy with an almost full recovery of the neural anatomy. CONCLUSION: Our results suggest that the use of MSC combined with tubing technique yields a satisfactory recovery of motor function and nervous regeneration.