RESUMO
During the manufacture of cell- and tissue-based products, such as engineered cartilage for autologous chondrocyte implantation, maximizing the number of cells isolated from donor tissue substantially improves the productivity of these products. The method used for agitating tissues with digestive fluid and enzymes can considerably affect both the quality and quantity of isolated cells. This study aimed to investigate the effectiveness of a rotation/revolution-type agitator for chondrocyte isolation following the enzymatic digestion of rat costal cartilage. Cartilage tissue cut into 1 mm3-thick sections was equally divided between two groups and placed in 50-mL conical tubes; sections in both groups were digested using 0.1 mg/mL liberase TH (collagenase/thermolysin) at 37 °C for 4 h with either rotation/revolution or conventional orbital agitation method. Compared with using conventional orbital agitator, using the rotation/revolution-type agitator resulted in a significant (>two-fold) increase in the number of isolated cells. In subsequent primary cultures, chondrocytes obtained by rotation/revolution agitation showed superior initial attachment to tissue culture dish on day 1 and 2 compared with those obtained by conventional agitation; however, no differences in cell proliferation or cartilage-related molecule expression patterns were observed between cells derived from either method after 3 days of subculture. These findings suggested that there are no disadvantages to the proposed rotation/revolution agitation method. Rotation/revolution-type agitators are a promising apparatus for preparing chondrocytes for primary cultures and cartilage tissue engineering.
Assuntos
Cartilagem/fisiologia , Separação Celular/instrumentação , Condrócitos/citologia , Rotação , Técnicas de Cultura de Tecidos/instrumentação , Engenharia Tecidual , Animais , Cartilagem/citologia , Cartilagem/crescimento & desenvolvimento , Proliferação de Células , Separação Celular/métodos , Células Cultivadas , Colagenases/metabolismo , Desenho de Equipamento , Ratos , Ratos Sprague-Dawley , Costelas/citologia , Termolisina/metabolismo , Técnicas de Cultura de Tecidos/métodos , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodosRESUMO
A novel hyaluronan (HA) derivative, poly(L-glutamic acid)-grafted hyaluronan (PGA-g-HA), was synthesized to improve the durability of conventional HA products for intra-articular injection. The purpose of this study was to investigate the characteristics of the novel HA derivative in terms of viscoelasticity, degradation behavior, non-immunogenicity, and bioactivity using preliminary in vitro and in vivo experiments. The storage modulus (G') and loss modulus (Gâ³) of PGA-g-HA were similar to those of HA80 (approximately 8.0 × 105 Da) rather than those of original HA200 (approximately 2.0 × 106 Da). PGA-g-HA showed strong resistance against hyaluronidase hydrolysis compared to unmodified HA200. The immunogenicity resulting from grafting PGA to HA200 was not detected in bone marrow derived dendritic cells. The anti-inflammatory activity of PGA-g-HA was confirmed in IL-1ß-stimulated chondrocytes. In addition, compared to unmodified HA200, the intra-articular injection of PGA-g-HA produced greater chondroprotective effects on a monoiodoacetic acid-induced model of rat knee osteoarthritis at two weeks after a single treatment. Therefore, PGA-g-HA is expected to be a promising medicine and biomedical device for intra-articular injection. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3006-3016, 2017.
Assuntos
Anti-Inflamatórios/uso terapêutico , Ácido Hialurônico/uso terapêutico , Osteoartrite do Joelho/tratamento farmacológico , Ácido Poliglutâmico/uso terapêutico , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/química , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/imunologia , Elasticidade , Ácido Hialurônico/administração & dosagem , Ácido Hialurônico/química , Injeções Intra-Articulares , Masculino , Osteoartrite do Joelho/imunologia , Ácido Poliglutâmico/administração & dosagem , Ácido Poliglutâmico/química , Ratos Sprague-Dawley , ViscosidadeRESUMO
A stereodivergent strategy for the synthesis of skipped dienes is developed. The method consists of hydroboration of allenes and Migita-Kosugi-Stille coupling, which allows for access to all four possible stereoisomers of the skipped dienes. The hydroboration is especially useful for providing both E-allylic and Z-allylic alcohols from the same allene by simply changing the organoborane reagent. The strategy was successfully applied to a unified total synthesis of the madangamine alkaloids via a common ABCE-tetracyclic intermediate with a (Z,Z)-skipped diene. The late-stage variation of the D-ring enabled the supply of synthetic madangamines A, C, and E for the first time.
Assuntos
Compostos Heterocíclicos de 4 ou mais Anéis/síntese química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Estrutura Molecular , EstereoisomerismoRESUMO
A general synthetic route toward a diazatricyclic core common to the madangamine family is described. Ring-closing metathesis and palladium-catalyzed cycloisomerization provided the cis-fused diazadecalin structure, accompanied by formation of the N-Boc-enamine, which was utilized as an N-acyliminium ion equivalent. Direct cyclization from the N-Boc-enamine was achieved through the in situ formation of an N,O-acetal.
Assuntos
Compostos Aza/síntese química , Compostos Heterocíclicos de 4 ou mais Anéis/síntese química , Compostos Aza/química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Estrutura Molecular , EstereoisomerismoRESUMO
While the synthesis of amide bonds is now one of the most reliable organic reactions, functionalization of amide carbonyl groups has been a long-standing issue due to their high stability. As an ongoing program aimed at practical transformation of amides, we developed a direct nucleophilic addition to N-alkoxyamides to access multisubstituted amines. The reaction enabled installation of two different functional groups to amide carbonyl groups in one pot. The N-alkoxy group played important roles in this reaction. First, it removed the requirement for an extra preactivation step prior to nucleophilic addition to activate inert amide carbonyl groups. Second, the N-alkoxy group formed a five-membered chelated complex after the first nucleophilic addition, resulting in suppression of an extra addition of the first nucleophile. While diisobutylaluminum hydride (DIBAL-H) and organolithium reagents were suitable as the first nucleophile, allylation, cyanation, and vinylation were possible in the second addition including inter- and intramolecular reactions. The yields were generally high, even in the synthesis of sterically hindered α-trisubstituted amines. The reaction exhibited wide substrate scope, including acyclic amides, five- and six-membered lactams, and macrolactams.