RESUMO
We synthesized a carboxymethylcellulose with phenol moieties by covalently incorporating tyramine into carboxymethylcellulose using aqueous-phase carbodiimide activation chemistry. The resulting hydrogel was obtained from an aqueous solution of the conjugate via the horseradish peroxidase-catalyzed oxidation reaction of phenols by consuming H(2)O(2), where the gelation speed depended on the concentrations of enzyme and H(2)O(2). The viability of the mammalian cells enclosed within the hydrogel prepared from 1.5% (w/v) conjugate solution containing 5 units/ml horseradish peroxidase and 1 mM H(2)O(2), was 80% after 24 h. These results demonstrate that this carboxymethylcellulose with phenol moieties has potential for biomedical applications including tissue-engineering.
Assuntos
Materiais Biocompatíveis/química , Carboximetilcelulose Sódica/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Fenol/química , Engenharia Tecidual , Animais , Materiais Biocompatíveis/metabolismo , Materiais Biocompatíveis/toxicidade , Carboximetilcelulose Sódica/metabolismo , Carboximetilcelulose Sódica/toxicidade , Peroxidase do Rábano Silvestre/química , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/toxicidade , Teste de Materiais , Tiramina/químicaRESUMO
In situ gelation of an aqueous solution of carboxymethylcellulose derivative bearing phenolic hydroxyl groups (CMC-Ph) that contained suspended adipose-derived stem cells (ASCs) was studied in vitro and in vivo for evaluating feasibility in adipose tissue-engineering strategies. The rat ASCs that were enclosed in the CMC-Ph gels through a horseradish peroxidase-catalysed reaction showed 92.8% viability, good proliferation and adipogenic differentiation in vitro. Ten weeks after the subcutaneous injection of ASCs-suspending CMC-Ph for in situ gelation, clearly visible new vascularized adipose tissue formed at the injection site. The number of blood vessels and the area occupied by adipose tissues were five and eight times larger, respectively, than those found in the implanted acellular gel. The adipogenesis and neovascularization were further enhanced by incorporation of fibroblast growth factor into the CMC-Ph gel containing ASCs.
Assuntos
Tecido Adiposo/citologia , Carboximetilcelulose Sódica/farmacologia , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Células-Tronco/citologia , Engenharia Tecidual/métodos , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Imobilizadas/citologia , Células Imobilizadas/efeitos dos fármacos , Injeções , Injeções Subcutâneas , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Fenóis/química , Ratos , Ratos Wistar , Regeneração/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
An aqueous solution of carboxylmethylcellulose with phenolic hydroxyl groups (CMC-Ph) is gellable within 1 min via a peroxidase-catalyzed oxidative reaction under mild conditions suitable for mammalian cells. In this research, we evaluated cellular adhesion and proliferation on the resultant hydrogel, and the feasibility of the hydrogel as a substrate for cell sheet technology. Within 4 h of seeding, 76.9% of L929 fibroblast cells adhered to the gel and showed similar morphology of spreading to that on cell culture dish. Subsequently, the adherent cells proliferated on the gel and formed a confluent monolayer after 168 h of culture. From the confluent monolayer we could harvest a cell sheet after about 5 min of digestion of the gel using cellulase dissolved in medium at 5 U ml(-1). The cells in the cell sheet showed well-preserved morphology similar to that shown before they were detached from the gel. In addition, the harvested cell sheet readhered and proliferated after being transferred to another culture dish. These results demonstrate that CMC-Ph gel is a good candidate material for obtaining cell sheets.
Assuntos
Carboximetilcelulose Sódica/química , Adesão Celular , Hidrogéis , Tiramina/química , Animais , Linhagem Celular , Proliferação de Células , Estudos de ViabilidadeRESUMO
The effect of Ph-OH group content on gelation time, mechanical properties, hydrophobicity, and cellular adhesiveness of hydrogels produced from carboxymethylcellulose derivatives is investigated. A higher Ph-OH group content induces faster gelation and yields more brittle and hydrophobic gels. After 4 h of seeding, a larger number of L929 fibroblasts adhere to the hydrogel of the CMC-Ph that contains 15.4 Ph-OH groups per 100 repeat units of uronic acid (97% adhesion rate) than to the gel of CMC-Ph with only 8.4 Ph-OH groups (62% adhesion rate). The results demonstrate that controlling the Ph-OH group content is an effective and useful way to control cellular adhesion and proliferation on the hydrogels, as well as gelation time and mechanical properties of the gels.
Assuntos
Carboximetilcelulose Sódica/química , Fibroblastos/citologia , Hidrogéis/síntese química , Fenóis/química , Alicerces Teciduais/química , Animais , Adesão Celular , Linhagem Celular , Proliferação de Células , Hidrogéis/química , Cinética , Camundongos , PeroxidasesRESUMO
A phenolic hydroxyl group was incorporated into gelatin, using aqueous-phase carbodiimide activation chemistry, to obtain in situ gellable and injectable protein-based materials for drug delivery and tissue engineering applications. By this means, gelatin derivatives that were gellable via a peroxidase-catalyzed reaction were obtained. The enzymatically cross-linked gelatin gels did not melt at 37 degrees C and showed tunable proteolytic degradability. The time necessary for gelation decreased with increasing content of the phenolic hydroxyl (Ph) group, peroxidase concentration and decreasing H(2)O(2) concentration. Resistance to gel compression also depended on the content of Ph groups, with the gel containing the lowest Ph group content showing the greatest resistance to compression. We encapsulated L929 fibroblast cells in gelatin gels under conditions that induced gelation in about 10 s. The encapsulated cells showed about 95% viability. In addition, L929 cells seeded on the gels showed the same growth profiles as those seeded on an unmodified gelatin-coated dish. Subcutaneous rodent injection experiments demonstrated successful in situ formation of gels at the injected site.
Assuntos
Materiais Biocompatíveis/química , Sistemas de Liberação de Medicamentos , Gelatina , Géis/química , Fenóis/química , Engenharia Tecidual/métodos , Animais , Linhagem Celular , Gelatina/administração & dosagem , Gelatina/química , Peróxido de Hidrogênio/metabolismo , Teste de Materiais , Camundongos , Estrutura Molecular , Oxidantes/metabolismo , Peroxidases/metabolismoRESUMO
Microcapsules with a single, spherical hollow core less than 150 microm in diameter were developed to obtain multicellular spheroids with well-defined sizes of less than 150 microm in diameter. An aqueous solution of phenolic hydroxyl derivative of carboxymethylcellulose (CMC-Ph) containing human hepatoma cell line (HepG2) cells and horse radish peroxidase (HRP) was injected into a coflowing stream of liquid paraffin, containing H(2)O(2), resulting in cell-enclosing CMC-Ph microparticles, 135 microm in diameter, via a peroxidase-catalyzed crosslinking reaction. The CMC-Ph microparticles were then coated with a phenolic hydroxyl derivative of alginate (Alg-Ph) gel membrane several dozen micrometers in thickness, crosslinked via the same enzymatic reaction process, followed by further crosslinking between the carboxyl groups of alginate by Sr(2+). A hollow core structure was achieved by immersing the resultant microcapsules in a medium containing cellulase, which degrades the enclosed CMC-Ph microparticles. The HepG2 cells in the microcapsules then grew and completely filled the hollow core. Multicellular spheroids the same size as the CMC-Ph microparticles, with living cells at their outer surface, were collected within 1 min by soaking them in a medium containing alginate lyase to degrade the Alg-Ph gel microcapsule membrane.