RESUMO
This study aims to investigate the bioactivity of collagen II/hyaluronan/chondroitin-6-sulfate tri-copolymer as bionic scaffold for nucleus pulposus (NP) tissue engineering. Collagen II (C II) (pH 1-2) was mixed with hyaluronan (HyA) and lyophilized to prepare C II/HyA matrices. Chondroitin 6-sulfate (6-CS) was covalently attached to the C II/HyA matrices using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Then, cells were expanded from rabbit NP and seeded in the tri-copolymer scaffold. Cell-scaffold hybrids were maintained for up to 28 days in culture. Cell viability/proliferation, extracellular matrix (ECM)-related gene expression, and the content of sulfated glycosaminoglycans (s-GAG) were evaluated. Our results are as following: when cultured for 28 days, the cell-scaffold hybrids maintained active cell viability/proliferation and exhibited a significantly increased s-GAG content. In addition, rabbit NP cells cultured in the scaffold demonstrated a significantly higher level of C II and aggrecan gene expression and a significantly lower level of Collagen I (C I) gene expression when compared with that of monolayer cells. Histological studies and scanning electron microscopy (SEM) further indicated newly secreted ECM deposits in the scaffolds. In conclusion, the C II/HyA-CS scaffold may be an alternative material for NP tissue engineering due to its satisfactory bioactivity, and it deserves further in vivo investigation.
Assuntos
Materiais Biocompatíveis , Sulfatos de Condroitina/química , Colágeno Tipo II/química , Ácido Hialurônico/química , Alicerces Teciduais/química , Animais , Bisbenzimidazol , Proliferação de Células , Sobrevivência Celular , Colágeno Tipo II/biossíntese , Colágeno Tipo II/genética , Corantes , DNA/biossíntese , DNA/genética , Matriz Extracelular/química , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Azul de Metileno/análogos & derivados , Microscopia Eletrônica de Varredura , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sincalida/metabolismoRESUMO
To construct a novel scaffold for nucleus pulposus (NP) tissue engineering, The porous type II collagen (CII)/hyaluronate (HyA)-chondroitin-6-sulfate (6-CS) scaffold was prepared using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) cross-linking system. The physico-chemical properties and biocompatibility of CII/HyA-CS scaffolds were evaluated. The results suggested CII/HyA-CS scaffolds have a highly porous structure (porosity: 94.8 +/- 1.5%), high water-binding capacity (79.2 +/- 2.8%) and significantly improved mechanical stability by EDC/NHS crosslinking (denaturation temperature: 74.6 +/- 1.8 and 58.1 +/- 2.6 degrees C, respectively, for the crosslinked scaffolds and the non-crosslinked; collagenase degradation rate: 39.5 +/- 3.4 and 63.5 +/- 2.0%, respectively, for the crosslinked scaffolds and the non-crosslinked). The CII/HyA-CS scaffolds also showed satisfactory cytocompatibility and histocompatibility as well as low immunogenicity. These results indicate CII/HyA-CS scaffolds may be an alternative material for NP tissue engineering due to the similarity of its composition and physico-chemical properties to those of the extracellular matrices (ECM) of native NP.
