RESUMO
Nowadays, intervertebral disc (IVD) degeneration is one of the principal causes of low back pain involving high expense within the health care system. The long-term goal is the development of a medical treatment modality focused on a more biological regeneration of the inner nucleus pulposus (NP). Hence, interest in the endoscopic implantation of an injectable material took center stage in the recent past. We report on the development of a novel polyurethane (PU) scaffold as a mechanically stable carrier system for the reimplantation of expanded autologous IVD-derived cells (disc cells) to stimulate regenerative processes and restore the chondrocyte-like tissue within the NP. Primary human disc cells were seeded into newly developed PU spheroids which were subsequently encapsulated in fibrin hydrogel. The study aims to analyze adhesion properties, proliferation capacity and phenotypic characterization of these cells. Polymerase chain reaction was carried out to detect the expression of genes specifically expressed by native IVD cells. Biochemical analyses showed an increased DNA content, and a progressive enhancement of total collagen and glycosaminoglycans (GAG) was observed during cell culture. The results suggest the synthesis of an appropriate extracellular matrix as well as a stable mRNA expression of chondrogenic and/or NP specific markers. In conclusion, the data presented indicate an alternative medical approach to current treatment options of degenerated IVD tissue.
Assuntos
Fibrina/química , Disco Intervertebral/citologia , Poliuretanos/química , Regeneração , Agrecanas/genética , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo II/genética , Proteínas da Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Transportador de Glucose Tipo 1/genética , Humanos , Disco Intervertebral/metabolismo , Disco Intervertebral/fisiologia , Metaloproteinase 2 da Matriz/genética , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9/genética , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
A titanium foam for spine fusion and other applications was tested by cell culture. Its high porosity and surface roughness should enable bone cells to grow through it, resulting in a better fixation of the vertebral body. The foam was tested by in vitro experiments with human osteoblasts under static culture conditions and in a perfused system. By means of cell number, viability, scanning electron microscopy and histological staining, cell proliferation could be observed. The expression of osteogenic genes like collagen-I, alkaline phosphatase and osteocalcin was proven by reverse transcription polymerase chain reaction (RT-PCR) as well as in the case of alkaline phosphatase with biochemical methods. The conducted experiments showed that human osteoblasts could grow through the interconnected porosity of the metal foam and that they expressed an osteoblast like phenotype. The results suggest that in vivo osteoblasts are likely to form a trabecular bone bridge through this titanium foam. Consequently, with this osteoconductive material, there may be a reduced need for autologous bone in spinal fusion procedures.
Assuntos
Proliferação de Células , Osteoblastos/fisiologia , Engenharia Tecidual/métodos , Titânio , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Materiais Biocompatíveis , Linhagem Celular , Colágeno/biossíntese , Colágeno/genética , Regulação da Expressão Gênica , Humanos , Microscopia Eletrônica de Varredura , Osteoblastos/citologia , Osteocalcina/biossíntese , Osteocalcina/genética , Osteogênese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fusão Vertebral/métodosRESUMO
The primary structure of mitochondrial aspartate aminotransferase from chicken is reported. The enzyme is a dimer of identical subunits. Each subunit contains 401 amino acid residues; the calculated subunit molecular weight of the apoform is 44,866. The degree of sequence identity with the homologous cytosolic isoenzyme from chicken is 46%. A comparison of the primary structures of the mitochondrial and the cytosolic isoenzyme from pig and chicken shows that 40% of all residues are invariant. The degree of interspecies sequence identity both of the mitochondrial and the cytosolic isoenzyme from chicken and pig (86% and 83%, respectively) markedly exceeds that of the intraspecies identity between mitochondrial and cytosolic aspartate aminotransferase in chicken (46%) or in pig (48%). Based on these values, the duplication of the aspartate aminotransferase ancestral gene is estimated to have occurred approximately 1000 million years ago, i.e. at the time of the emergence of eukaryotic cells. By sequence comparison it is possible to identify amino acid residues and segments of the polypeptide chain that have been conserved specifically in the mitochondrial isoenzyme during phylogenetic evolution. These segments comprise about a third of the total polypeptide chain and appear to cluster in a certain surface region. The cluster carries an excess of positively charged residues which exceeds the overall charge difference between the cytosolic (pI approximately 6) and the mitochondrial isoenzyme (pI approximately 9).
