RESUMO
Porous mineralized scaffolds are required for various applications in bone engineering. In particular, tube-like pores with controlled orientation inside the scaffold may support homogeneous cell seeding as well as sufficient nutrient supply and may facilitate blood vessel ingrowth. Scaffolds with parallely orientated tube-like pores were generated by diffusion-controlled ionotropic gelation of alginate. Incorporation of hydroxyapatite (HA) during the gelation process yielded stable scaffolds with an average pore diameter of approximately 90 microm. To evaluate the potential use of alginate-gelatine-HA scaffolds for bone tissue engineering, in vitro tests with human bone marrow stromal cells (hBMSCs) were carried out. We analysed biocompatibility and cell penetration into the capillary pores by microscopic methods. hBMSCs were also cultivated on alginate-gelatine-HA scaffolds for 3 weeks in the presence and absence of osteogenic supplements. We studied proliferation and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content and alkaline phosphatase (ALP) activity and found a 10-14-fold increase of cell number after 2 weeks of cultivation, as well as an increase of specific ALP activity for osteogenic-induced hBMSCs. Furthermore, the expression of bone-related genes [ALP, bone sialoprotein II (BSPII)] was analysed. We found an increase of ALP as well as BSPII expression for osteogenic-induced hBMSCs on alginate-gelatin-HA scaffolds.
Assuntos
Células da Medula Óssea/citologia , Alicerces Teciduais , Alginatos , Fosfatase Alcalina/metabolismo , Sequência de Bases , Fenômenos Biomecânicos , Células da Medula Óssea/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , DNA/genética , DNA/metabolismo , Primers do DNA/genética , Durapatita , Gelatina , Ácido Glucurônico , Ácidos Hexurônicos , Humanos , Hidrogéis , Sialoproteína de Ligação à Integrina , L-Lactato Desidrogenase/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Varredura , Osteogênese/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialoglicoproteínas/metabolismo , Células Estromais/citologia , Células Estromais/metabolismo , Engenharia TecidualRESUMO
AIM: Longitudinal course and genotype-phenotype correlation in patients and carriers with heterozygous mutations in hBEST1 (bestrophin). METHODS: Thirteen patients and seven possible carriers were characterised by mutation analysis with SSCPA and direct sequencing, clinical examination and fundus autofluorescence (AF). Electrophysiology (EOG and mfERG) and optical coherence tomography (OCT) were additionally performed whenever possible. RESULTS: We identified seven different heterozygous mutations in ten unrelated families with Best disease. I296del was the most frequent mutation. Five of nine individuals with I295del and two of three with N99K were asymptomatic carriers. One patient with I295del mutation had funduscopically unilateral Best disease. In three children (all with I295del), EOG initially showed a clearly present light peak that deteriorated during 5 years of follow-up in two of them. Increased AF corresponded well to funduscopically visible lesions. During 3-6 years of follow-up, the lesion area did not change significantly, but there were obvious changes in the inner structure of the lesion. CONCLUSION: In the present series I295del, the most frequent mutation in our study, and N99K showed reduced penetrance. EOG was normal in young patients even if prime signs were visible. The lesion area did not depend on the mutation and did not correlate with VA. Lower VA was associated with a more irregular AF pattern due to scarring or haemorrhage. Our results indicate a disease causing effect that is cumulative over time.
