Effects of low level laser irradiation on the osteogenic capacity of sodium alginate/gelatin/human adipose-derived stem cells 3D bio-printing construct / 北京大学学报(医学版)

OBJECTIVE@#To explore the effects of low level laser irradiation (LLLI) on the osteogenic capacity of three-dimensional (3D) structure by 3D bio-printing construct used human adipose-derived stem cells (hASCs) as seed cells.@*METHODS@#Using hASCs as seed cells, we prepared sodium alginate/gelatin/hASCs 3D bio-printing construct, and divided them into four groups: PM (proliferative medium), PM+LLLI, OM (osteogenic medium) and OM+LLLI, and the total doses of LLLI was 4 J/cm². Immunofluorescence microscopy was used to observe the viability of the cells, and analyze the expression of the osteogenesis-related protein Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN).@*RESULTS@#The 3D constructs obtained by printing were examined by microscope. The sizes of these 3D constructs were 10 mm×10 mm×1.5 mm. The wall thickness of the printed gelatin mold was approximately 1 mm, and the pores were round and had a diameter of about 700 μm. The cell viability of sodium alginate/gelatin/hASCs 3D bio-printing construct was high, and the difference among the four groups was not significant. On day 7, the expression of OCN from high to low was group OM+LLLI, PM+LLLI, OM and PM. There were significant differences among these groups (P<0.01), but there was no significant difference between group PM+LLLI and OM. On day 14, the expression of OCN in each group was higher than that on day 7, and there was no significant difference between group OM+LLLI and OM. The expression of Runx2 in group OM+LLLI was more than 90%, significantly higher than that in group OM (P<0.01). But the expression of Runx2 in group PM+LLLI and OM+LLLI were significantly lower than that in the non-irradiated groups. The expression of osteogenesis-related protein Runx2 and OCN were higher in OM groups than in PM groups. Furthermore, the irradiated groups were significantly higher than the non-irradiated groups.@*CONCLUSION@#LLLI does not affect the cell viability of sodium alginate/gelatin/hASCs 3D bio-printing construct, and may promote the osteogenic differentiation of hASCs.

Low-level laser irradiation promotes proliferation of cryopreserved adipose-derived stem cells / Laser de baixa intensidade promove proliferação de células-tronco derivadas de tecido adiposo criopreservadas

ABSTRACT Objective To evaluate the effect of low-level laser irradiation on proliferation and viability of murine adipose-derived stem cells previously submitted to cryopreservation. Methods Adipose-derived stem cells were isolated from inguinal fat pads of three mice, submitted to cryopreservation in fetal bovine serum with 10% dimethylsulfoxide for 30 days and then thawed and maintained in normal culture conditions. Culture cells were either irradiated or not (control) with an InGaAIP diode laser at zero and 48 hours, using two different energy densities (0.5 and 1.0J/cm2). Cell proliferation was evaluated by trypan blue exclusion method and MTT assay at intervals of zero, 24, 48, and 72 hours after the first laser application. Cell viability and apoptosis of previously cryopreserved cells submitted to laser therapy were evaluated by flow cytometry. Results The Irradiated Groups (0.5 and 1.0J/cm2) showed an increased cell proliferation (p<0.05) when compared to the Control Group, however no significant difference between the two energy densities was observed. Flow cytometry revealed a percentage of viable cells higher than 99% in all groups. Conclusion Low-level laser irradiation has stimulatory effects on the proliferation of adipose-derived stem cells previously submitted to cryopreservation.

RESUMO Objetivo Avaliar o efeito do laser de baixa intensidade na proliferação e na viabilidade de células-tronco derivadas de tecido adiposo murinas previamente submetidas à criopreservação. Métodos Células-tronco derivadas de tecido adiposo foram isoladas da região inguinal de três camundongos, submetidas à criopreservação em soro fetal bovino com 10% de dimetilsulfóxido por 30 dias e, depois, descongeladas e mantidas em condições normais de cultivo. As células cultivadas foram irradiadas ou não (controle) com um laser de diodo InGaAIP nos intervalos de zero e 48 horas, utilizando duas densidades de energia diferentes (0,5 e 1,0J/cm2). A proliferação celular foi avaliada pelo método de exclusão de azul de tripan e ensaio MTT, nos intervalos de zero, 24, 48 e 72 horas após a primeira aplicação do laser. A viabilidade celular e a apoptose das células previamente criopreservadas submetidas à laserterapia foram avaliadas por citometria de fluxo. Resultados Os Grupos Irradiados (0,5 e 1,0J/cm2) apresentaram aumento da proliferação celular (p<0,05) quando comparados ao Grupos Controle, porém não foi observada diferença significativa entre as duas densidades de energia. A citometria de fluxo revelou percentagem de células viáveis superior a 99% em todos os grupos. Conclusão O laser de baixa intensidade tem efeitos estimuladores sobre a proliferação de células-tronco derivadas de tecido adiposo previamente submetidas à criopreservação.