Fosfatasa Alcalina (ALP) y Runx2 en cultivos celulares de osteoblastos estimulados con campo eléctrico

Abstract

ResumenEl objeto de este estudio fue identificar el estímulo eléctrico que debe aplicarse en cultivos celulares de osteoblastos (Ob) para aumentar la expresión del gen de Fosfatasa Alcalina (ALP) y el factor de transcripción Runx2. Se cultivaron Ob de la American Type Culture Collection (ATCC) Ref. CRL 11372. Los cultivos se estimularon del día cinco, cuando las células presentaron confluencia, hasta el día ocho. El estímulo aplicado a cada grupo experimental fue de 100mV, 200mV, 300mV, 400mV y 500mV respectivamente, se cultivó un grupo control no estimulado con cada grupo experimental. El campo eléctrico se generó con corriente alterna (AC) y se aplicó mediante dos placas de aluminio ubicadas de forma lateral y paralela a los frascos de cultivo de 25cm 2 . Los niveles de expresión de mRNA se midieron con la técnica quantitative reverse transcription polymerase chain reaction (qRT-PCR). Con la aplicación de campo generado con AC aumentó la expresión del factor de transcripción RunX2 en proporción directa al aumento del voltaje aplicado. La expresión del gen de ALP fue inversamente proporcional a la aplicación del estímulo y se identificó una diferencia significativa entre la presencia y ausencia del estímulo, siendo mayor en ausencia del estímulo. El campo eléctrico generó una señal que puede aumentar o disminuir la expresión de los genes que median la formación de tejido óseo. En el caso de Runx2, favoreció la diferenciación de células mesenquimales a Ob con la consecuente actividad de remodelación y formación del tejido óseo.Palabras clave: Osteoblastos, fosfatasa alcalina, estímulo eléctrico, Runx2, expresión génica Abreviaciones: *FA, Fosfatasa Alcalina*; *Ob, Osteoblastos* ALKALINE PHOSPHATASE (ALP) AND Runx2 IN CELL CULTURES STIMULATED OSTEOBLASTS ELECTRIC FIELD AbstractThe purpose of this study was to identify the electrical fields to be applied in osteoblast (Ob) cell cultures, in order to increase the expression of Alkaline Phosphatase (ALP) gene and the transcription factor Runx2. Ob cultured where from the American Type Culture Collection (ATCC) Ref CRL 11372. Cell Cultures received stimulation at day five, when they showed a confluent monolayer organization until day eight. The stimulus applied to each experimental group was 100mV, 200mV, 300mV, 400mV and 500mV respectively, a control group was cultured without stimulation. The electric field is generated

Directing lineage specification of human mesenchymal stem cells by decoupling electrical stimulation and physical patterning on unmodified graphene

“…Additionally, osteopontin is known to carry a significant negative charge, thereby making it sensitive in carrying any bioelectrical signals 36 . With respect to RUNX2, a separate study found that under increasing AC current, RUNX2 expression increased in an exponential-like pattern, which suggests this osteogenic-associated protein may have other roles in responding to external stimuli such as electrical current, thereby matching the observation we saw in our experiments 37 . Finally, as our experimental setup had direct cell interface with graphene, the fact that we were able to observe similar responses mentioned in the aforementioned studies demonstrates the sensitivity these cell have on graphene and that only fractions of the electrical stimulation conditions are needed to elicit observable protein-level cell responses.…”

“…36 With respect to RUNX2, a separate study found that under increasing AC current, RUNX2 expression increased in an exponential-like pattern, which suggests this osteogenicassociated protein may have other roles in responding to external stimuli such as electrical current, thereby matching the observation we saw in our experiments. 37 Finally, as our experi-mental setup had direct cell interface with graphene, the fact that we were able to observe similar responses mentioned in the aforementioned studies demonstrates the sensitivity these cell have on graphene and that only fractions of the electrical stimulation conditions are needed to elicit observable proteinlevel cell responses. While electrical stimulation could cue initial osteogenic potential on unpatterned graphene, as indicated by the alterations in RUNX2, the lack of increased late stage osteogenic protein osteopontin suggests the hMSCs have not adopted a new phenotype; however, when stimulation is combined with physical patterning, a further progression towards a more mature osteogenic phenotype is promoted.…”

Biomineralization Process in Three-Dimensional Scaffolds Based on Bacterial Nanocellulose for Bone Tissue Engineering: Chemi-Cal and Morphological Features, and Stem Cell Differentiation

“…Then, as reported by various authors, after the start of mineralization, ALP is no longer needed and therefore the cellular levels of the enzyme decrease before a mature mineralized matrix is formed. Therefore, the decrease in the expression of ALP may be indicative of the initiation of biomineralization of pre-osteoblasts on the designed BNC scaffolds [62], [63]. The results of ALP expression of BM-MSC grown on the different biomineralized BNC scaffolds show us that biomineralized BNC scaffolds do promote the secretion of this protein by BM-MSC.…”