Retinoic acid increases the effect of bone morphogenetic protein type 2 on osteogenic differentiation of human adipose-derived stem cells

Abstract Bone morphogenetic protein type 2 (BMP-2) and retinoic acid (RA) are osteoinductive factors that stimulate endogenous mechanisms of bone repair which can be applied on management of osseous defects in oral and maxillofacial fields. Objective Considering the different results of RA on osteogenesis and its possible use to substitute/potency BMP-2 effects, this study evaluated the outcomes of BMP-2, RA, and BMP-2+RA treatments on in vitro osteogenic differentiation of human adipose-derived stem cells (ASCs) and the signaling pathway(s) involved. Material and Methods ASCs were treated every other day with basic osteogenic medium (OM) alone or supplemented with BMP-2, RA, or BMP-2+RA. Alkaline phosphatase (ALP) activity was determined using the r-nitrophenol method. Extracellular matrix mineralization was evaluated using von Kossa staining and calcium quantification. Expression of osteonectin and osteocalcin mRNA were determined using qPCR. Smad1, Smad4, phosphorylated Smad1/5/8, BMP-4, and BMP-7 proteins expressions were analyzed using western blotting. Signaling pathway was evaluated using the IPA® software. Results RA promoted the highest ALP activity at days 7, 14, 21, and 28, in comparison to BMP-2 and BMP-2+RA. BMP-2+RA best stimulated phosphorylated Smad1/5/8 protein expression at day 7 and Smad4 expression at days 7, 14, 21, and 28. Osteocalcin and osteonectin mRNA expressions were best stimulated by BMP-2+RA at day 7. Matrix mineralization was most improved by BMP-2+RA at days 12 and 32. Additionally, BMP-2+RA promoted the highest BMP signaling pathway activation at days 7 and 14, and demonstrated more activation of differentiation of bone-forming cells than OM alone. Conclusions In summary, RA increased the effect of BMP-2 on osteogenic differentiation of human ASCs.

Stanozolol promotes osteogenic gene expression and apposition of bone mineral in vitro

Abstract Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. Objective: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. Material and Methods: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. Results: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. Conclusions: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.

Effects of melatonin on tibia bone defects in rats / Efectos de la melatonina sobre defectos óseos en la tibia de ratas

The aim of this study was to evaluate the effects of melatonin healing in a tibial bone defect model in rats by means of histopathological and immunohistochemistry analysis. Twenty one male Wistar albino rats were used in this study. In each animal, bone defects (6 mm length ) were created in the tibias. The animals were divided into three groups. In group 1 control group (rats which tibial defects). Group 2 melatonin (10 mg/kg) + 14 days in the tibial defect group) was administered intraperitoneally to rats. Group 3 melatonin (10 mg/kg) + 28 days in the tibial defect group) was administered intraperitoneally to rats. Histopathological analysis of samples was performed to evaluate the process of osteoblastic activity, matrix formation, trabecular bone formation and myeloid tissue in bone defects. Immunohistochemical and immunoblot analysis demonstrated non-collagenous proteins (osteopontin and osteonectin) differences in tibial bone defects. The expression of osteopontin on tibia was increased by 14 days melatonin treatment. The expression of osteonectin on tibia was dramatically increased by 14 days melatonin treatment.

El objetivo fue evaluar por medio de análisis histopatológico e inmunohistoquímico los efectos cicatrizantes de la melatonina en un modelo de defecto óseo tibial en ratas. Se utilizaron 21 ratas albinas Wistar macho. En cada animal, se crearon defectos óseos en las tibias de 6 mm de longitud. Los animales se dividieron en tres grupos. El Grupo 1 correspondió al grupo control (defectos tibiales sin tratamiento). Al Grupo 2 se administró melatonina por vía intraperitoneal (10 mg/kg) 14 días posteriores al defecto tibial. Al Grupo 3 se administró melatonina por vía intraperitoneal (10 mg/kg) 28 días posteriores al defecto tibial. Se realizó un análisis histopatológico para evaluar los procesos de actividad osteoblástica, formación de matriz, formación de hueso trabecular y tejido mieloide en los defectos óseos. Los análisis inmunohistoquímicos y de inmunotransferencia mostraron diferencias de proteínas no colágenas (osteopontina y osteonectina). La expresión de osteopontina en defectos óseos tibiales se incrementó en el Grupo 2. La expresión de osteonectina en la tibia se incrementó fuertemente bajo el tratamiento con melatonina por 14 días.

Immunohistochemical expression of TGF-ß1 and osteonectin in engineered and Ca(OH)2-repaired human pulp tissues

Abstract The aim of the present study was to evaluate the expression of transforming growth factor-β1 (TGF-β1) and osteonectin (ON) in pulp-like tissues developed by tissue engineering and to compare it with the expression of these proteins in pulps treated with Ca(OH)2 therapy. Tooth slices were obtained from non-carious human third molars under sterile procedures. The residual periodontal and pulp soft tissues were removed. Empty pulp spaces of the tooth slice were filled with sodium chloride particles (250-425 µm). PLLA solubilized in 5% chloroform was applied over the salt particles. The tooth slice/scaffold (TS/S) set was stored overnight and then rinsed thoroughly to wash out the salt. Scaffolds were previously sterilized with ethanol (100-70°) and washed with phosphate-buffered saline (PBS). TS/S was treated with 10% EDTA and seeded with dental pulp stem cells (DPSC). Then, TS/S was implanted into the dorsum of immunodeficient mice for 28 days. Human third molars previously treated with Ca(OH)2 for 90 days were also evaluated. Samples were prepared and submitted to histological and immunohistochemical (with anti-TGF-β1, 1:100 and anti-ON, 1:350) analyses. After 28 days, TS/S showed morphological characteristics similar to those observed in dental pulp treated with Ca(OH)2. Ca(OH)2-treated pulps showed the usual repaired pulp characteristics. In TS/S, newly formed tissues and pre-dentin was colored, which elucidated the expression of TGF-β1 and ON. Immunohistochemistry staining of Ca(OH)2-treated pulps showed the same expression patterns. The extracellular matrix displayed a fibrillar pattern under both conditions. Regenerative events in the pulp seem to follow a similar pattern of TGF-β1 and ON expression as the repair processes.