Osteogenic Potential and Bioactive Profiles of Piper sarmentosum Ethanolic Extract-Treated Stem Cells.

Piper sarmentosum is a well-known traditional herbal plant in various diseases treatments. Multiple scientific studies have also reported various biological activities exhibited by the plant's extract, such as antimicrobial, anticarcinogenic and antihyperglycemic activities, and, in addition, a bone protective effect in ovariectomized rats has been reported. However, no known Piper sarmentosum extract is involved in osteoblast differentiation using stem cells. Our study aims to identify the potential of P. sarmentosum ethanolic extract to induce osteoblast differentiation of human peripheral blood stem cells. Prior to the assay, the proliferation ability of the cells was observed for 14 days and the presence of hematopoietic stem cells in the culture was determined by the expression of SLAMF1 and CD34 genes. During the differentiation assay, the cells were treated with P. sarmentosum ethanolic extract for 14 days. Osteoblast differentiation was examined using an (alkaline phosphatase) ALP assay, by monitoring the expression of osteogenic gene markers and by von Kossa staining. The untreated cells served as the negative control, while cells treated with 50 µg/mL ascorbic acid and 10 mM ß-glycerophosphate acted as the positive control. Finally, the determination of the compound profile was performed using a gas chromatography-mass spectrometry (GC-MS) analysis. The isolated cells were able to proliferate for 14 days during the proliferation assay. The expression of hematopoietic stem cell markers was also upregulated during the 14 days assay. Following the differentiation induction, the ALP activity exhibited a significant increase (p < 0.05) from day 3 of the differentiation assay. A molecular analysis also showed that the osteogenic markers ALP, RUNX2, OPN and OCN were upregulated compared to the positive control. The presence of mineralized cells with a brownish-stained morphology was observed, indicating the mineralization process increased in a time-dependent manner regardless of the concentration used. There were 54 compounds observed in the GC-MS analysis, including ß-asarones, carvacrol and phytol, which have been shown to possess osteoinductive capacities. Our results demonstrate that the ethanolic extract of P. sarmentosum can induce osteoblast differentiation of peripheral blood stem cells. The extract contains potent compounds which can potentially induce the differentiation of bone cells, i.e., osteoblasts.

Label-free Quantitative Proteomic Analysis of Ascorbic Acid-induced Differentially Expressed Osteoblast-related Proteins in Dental Pulp Stem Cells from Deciduous and Permanent Teeth.

BACKGROUND: Proteomic is capable of elucidating complex biological systems through protein expression, function, and interaction under a particular condition. OBJECTIVE: This study aimed to determine the potential of ascorbic acid alone in inducing differentially expressed osteoblast-related proteins in dental stem cells via the liquid chromatography-mass spectrometry/ mass spectrometry (LC-MS/MS) approach. METHODS: The cells were isolated from deciduous (SHED) and permanent teeth (DPSC) and induced with 10 µg/mL of ascorbic acid. Bone mineralisation and osteoblast gene expression were determined using von Kossa staining and reverse transcriptase-polymerase chain reaction. The label-free protein samples were harvested on days 7 and 21, followed by protein identification and quantification using LC-MS/MS. Based on the similar protein expressed throughout treatment and controls for SHED and DPSC, overall biological processes followed by osteoblast-related protein abundance were determined using the PANTHER database. STRING database was performed to determine differentially expressed proteins as candidates for SHED and DPSC during osteoblast development. RESULTS: Both cells indicated brownish mineral stain and expression of osteoblast-related genes on day 21. Overall, a total of 700 proteins were similar among all treatments on days 7 and 21, with 482 proteins appearing in the PANTHER database. Osteoblast-related protein abundance indicated 31 and 14 proteins related to SHED and DPSC, respectively. Further analysis by the STRING database identified only 22 and 11 proteins from the respective group. Differential expressed analysis of similar proteins from these two groups revealed ACTN4 and ACTN1 as proteins involved in both SHED and DPSC. In addition, three (PSMD11/RPN11, PLS3, and CLIC1) and one (SYNCRIP) protein were differentially expressed specifically for SHED and DPSC, respectively. CONCLUSION: Proteome differential expression showed that ascorbic acid alone could induce osteoblastrelated proteins in SHED and DPSC and generate specific differentially expressed protein markers.

Gene expression profiles for in vitro human stem cell differentiation into osteoblasts and osteoclasts: a systematic review.

Background: There have been promising results published regarding the potential of stem cells in regenerative medicine. However, the vast variety of choices of techniques and the lack of a standard approach to analyse human osteoblast and osteoclast differentiation may reduce the utility of stem cells as a tool in medical applications. Therefore, this review aims to systematically evaluate the findings based on stem cell differentiation to define a standard gene expression profile approach. Methods: This review was performed following the PRISMA guidelines. A systematic search of the study was conducted by retrieving articles from the electronic databases PubMed and Web of Science to identify articles focussed on gene expression and approaches for osteoblast and osteoclast differentiation. Results: Six articles were included in this review; there were original articles of in vitro human stem cell differentiation into osteoblasts and osteoclasts that involved gene expression profiling. Quantitative polymerase chain reaction (qPCR) was the most used technique for gene expression to detect differentiated human osteoblasts and osteoclasts. A total of 16 genes were found to be related to differentiating osteoblast and osteoclast differentiation. Conclusion: Qualitative information of gene expression provided by qPCR could become a standard technique to analyse the differentiation of human stem cells into osteoblasts and osteoclasts rather than evaluating relative gene expression. RUNX2 and CTSK could be applied to detect osteoblasts and osteoclasts, respectively, while RANKL could be applied to detect both osteoblasts and osteoclasts. This review provides future researchers with a central source of relevant information on the vast variety of gene expression approaches in analysing the differentiation of human osteoblast and osteoclast cells. In addition, these findings should enable researchers to conduct accurately and efficiently studies involving isolated human stem cell differentiation into osteoblasts and osteoclasts.

The effects of Piper sarmentosum aqueous extracts on zebrafish (Danio rerio) embryos and caudal fin tissue regeneration.

In Malaysia, Piper sarmentosum or 'kaduk' is commonly used in traditional medicines. However, its biological effects including in vivo embryonic toxicity and tissue regenerative properties are relatively unknown. The purpose of this study was to determine zebrafish (Danio rerio) embryo toxicities and caudal fin tissue regeneration in the presence of P. sarmentosum aqueous extracts. The phytochemical components and antioxidant activity of the extract were studied using GC-MS analysis and DPPH assay, respectively. Embryo toxicity tests involving survival, heartbeat, and morphological analyses were conducted to determine P. sarmentosum extract toxicity (0-60 µg/mL); concentrations of 0-400 µg/mL of the extract were used to study tissue regeneration in the zebrafish caudal fin. The extract contained several phytochemicals with antioxidant activity and exhibited DPPH scavenging activity (IC50 = 50.56 mg/mL). Embryo toxicity assays showed that a concentration of 60 µg/mL showed the highest rates of lethality regardless of exposure time. Slower embryogenesis was observed at 40 µg/mL, with non-viable embryos first detected at 50 µg/mL. Extracts showed significant differences (p < 0.01) for tissue regeneration at all concentrations when compared to non-treated samples. In conclusion, Piper sarmentosum extracts accelerated tissue regeneration, and extract concentrations at 60 µg/mL showed the highest toxicity levels for embryo viability.