Petroleum ether extract of Cissus quadrangularis Linn. Email: kummigames yahoo. OBJECTIVE: To evaluate the effects of the petroleum ether extract of Cissus quadrangularis on the proliferation rate of bone marrow mesenchymal stem cells, the differentiation of marrow mesenchymal stem cells into osteoblasts osteoblastogenesis and extracellular matrix calcification. This study also aimed to determine the additive effect of osteogenic media and Cissus quadrangularis on proliferation, differentiation and calcification.
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Petroleum ether extract of Cissus quadrangularis Linn. Email: kummigames yahoo. OBJECTIVE: To evaluate the effects of the petroleum ether extract of Cissus quadrangularis on the proliferation rate of bone marrow mesenchymal stem cells, the differentiation of marrow mesenchymal stem cells into osteoblasts osteoblastogenesis and extracellular matrix calcification.
This study also aimed to determine the additive effect of osteogenic media and Cissus quadrangularis on proliferation, differentiation and calcification. Extracellular matrix calcification was confirmed by Von Kossa staining. Cells grown in osteogenic media containing Cissus quadrangularis exhibited higher proliferation, differentiation and calcification rates than did control cells. Keywords: Cissus quadrangularis; Mesenchymal stem cells; Osteoblastogenesis; Osteoporosis; Osteoblast.
The repair of bone defects secondary to trauma, osteoporosis, osteomyelitis and nonunion fracture poses a significant problem for many clinicians, particularly orthopedic, head and neck, and plastic surgeons. Several strategies have been employed to promote the bone-healing process. Bone marrow mesenchymal stem cells MSCs have recently received widespread attention due to their potential use in tissue engineering applications.
MSCs are defined as self-renewable, multipotent progenitor cells with the capacity to differentiate into several distinct mesenchymal lineages and are thus excellent candidates for tissue engineering. As a part of our continuing search for biologically active natural anti-osteoporotic agents, we have extensively evaluated the efficacy of petroleum ether extract of Cissus quadrangularis Linn. CQ on osteomodulation in a rat model. CQ Veldt Grape or Winged Treebine , a vine that belongs to the family Vitaceae, is one of the most frequently used medicinal plants in India.
Extracts of this plant are reported to contain phytoestrogenic steroids, ascorbic acid, carotene, calcium and anabolic steroids. CQ was also recently shown to have significant effects in periodontal regenerative therapy. Phytochemical analyses of CQ have revealed high contents of ascorbic acid, carotene, anabolic steroidal substances and calcium.
Although different uses of CQ have been investigated, the exact mechanism of its bone-healing properties has not been evaluated.
Therefore, in the present study, we aimed to evaluate the effects of petroleum ether extract of CQ on bone marrow mesenchymal stem cell proliferation and osteogenic differentiation.
Plant extract: The stems of Cissus quadrangularis CQ were collected from the Nalgonda District of Andhra Pradesh, India, and were identified and authenticated by a botanist. A voucher specimen was deposited in the pharmacology department of Manipal University, Manipal. The fleshy stems 2. The total ethanol extract was concentrated in a vacuum, after which the extract was dissolved in water and the solution was partitioned with petroleum ether.
A total of The rats were housed in sanitized polypropylene cages containing sterile paddy husks as bedding. All animals were allowed free access to distilled water and commercial diet. Isolation of bone marrow cells: Three male Wistar rats of the albino strain were anesthetized with ether.
The bones were cut at both ends, and the bone marrow was flushed with 5 mL of media. Bone marrow cells were collected separately from three different animal bones. The cells were mixed gently and filtered through a membrane filter to remove debris. The filtrate was centrifuged at 3, rpm for 5 min, and the cells were washed with DMEM and used to seed cultures. After three days, the floating cells were discarded; the media were changed every third day thereafter.
These three cell lines were independently subjected to all of the assays in this project. Once monolayer cells were confluent, they were trypsinized and subcultured as required.
After fixation, cells were washed three times with distilled water and then air-dried for 10 min. ALP-positive cells were stained pink. Cells were then washed with distilled water, treated with 2. The calcified extracellular matrix appeared as black nodules. Then, the formazan crystals were dissolved in DMSO for 1 hr at room temperature. The absorbance of the colored solution was measured using an ELISA plate reader at a wavelength of nm.
Data were analyzed using the Student's t -test in GraphPad. The represented data are the average value of three different assays. Furthermore, treatment of the MSCs grown in osteogenic media with plant extract resulted in a 3-fold increase in the cell proliferation rate in comparison with cells grown in basal media alone Figure 1B.
Thus, our results from three independent assays show that treatment of undifferentiated MSCs with the plant extract alone has a significant influence on proliferation, while an additive effect was seen when cells were also treated with osteogenic media. Furthermore, MSCs grown in basal media with different concentrations of CQ extract began to show ALP activity by the 15 th day of treatment; most cells were strongly positive for ALP by the end of the 20 th day of treatment.
It has been shown that untreated MSCs must be cultured in osteogenic media for 28 days before ALP-positive, differentiated osteoblasts can be identified. The figures are representative photographs of three independent experiments that showed similar results. Effect of CQ extract on the mineralization of extracellular matrix: MSCs grown in basal media alone did not show any signs of mineralization in the extracellular matrix Figure 4A even after 28 days. Thus, our result suggests that CQ extract not only induces the differentiation of MSCs into osteoblasts but also causes the differentiated osteoblasts to deposit calcium salts in the extracellular matrix.
Additionally, undifferentiated MSCs can be differentiated into functional osteoblasts by treatment with CQ extract alone, without any other stimulator in the culture media. The figures are representative photographs of three similar independent experiments. Human bone is composed of a mineralized organic matrix and bone cells. Osteoblasts are active mature bone cells that synthesize the organic matrix and regulate the mineralization process.
After mineralization, the complete bone becomes hard and rigid with the mechanical properties necessary to withstand external forces, support the body and protect the internal organs.
The proliferation rate and biological activity of the osteoblasts controls the rate of bone formation, and accelerated osteoblast growth is the key factor for efficient bone repair. The reduction in bone mass in osteoporosis is due to an imbalance between bone resorption and bone formation in which the rate of resorption exceeds that of formation. The most important risk factor for osteoporosis is advanced age, in both men and women. In women, estrogen deficiency following menopause is correlated with a rapid reduction in BMD bone mineral density.
In men, a decrease in testosterone levels has similar but less pronounced effects. Other causes of osteoporosis include tobacco-smoking, low body mass index, malnutrition, alcoholism, insufficient physical activity and exposure to heavy metals like cadmium. Bone marrow mesenchymal stem cells are the source of bone-forming osteoblasts. This activity of the plant extract is increased further in the presence of osteogenic media.
The plant extract also facilitated extracellular matrix mineralization, which was more pronounced in the presence of osteogenic media. Finally, the presence of plant extract in the control and osteogenic media stimulated the proliferation rate of MSCs. Our results clearly show that the CQ plant extract enhances the proliferation and differentiation ability of MSCs into osteoblasts. ALP activity, the most widely recognized biomarker for osteoblast activity, 25 was enhanced by a short treatment with CQ.
Our findings are in line with several previous in vivo experiments that have demonstrated that CQ promotes ALP activity and enhances collagen synthesis in the fracture-healing process. The phytogenic steroids found in CQ 7 may be involved in stimulating osteoblastogenesis and may act on estrogen receptors of bone cells.
The exact molecular mechanism involved in CQ-promoted osteogenesis remains to be explored. However, some evidence suggests that Wnt signaling may be involved. This pathway has been shown to play a significant role in the control of osteoblastogenesis and bone formation.
Mutations in these signaling molecules are strongly associated with changes in bone mineral density and fractures. Loss-of-function mutations in LRP5 low-density lipoprotein receptor-related protein receptors cause osteoporosis-pseudoglioma syndrome, while gain-of-function mutations in the same group lead to high bone mass phenotypes. Recently, it has been shown that the CQ-mediated increase in osteoblast activity may be mediated through a MAPK-dependent pathway.
Our in vitro and in vivo data suggest that CQ can be effectively used to treat various bone disorders and can also be used as a preventive measure for disorders that lead to decreased bone mineral density.
We sincerely thank Dr. We thank Ms. Rekha Nayak for her technical help during this project. Pongboonrod S. Kaeseam- Bunnakit Printing. Sivaranjan II, Balachandran I. Ayurvedic drugs and their plant sources. Oxford and India book house publishing Co. Pvt Ltd, Biomechanical and Calcium 45 studies on the effect of Cissus quadrangularis in fracture repair. Ind J Med Res. Madan Nayar. Pharmalogical study of the stem of Cissus Quadrangularis Linn.
J Sci Ind Res. Biochemical and Ca 45 studies on the effect of Cissus quadrangularis in fracture healing. Sen SP. Preliminary clinical studies of Cissus quadrangularis. J Med Res. Studies on Cissus quadrangularis Linn. Experimental studies of the use of 'Cissus quadrangularis' in healing of fractures. Ind J Med Sci. Studies on "Cissus Quadrangularis" in fracture by using phosphorus Studies on Cissus quadrangularis in experimental fracture repair: Effect of chemical parameters in blood.
Studies on fracture healing in vivo by using radioactive strontium.
Cissus quadrangularis is a plant that has been revered for its medicinal properties for thousands of years. Historically, it has been used to treat many conditions, including hemorrhoids, gout, asthma, and allergies. However, recent research has found that this power-packed plant may also help promote bone health, relieve joint pain, and protect against chronic conditions like heart disease, diabetes, and stroke. This article reviews the uses, benefits, and side effects of Cissus quadrangularis , as well as its dosage information.
Osteogenic potential of cissus qudrangularis assessed with osteopontin expression
Fracture healing involves complex processes of cell and tissue proliferation and differentiation. Many factors are involved, including growth factors, inflammatory cytokines, antioxidants, bone breakdown osteoclast and bone building osteoblast cells, hormones, amino acids, and uncounted nutrients. We studied the osteogenic potential of Cissus quadrangularis CQ , a plant that has been customarily used in the Indian subcontinent to hasten the process of healing in bone fractures. Total of 60 patients age, years of mandible fracture was divided in two groups.