• 한국동물위생학회지
• /
• 제34권4호
• /
• pp.397-401
• /
• 2011

Aspiration of lytic bone lesions is an excellent diagnostic test in the initial evaluation of primary bone tumor. However, cytologically, it can be difficult to differentiate osteosarcoma (OSA) from other bone neoplasms, including fibrosarcoma, chondrosarcoma, synovial cell sarcoma, malignant fibrous histiocytoma and malignant peripheral nerve sheath tumor. The purpose of this study is to introduce alkaline phosphatase (ALP) staining to differentiate OSA from other mesenchymal tumors. Tumors actively producing bone are specifically positive for ALP staining. Unstained, cytologic specimens were incubated for 10 minutes with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt-phosphatase substrate. Among 20 cases of cytology specimen, 14 were positive for ALP staining and histopathology, 6 were negative for ALP staining and histopathology. ALP staining was 100% sensitive and specificity for the diagnosis of OSA. Aspirate cytology with ALP staining was a simple, fast, safe and accurate diagnostic test for the evaluation of suspected OSA lesions in dogs.

• Nutritional Sciences
• /
• 제8권4호
• /
• pp.242-249
• /
• 2005

It is well established that zinc plays an important role in bone metabolism and mineralization. The role of zinc in bone formation is well documented in animal models, but not much reported in cell models. In the present study, we evaluated zinc deficiency effects on osteoblastic cell proliferation, alkaline phosphatase activity and expression, and extracellular matrix bone nodule formation and bone-related gene expression in osteoblastic MC3T3-E1 cells. To deplete cellular zinc, chelexed-FBS and interpermeable zinc chelator TPEN were used. MC3T3-E1 cells were cultured in zinc concentration-dependent (0-15 ${\mu}M\;ZnCl_2$) and time-dependent (0-20 days) manners. MC3T3-E1 cell proliferation by MTT assay was increased as medium zinc level increased (p<0.05). Cellular Ca level and alkaline phosphatase activity were increased as medium zinc level increased (p<0.05). Alkaline phosphatase expression, a marker of commitment to the osteoblast lineage, measured by alkaline phosphatase staining was increased as medium zinc level increased. Extracellular calcium deposits measured by von Kossa staining for nodule formation also appeared higher in Zn+(15 ${\mu}M\;ZnCl_2$) than in Zn-(0 ${\mu}M\;ZnCl_2$). Bone formation marker genes, alkaline phosphatase and osteocalcin, were also expressed higher in Zn+ than in Zn-. The current work supports the beneficial effect of zinc on bone mineralization and bone-related gene expression. The results also promote further study as to the molecular mechanism of zinc deficiency for bone formation and thus facilitate to design preventive strategies for zinc-deficient bone diseases.

• 한국가축번식학회지
• /
• 제19권1호
• /
• pp.55-63
• /
• 1995

The putative totipotency germ cells has a relative abundance of alkaline phosphatases. Thus, histological staining of AP activity offers a new route to isolate totipotent cells and also provides insights into culture systems of these cells. Furthermore, the AP staining technique is simple and fast, requires only the napthol AS/MS substrate in combination with trapping diazonium salts such as fast red or fast blue. However, our unexpected finding was that AP staining of mouse ES cells were detected in the undifferentiaed epiblast-derived cells as well as several types of differentiating cells. This findings are different from results of Talbot et al. (1993) reported usefulness of the AP staining and implies that histological staining of AP may not by useful to determine undifferentiaed state or totipotency of ES cells. Thus, we have investigated the patterns of AP expression by RT-PCR in order to identify a marker of undifferentiated ES/primordial germ (PG) cells. In RT-PCR analysis, embryonic (E)-AP was detected only in undifferentiated ES cells, but intestinal(I)-AP was not detected in all of the examined ES and PG cells. In addition, nonspecific (NS)-AP wasdetected in undifferentiated PG cell from day 7, 5 to 13 of gestation. Histological activity of AP in ES cells was completely suppressed by addition of L-phenylalanine (Phe), L-homoarginine (Har), and L-phenylalanylglycylglycine (PheGlyGly) as an inhibitor, but RT-PCR showed the same results as in the absence of an inhibitors. Our findings suggested that expression of E-AP and NS-AP may use as a marker to determine the undifferentiated status in ES and PG cells.

• 한국동물학회지
• /
• 제36권3호
• /
• pp.425-432
• /
• 1993

Culex pipiens pallens에 존재하는 Alkaline phosphatase의 연구를 위한 적정 분석조건과 우화 후, 시간 경과에 따른 Alkaline phosphatase 활성 경향에 대해서 연구하였다. C. pipiens에 존재하는 Alkaline phosphatase의 활성은 우화 직후부터 지속적으로 감소하다가, 흡혈 자극에 의해서 급격하게 증가한다. 흡혈 후 30시간이 경과했을 때, 최대의 활성도를 보이고 감소하나, 흡혈 48시간 이후에는 다시 증가하여 지속적으로 유지됨을 알 수 있다. 기관별 분석에서 첫번째 활성 증가는 중장에서 일어나고, 두번째 활성 증가는 난소에서 일어남을 알 수 있다. 그리고 흡혈 후 30시간된 성체에서는 5개의 동위효소 밴드가 보이는데, 난소에서 ALP-1와 ALP-2가 나타나고, 가슴에서는 ALP-3, ALP-4와 ALP-5가 보인다. 지방체에서는 ALP-4와 ALP-5가, 중장에서는 ALP-3, ALP-4와 ALP-5가 나타남을 알 수 있다. 그리고 흡혈 후 72시간된 성체에서, ALP-1, ALP-2가 동일하게 존재함을 알 수 있다.

• 동의생리병리학회지
• /
• 제16권5호
• /
• pp.1042-1047
• /
• 2002

Recently, many natural medicines, which have advantage of less side effects and possibility of long-term use, have been studied for their capacity and effects of anti-bacterial, anti-inflammatory and regenerative potential for periodontal tissues. Cortex Eucommiae, Eupoly phaga, Semen Cuscutae, Halloysitum Rubrum have been traditionally used as medicines for treatment of bone disease in Korea. The objective of the present study is to examine the ability of alkaline phosphatase (ALP) activity in human fetal osteoblast cell line (hFOB1) with several natural medicines. hFOB1 added DMEM/F-12 were cultured with dexamethasone as a positive control, and with each natural medicine. ALP activity was measured by spectrophotometer for enzyme activity and naphthol AS-Bl staining was performed for morphometry. All of the natural medicines induced a higher ALP activity compared to negative control, especially, Cortex Eucommiae increased an ALP activity in all experimental groups (p<0.05). In naphthol AS-Bl staining, all of the natural medicines of this study increased the stained area compared to negative control. Especially, Cortex Eucommiae and Eupoly phaga showed statistical significance compared to negative control (p<0.05). These results indicate that Cortex Eucommiae, Eupoly phaga, Semen Cuscutae, Halloysitum Rubrum have an inducing ability of ALP synthesis on osteoblasts.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• 제30권2호
• /
• pp.133-141
• /
• 2008

Stem cells have self-renewal capacity, long-term viability, and multiline age potential. Adult bone marrow contains mesenchymal stem cells. Bone marrow-derived mesenchymal stem cells (BMSCs) are progenitors of skeletal tissue components and can differentiate into adipocytes, chondrocytes, osteoblasts, and myoblasts in vitro and undergo differentiation in vivo. However, the clinical use of BMSCs has presented problems, including pain, morbidity, and low cell number upon harvest. Recent studies have identified a putative stem cell population within the adipose tissue. Human adipose tissue contains pluripotent stem cells simillar to bone marrow-derived stem cells that can differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. Human adipose tissue-derived stem cells (ATSCs) could be proposed as an alternative source of adult bone marrow stem cells, and could be obtained in large quantities, under local anesthesia, with minimal discomfort. Human adipose tissue obtained by liposuction was processed to obtain ATSCs. In this study, we compared the osteogenic differentiation of ATSCs in a specific osteogenic induction medium with that in a non-osteogenic medium. ATSCs were incubated in an osteogenic medium for 28 days to induce osteogenesis respectively. Osteogenic differentiation was assessed by von Kossa and alkaline phosphatase staining. Expression of osteocyte specific bone sialoprotein, osteocalcin, collagen type I and alkaline phosphatase, bone morphogenic protein 2, bone morphogenic protein 6 was confirmed by RT-PCR. ATSCs incubated in the osteogenic medium were stained positively for von Kossa and alkaline phosphatase staining. Expression of osteocyte specific genes was also detected. Since this cell population can be easily identified through fluorescence microscopy, it may be an ideal source of ATSCs for further experiments on stem cell biology and tissue engineering. The present results show that ADSCs have an ability to differentiate into osteoblasts. In the present study, we extend this approach to characterize adipose tissue-derived stem cells.

• 대한한의학회지
• /
• 제38권4호
• /
• pp.1-10
• /
• 2017

Objectives: The aim of the present study is to evaluate the effects of the extract of Pongamia pinnata on the morphology, viability, and differentiation potential of human stem cells derived from the gingiva. Methods: Stem cells obtained from gingivae were cultured in an osteogenic medium in the presence of methanol extract of Pongamia pinnata (PPT) at concentrations ranging from 0.001 to 1%. Evaluations of cell morphology and cellular viability were done at Day 1. Alkaline phosphatase activity assays and Alizarin red S staining were performed to evaluate the osteogenic differentiation of stem cells. Results: The morphology of stem cells in the presence of PPT at final concentrations of 0%, 0.001%, 0.01%, 0.1%, and 1% did not produce any noticeable changes when compared with the untreated control group. Application of PPT produced a significant increase in alkaline phosphatase activity when compared to the control group. The results of the Alizarin Red S staining showed a significant increase of absorbance with the 0.001% group. Conclusions: Based on these findings, it was concluded that PPT could produce beneficial effects on mesenchymal stem cells with enhanced osteogenic differentiation.

• Journal of Yeungnam Medical Science
• /
• 제37권3호
• /
• pp.217-225
• /
• 2020

Background: This study was performed to provide a long-term bacterial seal through the formation of reparative dentin bridge, calcium silicate-based pulp capping materials have been used at sites of pulpal exposure. The aim of this study was to evaluate the mineralization-inducing potentials of calcium silicate-based pulp capping materials (ProRoot MTA [PR], Biodentine [BD], and TheraCal LC [TC]) in human dental pulp cells (HDPCs). Methods: Specimens of test materials were placed in deionized water for various incubation times to measure the pH variation and the concentration of calcium released. The morphology of HDPCs cultured on the specimens was examined using a confocal laser scanning microscope (CLSM). Alizarin red S staining and alkaline phosphatase assays were used to evaluate mineralization-inducing potentials of the capping materials. Results: BD showed the highest calcium release in all test periods, followed by PR and TC. (p<0.05). All experimental groups showed high alkalinity after 1 day, except at 14 days. BD showed the highest cell viability compared with PR and TC after 1 and 3 days, while TC showed the lowest value (p<0.05). The CLSM analysis showed that cells were well adhered and expressed actin filaments for all pulp capping materials. Mineralization by PR and BD groups was higher than that by TC group based on alizarin red S staining. BD showed significantly higher alkaline phosphatase activity than PR and TC, while TC showed the lowest value (p<0.05). Conclusion: Within the limitations of the in vitro study, BD had higher mineralization-inducing potential than PR and TC.

• 대한치과교정학회지
• /
• 제30권2호
• /
• pp.205-214
• /
• 2000

Nd-Fe-B 자석의 정자기장 (Static Magnetic Field)이 MC3T3-E1 세포의 alkaline phosphase (ALP) 활성도에 미치는 영향을 알아보기 위하여, MC3T3-El세포를 12 well 세포배양접시의 1열과 3열에 접종하고 1열과 3열의 첫째 칸 하방에 Nd-Fe-B 자석을 가하여 7일, 13일, 19일, 25일간 배양한 후 세포의 ALP 활성도를 측정하였으며, 100 mm 세포배양접시의 한 쪽 가장자리 하방에 Nd-Fe-B 자석을 가하여 7일, 13일, 19일, 25일간 배양한 후 ALP 염색을 하여 다음과 같은 결과를 얻었다. 1. 정자기장을 가한 13, 19, 25일에 각각 100 mT에서는 대조군에 비해 ALP활성도가 감소된 반면 4.6 mT와 0.5 mT에서는 대조군에 비해 ALP 활성도가 증가되었다 (P<0.01). 2. ALP염색에서 전체적으로는 19일까지 ALP가 증가되었다가 25일에 다소 감소되는 양상을 나타내었으며, 각 세포배양접시에서는 7일에는 뚜렷한 차이를 보이지 않았으나 13, 19, 25일에는 자석이 놓인 부위 (100 mT) 가 그 반대편(0.5 mT)에 비해 ALP가 감소되었음을 육안으로나 도립위상차현미경으로 관찰할 수 있었다. 이상의 결과 0.5 mIT와 4.6 mT의 낮은 자기장에서는 ALP 활성도가 증가되었으며 100 mT의 높은 자기장에서는 ALP활성도가 감소되어, Nd-Fe-B자석의 정자기장이 MC3T3-E1 세포의 ALP 활성도에 영향을 미쳐 골조직의 형성 및 개조에 영향을 미칠 수 있을 것으로 생각된다.

• Journal of Periodontal and Implant Science
• /
• 제33권1호
• /
• pp.49-60
• /
• 2003

Several growth factors and polypeptides are not commonly yet used for regenerators of bone tissue or alveolar bone because of the insufficiency of studies on their side effects, genetic engineering for mass production and stability for clinical application. Recently, many herbal medicines, which have advantage of less side effects and possibility of long-term use, have been studied for their capacity and effects of anti-bacterial, antiinflammatory and regenerative potential of periodontal tissues. Morindae Radix, Cibotium Barometz (L.), Albizziae Cortex, Cistandhis Herba have been traditionally used as medicines for treatment of bone disease in Eastern medicine. The objective of the present study is to examine the ability of alkaline phosphatase (ALP) activity of human fetal osteoblast (hFOB1) when several natural medicines were supplemented. hFOB1 were cultured with Dulbecuo's Modified Eagle's Medium Nutrient Mixture F-12 HAM ( DMEM/F-12 1:1 Mixture, Sigma, USA) and negative control, dexamethasone (positive control), and each natural medicines for 3 days. And then ALP activity was measured by spectrophotometer for enzyme activity and Alizarin red S staining for morphometry. Among the natural medicines of this study, Morindae Radix, Cibotium Barometz (L.) and Cistanchis Herba induced higher activity of ALP synthesis than negative controls in all experimental group. Albizziae Cortex showed mild increases than negative control group. According to measurement of positively stained area, all of the natural medicines of this study increased compared to negative control. Especially, Cibotium Barometz (L.) and Cistanchis Herba showed statistical significance compared to negative control (p<0.05). These results indicate that Morindae Radix, Cibotium Barometz (L.), Albizziae Cortex, Cistandhis Herba have an inducing ability of ALP synthesis on osteoblast.