Alkaline Phosphatase Type Research Articles

7949 Unraveling a Unique Presentation of Paget’s Disease of Bone

Abstract Disclosure: S. Puri: None. L. Bovee: None. R. Narla: None. Background: Paget's Disease of Bone (PDB) is a disorder characterized by rapid bone destruction and repair, which results in distortion of bone architecture. PDB occurs most commonly in adults over 55 and of European descent. PDB presents with both lytic and sclerotic lesions, predominantly in the axial skeleton. Serum alkaline phosphatase (ALP) levels and bone turnover markers (BTMs) are typically elevated in PDB. Clinical Case: A 53-year-old African-American woman with a history of breast cancer, PTSD, depression, and obesity (BMI 37) presented with worsening back pain over the past six months. Her pain would start at the center of her back and radiate to her left lower extremity. She endorsed rapid weight loss and night sweats and had lumbar bony tenderness on exam, prompting a spinal MRI. Spinal MRI was significant for diffuse sclerosis of the L3 vertebral body only. The radiology report provided a broad differential for this finding, including metastases, lymphoma, infection, PDB, and hemangioma. Due to progression of the patient’s back pain and concern for possible malignancy, a whole-body SPECT was completed which showed isolated tracer uptake into the L3 vertebral body extending into the pedicles and spinous process. Similar findings were noted on bone scans dating back to 2001. Per the radiology report, the presence of sclerosis with minimal expansion is most suggestive of PDB. Endocrinology was consulted for guidance on treatment and surveillance imaging for this patient. The endocrinology team discussed the case with radiology, who confirmed that the patient’s imaging findings had classic features of PDB. Bone specific ALP (B-ALP) and procollagen type 1 amino-terminal propeptide (P1NP) as bone turnover markers (BTMs) were within normal limits. A DEXA scan is pending to evaluate for concurrent osteoporosis. Clinical Lessons: PDB was favored as the most likely diagnosis primarily due to the sclerotic appearance of the patient’s vertebral lesion on imaging, radicular symptoms, and exam finding of bony point tenderness. Other diagnoses, such as metastatic disease, were considered and were thought to be unlikely given that the vertebral lesion is monostotic and stable. This case highlights the necessity of carefully evaluating radiographic findings and clinical history in the diagnosis of PDB. In this case, the patient was from a demographic group unlikely to have PDB and, unexpectedly, did not have elevated BTMs. It is important to consider PDB on the differential diagnosis in cases such as this because PDB can present in myriad ways and in a variety of patient populations. Presentation: 6/2/2024

Low bone mineral density and reduced bone-specific alkaline phosphatase in 5q spinal muscular atrophy type 2 and type 3: A 2-year prospective study of bone health.

Individuals with spinal muscular atrophy (SMA) are at risk of developing skeletal problems. This study investigated bone mineral density (BMD), bone turnover markers and motor function in children and adolescents with SMA type 2 and type 3 over a two-year period. The effect of nusinersen was studied in a subgroup. Single-centre study, including 20 patients, 2-18 years, of whom ten patients received nusinersen treatment. BMD was measured by dual-energy X-ray absorptiometry. All patients had low BMD levels at baseline; mean Z-score -2.3 for total body less head (TBLH) and -2.9 for total hip left (THL). Significant correlations were found both at baseline and for the follow-up change for motor function and Z-scores (TBLH and THL). For the whole study group, reduced bone formation and unchanged bone resorption, assessed by bone-specific alkaline phosphatase (BALP) (p = 0.0006, ES = -0.83) and C-terminal cross-linking telopeptide of type I collagen (CTX), respectively, were found over the study period. However, BALP decreased less in the nusinersen treatment group, which suggests a positive development on bone mass in these patients. Bone health evaluation is important in follow-up programmes for SMA patients. Further investigations are warranted for individuals on survival motor neuron-targeted treatments.

Study of association of C-reactive protein and alkaline phosphatase in type 2 diabetes mellitus patients

The prevalence and incidence of type 2 diabetes are rising rapidly worldwide, especially in Asia. Diabetes has been linked to a shorter life expectancy mainly because of its complications, including heart disease, strokes, eye disease, inflammation and bone disease. The aim of the study was to examine the relationship between high sensitive C reactive protein (hsCRP) and alkaline phosphatase (ALP) in type 2 diabetes patients. Furthermore, we investigated correlation between serum hsCRP and ALP level with glycaemic triad (FBS, PPBS, HbA1c) in case and control group. A cross sectional study consists of 200 patients out of which 100 normal healthy control (Group I), case - 100 patients having type 2 DM (Group II). FBS, PPBS, HbA1c, hsCRP and ALP were measured. Mean serum hsCRP and ALP level were statistically significantly higher in case group compared to control group. Moreover, significant positive correlation was observed between hsCRP and ALP level as well as both with FBS, PPBS and HbA1c. Oxidative stress and inflammation appears to be a key component and also associated with poor glycaemic control and further pathogenesis of diabetes and its complications.

Correlation of Glycemic Control with Calcium, Inorganic Phosphate, and Alkaline Phosphatase in Type 1 Diabetes Mellitus

Context: Type 1 diabetes mellitus (T1DM) accounts for over 90% of diabetic cases with a prevalence of 0.33/1000 children in the African subregion. Hyperglycemia which is the major characteristic of T1DM may have a direct toxicity on osteoblasts and could lead to increased bone fragility and fractures in patients with T1DM. However, long-term glucose control can be monitored effectively with the measurement of glycated hemoglobin (HbA1c), while alkaline phosphatase (ALP), serum calcium, and inorganic phosphate are simple ways of assessing bone mineral density. Aim: This study aimed to evaluate the association between HbA1c and serum calcium, inorganic phosphate, and ALP. Subjects and Methods: This was a prospective cross-sectional study with a total of 26 T1DM patients and 20 apparently well children within the age range of 1–18 years. Blood samples were collected from the patients for measurement of HbAIc, serum ALP, serum calcium, and inorganic phosphate at the beginning of the study and after 3 months of insulin therapy. Results: The baseline mean HbA1c was significantly higher in the T1DM patients than in the controls (P = 0.00) and there was no significant decrease in HbA1c after 3 months of insulin therapy (P = 0.13) although HbA1c tended to be lower (12.57 ± 0.86% [baseline], 10.12 ± 0.74% [3 months postinsulin therapy]). There was a statistically significant reduction in ALP (P = 0.00). There was also a statistically significant correlation between ALP and mean HbA1c (r = 0.79, P = 0.00). Conclusion: Patients with T1DM often exhibit disorders related to calcium, inorganic phosphate, and ALP homeostasis with associated poor bone metabolism which may improve with adequate glycemic control and the addition of calcium supplements to their therapy.

High Sensitive C Reactive Protein (hsCRP) and Alkaline Phosphatase (ALP) in Type 2 Diabetes Patients

Background: Diabetes is a group of metabolic disorders, characterized by hyperglycemia which results from defects in insulin secretion, insulin action, or both. The prevalence and incidence of type 2 diabetes are rising rapidly worldwide. Diabetes has been linked to a shorter life expectancy mainly because of its complications, including heart disease, strokes, retinopathy, and chronic kidney disease and bone disease. Objective: To examine the relationship between high sensitive C reactive protein (hsCRP) and alkaline phosphatase (ALP) in type 2 diabetic patients. Methods: This study was a hospital based cross sectional study conducted at Dept. of Biochemistry, Ashiyan Medical College Hospital from January to June 2020. The study consists of 180 patients out of which 90 were normal healthy controls (Group I) and 90 patients having type 2 DM were included in case (Group II). FBS, PPBS, HbA1c, hsCRP and ALP of all subjects were measured. Results: Mean serum hsCRP and ALP level were statistically significant higher in case group compared to control group. Moreover, significant positive correlation was observed between hsCRP and ALP level as well as both with FBS, PPBS and HbA1c. Conclusions: Oxidative stress and inflammation appears to be a key component and also associated with poor glycaemic control and further pathogenesis of diabetes and its complications. All our finding suggest a link between oxidative stress, inflammation and glycaemic control in patient with type 2 diabetes mellitus.

Effect of Arabinogalactan on Intestinal Alkaline Phosphatase, Bacterial Endotoxin and Serum Cytokines in Type 2 Diabetic Rats

In the experiment, a low-dose intraperitoneal injection of streptozotocin (30 mg/kg) was used to establish a diabetic rat model. After the modeling being successful, the mice were continuously administered arabinogalactan aqueous solution (100 mg/kg·d, 500 mg/kg·d), and the changes in rat body mass and blood sugar were determined. Also, the activity of intestinal alkaline phosphatase (IAP) in feces and the changes in bacterial endotoxin lipopolysaccharide (LPS) concentration, and the serum cytokine concentration changes by MSD multi-factor method were detected. The results showed that compared with the model group and the positive control group, the fasting blood glucose concentration of the arabinogalactan polysaccharide dose group was significantly reduced (P<0.05), and the IAP activity was significantly increased (P<0.05). Besides, the LPS concentration was significantly reduced (P<0.05), and the concentration of Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1 (IL-1β) and growth-regulating oncogene α (KC/GRO) was significantly reduced in the intestine (P<0.05), while the concentration of the anti-inflammatory factors such as interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-10 (IL-10) and interleukin-13 (IL-13) increased significantly (P<0.05). Therefore, it can be proved that arabinogalactan can improve the activity of IAP, reduce the concentration of LPS, and regulate rat cytokines. Furthermore, arabinogalactan has a certain improvement effect on the immunity of type 2 diabetic rat model.

Groundnut (Apios americana Medik) Extract Enhances the Osteoblast Differentiation of MC3T3-E1 Cells

The effects of groundnut ( Apios americana Medik) extract on osteoblast differentiation were examined using MC3T3-E1 cells. MC3T3-E1 cells were treated with the crude extract along with other differentiating reagents. The alkaline phosphatase (ALP) activity of cells cultured in a differentiation medium supplemented with 0.01% crude groundnut extract was 1.5‐1.6 times higher than that of cells cultured in a differentiation medium without the extract. Crude groundnut extract was further separated into aqueous and methanol fractions. The methanol fraction enhanced ALP activity, osteocalcin, integrin-binding sialoprotein, and type I collagen expression, and calcium mineralization. Conversely, the aqueous fraction did not show such effects. Groundnut extract may enhance osteoblast differentiation, and this effect is likely conferred by water insoluble substance(s).

Hypoxia Enhanced Bone Regeneration Through the HIF-1α/β-Catenin Pathway in Femoral Head Osteonecrosis

BackgroundOsteonecrosis of the femoral head (ONFH) is a common disease. Transplantation of bone marrow stem cells (BMSCs) is a promising method to treat ONFH but is impeded by the low survival rate and deficiency of cell bioactivity. MethodsWe performed hypoxic preprocessing to treat BMSCs and assessed cell viability, apoptosis, differentiation, and growth factor expression in vitro. Subsequently, we constructed the ONFH model and delivered hypoxia-pretreated BMSCs to the rabbit femoral head after core decompression surgery, evaluating its effects on bone regeneration and ONFH repair. Six weeks later, micro-computed tomography (CT) and histopathology were performed to evaluate ONFH repair. ResultsOur findings demonstrated that hypoxic preprocessing promoted the viability of BMSCs, increased the expression of hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF), alkaline phosphatase (ALP), calcium deposition, and enhanced the formation of vessels-shaped structures. In an in vivo study, micro-CT observations demonstrated that the bone volume was increased in the hypoxia BMSCs group. Histological examination revealed reduced cellular apoptosis, lower empty lacunae rate, enhanced bone formation, and stronger trabecular bone in the hypoxia BMSCs group when compared with those transplanted with normoxia treated BMSCs. Additionally, immunological assessment of the hypoxia BMSCs group demonstrated increased expression of HIF-1α and β-catenin, as well as increased VEGF, ALP, osteocalcin (OCN), and collagen type I (Col-1). ConclusionsCollectively, our findings indicated that hypoxia stimulated angiogenesis and bone regeneration via the HIF-1/β-catenin pathway in BMSCs and that the delivery of hypoxia-pretreated BMSCs contributed to the treatment of early ONFH.

Effects of Er:YAG laser irradiation of different titanium surfaces on osteoblast response

The aim of this in vitro study was to evaluate the effects of erbium-doped yttrium aluminum garnet (Er:YAG) laser irradiation on titanium surface topography and the proliferation and differentiation of osteoblasts using standard clinical treatment settings. Er:YAG laser irradiation at two levels ((1): 160 mJ, pulse at 20 Hz; (2): 80 mJ, pulse at 20 Hz) was applied to moderately rough and smooth titanium disks before MG-63 osteoblast-like cells were cultured on these surfaces. Titanium surface and cell morphology were observed by scanning electron microscopy. Cell proliferation/viability was measured by CCK-8 test. Gene expression of alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa-B ligand (RANKL), and collagen type 1 was measured by qPCR, and OPG and OC protein production was determined by enzyme-linked immunosorbent assay. Treatment with Er:YAG laser at 160 mJ/20 Hz markedly caused heat-induced fusion of titanium and cell condensation on moderately rough surfaces, but not in smooth surfaces. MG-63 proliferation/viability decreased after 5 days in moderately rough surfaces. The expression of ALP, OC, OPG, and collagen type 1 was unaffected by laser treatment at 160 mJ/20. Laser irradiation at 80 mJ/20 Hz enhanced RANKL gene expression after 5 days in moderately rough surfaces. Study results suggest that Er:YAG laser irradiation at clinically relevant setting has no essential effect on osteogenic gene and protein expression of osteoblasts. However, surface structure, cell attachment, and proliferation are influenced by both treatment protocols, which implies that caution should be taken in the clinical treatment of peri-implant diseases when Er:YAG laser is used.

Effects of DLX3 on the osteogenic differentiation of induced pluripotent stem cell‑derived mesenchymal stem cells.

Osteoporosis is a disease characterized by the degeneration of bone structure and decreased bone mass. Induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) have multiple advantages that make them ideal seed cells for bone regeneration, including high-level proliferation, multi-differentiation potential and favorable immune compatibility. Distal-less homeobox (DLX)3, an important member of the DLX family, serves a crucial role in osteogenic differentiation and bone formation. The present study aimed to evaluate the effects of DLX3 on the proliferation and osteogenic differentiation of human iPSC-MSCs. iPSC-MSCs were induced from iPSCs, and identified via flow cytometry. Alkaline phosphatase (ALP), Von Kossa, Oil Red O and Alcian blue staining methods were used to evaluate the osteogenic, adipogenic and chondrogenic differentiation of iPSC-MSCs. DLX3 overexpression plasmids were constructed and transfected into iPSC-MSCs to generate iPSC-MSC-DLX3. iPSC-MSC-GFP was used as the control. Reverse transcription-quantitative PCR (RT-qPCR) and western blotting were performed to measure the expression of DLX3 2 days after transfection. Subsequently, cell proliferation was assessed using a Cell Counting Kit-8 assay on days 1, 3, 5 and 7. RT-qPCR and western blotting were used to analyze osteogenic-related gene and protein expression levels on day 7. ALP activity and mineralized nodules were assessed via ALP staining on day 14. Statistical analysis was performed using an unpaired Student's t-test. Flow cytometry results demonstrated that iPSC-MSCs were positive for CD73, CD90 and CD105, but negative for CD34 and CD45. iPSC-MSC-DLX3 had significantly lower proliferation compared with iPSC-MSC-GFP on days 5 and 7 (P<0.05). mRNA expression levels of osteogenic markers, such as ALP, osteopenia (OPN), osteocalcin (OCN) and Collagen Type I (COL-1), were significantly increased in iPSC-MSC-DLX3 compared with iPSC-MSC-GFP on day 7 (P<0.05). Similarly, the protein expression levels of ALP, OCN, OPN and COL-1 were significantly increased in iPSC-MSC-DLX3 compared with iPSC-MSC-GFP on day 7 (P<0.05). The number of mineralized nodules in iPSC-MSC-DLX3 was increased compared with that in iPSC-MSC-GFP on day 14 (P<0.05). Thus, the present study demonstrated that DLX3 serves a negative role in proliferation, but a positive role in the osteogenic differentiation of iPSC-MSCs. This may provide novel insight for treating osteoporosis.

Hypoglycaemic and anti-hyperglycaemic activity of Tabernanthe iboga aqueous extract in fructose-fed streptozotocin type 2 diabetic rats

Root bark preparations of the Gabonese plant Tabernanthe iboga (T. iboga) has long been used in traditional medicine in Central and West African regions for the management of type 2 diabetes (T2D). This study is the first investigation of in vivo hypoglycaemic activity in healthy rats and anti-hyperglycaemic activity of T. iboga in a 10% fructose-fed (40 mg/kg i.p.) streptozotocin (STZ) injected type 2 diabetic rat model. T. iboga at 50 to 200 mg/kg induced hypoglycaemia activity over 3 h fasted glucose tolerance in healthy Wistar rats and anti-hyperglycaemic effects on non-fasted and fasted blood glucose in fructose-fed STZ T2D rats with no toxicity. Fructose-fed STZ T2D rats developed characteristic type 2 diabetic complications over 6 weeks exhibiting significantly elevated fasting and non-fasting blood glucose, polydipsia, reduced body weight gain and glucose and insulin tolerance compared with STZ alone and normal control rats. T. iboga (50 mg/kg and 200 mg/kg bw) administered p.o. once daily for 4 weeks significantly improved diabetic symptoms of polydipsia, reduced body weight, hyperglycaemia, glucose and insulin tolerance (as AUC) compared with fructose-fed STZ T2D rats. T. iboga aqueous extract (50 mg/kg and 200 mg/kg) also significantly reversed altered actions of marker enzymes of liver including alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), creatinine, HbA1c and elevated triglycerides in fructose-fed STZ type 2 diabetic rats. Our outcomes show that daily oral provision of T. iboga improves type 2 diabetes complications, superior to glibenclamide, in rat fructose-fed STZ model and offers the potential for safe clinical management of T2D in Gabon.

β-mercaptoethanol promotes osteogenesis of human mesenchymal stem cells via sirt1-ERK pathway.

Human umbilical cord-derived mesenchymal stem cells (hUMSCs) hold strong self-renewal capacity and low immunogenicity, which have attracted attention as potential candidates for bone repair and regeneration. However, insufficient osteogenic differentiation markedly hinders the clinical applications of hUMSCs. In the present study, the effect of β-mercaptoethanol (BME), a small molecule antioxidant which has been identified to regulate cell proliferation and differentiation, on osteogenic differentiation of hUMSCs and underlying signaling mechanism were investigated. The results indicated that under osteogenic induction conditions, BME treatment increased the alkaline phosphatase (ALP) activity and promoted calcium mineralization in hUMSCs. The gene and protein expression of osteogenesis-related markers such as ALP, osteopontin (OPN), osteocalcin (OCN) and collagen type I (COLI) were also significantly up-regulated. Besides, BME promoted the protein expression of silent information regulator type 1 (sirt1) and stimulated the activation of extracellular signal-related kinase (ERK), contributing to increased Runx2 expression. Furthermore, blocking the expression of sirt1 attenuated BME-enhanced ERK phosphorylation and osteogenic differentiation of hUMSCs. These results indicated that BME accelerated osteogenic differentiation of hUMSCs by activating the sirt1-ERK signaling pathway, thereby providing insights into the development of MSCs-based bone regeneration strategies.

P1485SEVELAMER USE IS ASSOCIATED WITH DECREASED VITAMIN K LEVELS IN HEMODIALYSIS PATIENTS: RESULTS FROM VITAMIN K ITALIAN (VIKI) STUDY

Abstract Background and Aims Sevelamer (S) is a phosphate binding drug used to treat hyperphosphatemia in patients with CKD. Our aim was to evaluate the hypothesis that the use of (S) could interfere with Vitamin K absorption in hemodialysis (HD) patients of VIKI study. Method We tested this hypothesis in VIKI, a cross-sectional study of 387 hemodialysis patients, we established the prevalence of vitamin K deficiency and to assessed the relationship between vitamin K status, vertebral fractures, vascular calcification. We determined serum concentrations of vitamin 25(OH)D; alkaline phosphatase (ALP); vitamers K1, MK4, MK5, MK6, MK7; osteocalcin (BGP) and Matrix Gla Protein (MGP). We highlighted that MK4 deficiency was the strongest predictor of aortic calcification (OR, 2.82; 95% CI, 1.14–7.01) while vitamin K1 deficiency was the strongest predictor of vertebral fractures fractures (OR: 2.94; 95% CI, 1.38–6.26). Results 163 of 387 patients (42.1%) were treated with Sevelamer. There were no differences in levels of 25(OH)D, K1, MK5, MK6 and MK7 among patients treated with and without Sevelamer. Remarkably, the prevalence of MK4 deficiency was higher in Sevelamer treated patients (13.5% vs 5.4%, p=0.005). Sevelamer treated patients also had higher median levels of ALK (89 UI/L vs 77.5 UI/L, p=0.001) and total BGP (210 mcg/L vs 152 mcg/L, p=0.002) and lower median levels of total MGP (16.4 nmol/L vs 20.3 nmol/L, p=0.037) (Table 1 and Figure 1). In multivariable logistic regression, the odds ratio of MK4 deficiency (dependent variable) in patients treated with compared to without Sevelamer was ∼3-fold higher (OR: 2.64, 95% CI: 1.25-5.58, p=0.011) after adjustment for confounders of Vitamin K levels, including older age, previous myocardial infarction, type of HD, ALP, PTH, MGP, BGP, cholesterol and albumin. Conclusion These data support the hypothesis that Sevelamer could interfere with MK4 absorption in HD patients. Longitudinal interventional studies are needed to prove the causal nature of these associations.

Correlation between Plasma Osteopontin and Alkaline Phosphatase in Type 2 Diabetes Mellitus Patients

Diabetes Mellitus (DM) is a chronic disease caused by pancreas the inability to produce insulin or ineffectively insulin use.Fracture risk in type 2 DM patients increases even though the bone density is normal. This study aimed to examine thecorrelation of osteopontin (OPN) and alkaline phosphatase (ALP) in type 2 DM patients. An observational analytical studywas conducted in 73 type 2 DM patients in Dr. Moewardi Hospital, Surakarta from October to November 2018. The subjectswere examined for blood pressure, fasting blood glucose, two hours postprandial blood glucose, HbA1c, OPN, and ALPlevels. P-value &lt;0.05 was statistically significant with a 95% confidence interval. Poorly controlled type 2 DM had higher OPNlevels than well-controlled (20.5±2.8 vs. 14.8±3.1 ng/mL, p &lt;0.001). The ALP concentration was also higher in poorlycontrolled type 2 DM patients (79.9±31.7 vs. 61.1±25 U/L, p=0.003). The levels of OPN and ALP were significantly correlatedin type 2 diabetes (r=0.273; p=0.020) and in well-controlled patients (r=0.353; p=0.047) but no correlation was found inpoorly controlled type 2 DM patients (r= -0.073; p= 0.652). In this study, a significant correlation was found between OPNand ALP in patients with type 2 DM and well-controlled. Further study involving healthy controls and bone ALPmeasurement is needed.

All-trans retinoic acid and vascular endothelial growth factor induced the directional osteogenic differentiation of mouse embryonic fibroblasts

To investigate the effect of all-trans retinoic acid (ATRA) and vascular endothelial growth factor (VEGF) on the osteogenic differentiation of mouse embryonic fibroblasts (MEFs). The fetal mice in the uterus of NIH pregnant mice (pregnancy 12-15 days) were collected, and the heads and hearts etc. were removed. Then MEFs were separated from the rest tissues of the fetal mice and cultured by trypsin digestion and adherent culture. HEK-293 cells were used to obtain recombinant adenovirus-red fluorescent protein (Ad-RFP) and Ad-VEGF by repeatedly freezing and thawing. Alkaline phosphatase (ALP) staining and quantitative detection were used to detect the changes of ALP activity in MEFs applied with ATRA or VEGF alone or combined use of ATRA and VEGF on the 3rd and 5th days. The cultured 3rd to 4th generation MEFs were divided into groups A, B, C, and D, and were cultured with DMSO plus Ad-RFP, ATRA, Ad-VEGF, ATRA plus Ad-VEGF, respectively. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the mRNA expressions of osteogenic markers including ALP, collagen type Ⅰ, osteopontin (OPN), osteocalcin (OCN), and angiogenic markers including VEGF, angiopoietin 1 (ANGPT1), and endomucin (EMCN) on the 3rd and 7th days. Immunohistochemical staining was used to detect the protein expressions of OPN and VEGF on the 3rd, 5th, and 7th days in each group. Alizarin red staining was used to detect calcium salt deposition levels in each group at 14 and 21 days after osteogenic induction. Fifteen athymic female nude mice aged 4 to 6 weeks were randomly divided into 3 groups and 5 mice in each group. Then MEFs treated with ATRA, Ad-VEGF, and ATRA plus Ad-VEGF were injected subcutaneously into the dorsal and ventral sides, respectively. X-ray observation, gross observation, and histological staining (Masson, HE, and Safranin O-fast green stainings) were performed at 5 weeks after implantation to observe the ectopic bone formation in nude mice in each group. MEFs were successfully isolated and cultured. The acquired Ad-RFP and Ad-VEGF were successfully transfected into MEFs with approximately 50% and 20% transfection rates. ALP activity tests showed that ATRA or Ad-VEGF could enhance ALP activity in MEFs ( P<0.05), and ATRA had a stronger effect than Ad-VEGF; and the combined use of ATRA and Ad-VEGF significantly enhanced the ALP activity in MEFs ( P<0.05). qRT-PCR test showed that the combined use of ATRA and Ad-VEGF also increased the relative mRNA expressions of early-stage osteogenesis-related markers ALP, OPN, and collagen type I ( P<0.05); the relative mRNA expressions of angiogenesis-related markers VEGF, EMCN, and ANGPT1 increased at 7 days ( P<0.05). Immunohistochemical staining showed that ATRA combined with Ad-VEGF not only enhanced OPN protein expression, but also increased VEGF protein expression on 7th day. Alizarin red staining showed that the application of ATRA or Ad-VEGF induced weak calcium salt deposition, and the combined use of ATRA and Ad-VEGF significantly enhanced the effect of calcium salt deposition in MEFs. The results of implantation experiments in nude mice showed that X-ray films observation revealed obvious bone mass in the ATRA plus Ad-VEGF group, and the bone was larger than that in other groups. Histological staining showed a large amount of collagen and mature bone trabeculae, bone matrix formation, and gray-green collagen bone tissue, indicating that the combined use of ATRA and Ad-VEGF significantly enhanced the osteogenic effect of MEFs in vivo. The combined use of ATRA and VEGF can induce the osteogenic differentiation of MEFs.

Study of Association of C - reactive protein and Alkaline Phosphatase in Type 2 Diabetes Mellitus Patients

Study of Association of C - reactive protein and Alkaline Phosphatase in Type 2 Diabetes Mellitus Patients

Discontinuation of Tyrosine Kinase Inhibitor in Children with Chronic Myeloid Leukemia (JPLSG STKI-14 study)

Kada: Bayer Yakuhin: Other: personal fees for a member of independent data monitoring committee of clinical trials, outside of the submitted work.. Imai:Juno Therapeutics: Patents & Royalties.

Analysis of the Basic Characteristics of Osteogenic and Chondrogenic Cell Lines Important for Tissue Engineering Implants.

We isolated and characterized cultures of bone and cartilage tissue cells of laboratory minipigs. The size and morphological features of adherent osteogenic and chondrogenic cells were specified. During long-term culturing under standard conditions, the studied cultures expressed specific markers that were detected by immunohistochemical staining: alkaline phosphatase and calcium deposits in osteoblasts and type II collagen and cartilage extracellular matrix in chondrogenic cells. Proliferative potential (mitotic index) of both cell types was 4.64% of the total cell number. Cell motility, i.e. the mean velocity of cell motion was 49 pixels/h for osteoblasts and 47 pixels/h for chondroblasts; the mean migration distance was 2045 and 2118 pixels for chondroblasts and osteoblasts, respectively. The obtained cell lines are now used as the control for evaluation of optimal biocompatibility of scaffold materials in various models. Characteristics of the motility of the bone and cartilage tissue cells can be used for modeling and estimation of the rate of cells population of 3D scaffolds made of synthetic and biological polymers with different internal structure and physicochemical properties during designing in vitro tissue implants.

Enamel matrix proteins regulate hypoxia-induced cellular biobehavior and osteogenic differentiation in human periodontal ligament cells

Hypoxia is a crucial microenvironment for inflamed periodontal tissue and periodontal wound healing. Enamel matrix proteins (EMPs) potentially can promote the formation of new periodontium. The effects of EMPs on periodontal ligament cells under hypoxia, however, remain unclear. We investigated the effects of EMPs on cellular biobehavior and osteogenic differentiation of human periodontal ligament cells (hPDLCs) under hypoxia. Under cobalt chloride (CoCl2)-induced hypoxia, cellular biobehavior of hPDLCs, including proliferation, attachment, spreading, and migration with or without EMPs, was evaluated by 3-(4, 5-dimethylthiazol- 2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), cell counting, spreading area measurement and wound scratch assay. The osteogenic activity of hPDLCs was assessed using alkaline phosphatase (ALP) and alizarin red S staining (ARS). The expressions of osteogenic genes including runt related transcription factor 2 (Runx2), ALP, osteocalcin (OCN) and collagen type I (Col-I) were detected using real time quantitative PCR, western blot and immunocytochemistry assays. The biobehavior and osteogenic differentiation of hPDLCs were inhibited significantly under hypoxia. EMPs have no effect on cell proliferation under mimicked hypoxia. EMPs partly reversed the inhibitory effects of hypoxia, however, for other cellular biobehavior including attachment, spreading and migration, and markedly up-regulated osteogenic differentiation activities including ALP, mineralization ability and the expressions of osteogenic genes such as Runx2, ALP, osteocalcin, and collagen type I in hPDLCs under hypoxia. EMPs attenuate the hypoxic injury to cellular biobehavior and osteogenic differentiation in hPDLCs under hypoxia.

Regulatory role of B-cell maturation antigen on the toxic effect of chromium ions on human SaOS-2 osteoblasts.

Metal prostheses of artificial joints undergo wear, producing numerous metal particles and ions, such as Cr3+ . Cr3+ is considered a key factor leading to aseptic loosening. Many studies focus on the effect of Cr3+ on osteoblasts; however, little is known about the effect of Cr3+ on the B-cell maturation antigen (BCMA) in the osteoblasts. In this study, we first demonstrated the BCMA expressed in human SaOS-2 osteoblasts through reverse transcriptase-PCR, Western blot, and immunocytochemical analyses. Cr3+ decreased alkaline phosphatase (ALP), osteocalcin (OC), cell mineralization, and collagen type I mRNA and protein expression. Moreover, Cr3+ has an inhibitive effect on the expression of the BCMA in human SaOS-2 osteoblasts. However, after we upregulated the expression of the BCMA, ALP, OC, cell mineralization, and collagen type I mRNA and protein expression were increased. Overall, this study demonstrates that the BCMA is involved in human SaOS-2 osteoblast osteogenetic metabolism and plays a regulatoryrole on the toxic effect of chromium ions on human SaOS-2 osteoblasts.