Effect of pamidronate administration on markers of bone turnover and disease activity in multiple myeloma

AIM: Bisphosphonates are potent inhibitors of osteoclastic activity and are used in the treatment of multiple myeloma (MM) in combination with chemotherapy. The effect of pamidronate on markers of bone resorption [cross-linked N-telopeptides of type I collagen (NTx)], markers of bone formation [serum alkaline phosphatase (BAP) and osteocalcin (OSC)], interleukin-6 (IL-6), beta2-microglobulin, CRP, paraprotein and disease-related pain and skeletal events has been evaluated in 62 newly diagnosed patients with MM. PATIENTS AND METHODS: The patients were randomly assigned to two groups: the first included 32 patients under chemotherapy and pamidronate (group I) and the second 30 patients on chemotherapy only (group II). Pamidronate was administered at a monthly dose of 90 mg iv, and the above parameters were evaluated at the beginning of this study and after 1, 3, 6, 9, 12 and 14 months of treatment. RESULTS: The addition of pamidronate to chemotherapy resulted in a significant reduction of NTx, IL-6 and paraprotein from the 3rd month and of beta2-microglobulin, CRP and pain from the 6th month of treatment. No changes of NTx, IL-6, beta2-microglobulin, CRP or skeletal events were observed in patients of group II, while paraprotein was significantly reduced after 6 months of treatment. The differences in NTx, IL-6, paraprotein and beta2-microglobulin were statistically significant between the two groups. Multivariate analysis revealed a significant correlation between changes of NTx, changes of IL-6 in both groups and reduction of pain and paraprotein in group I. CONCLUSIONS: These results suggest that pamidronate may have a synergistic action with chemotherapy in decreasing osteoclastic activity, in reducing markers of myeloma activity and myeloma related pain and in improving the quality of life in patients with MM.     

强骨饮联合益生菌对大鼠骨质疏松性骨折的愈合作用

目的 研究强骨饮联合益生菌对大鼠骨质疏松性骨折的愈合作用。方法 将♂SD大鼠50只随机分成5组：一般骨折对照组、骨质疏松性骨折模型组、强骨饮组、益生菌组、强骨饮联合益生菌组,每组10只。骨质疏松性骨折模型组和所有治疗组均摘除双侧卵巢制备骨质疏松模型。6周后,每组制备右侧股骨干中段横行骨折模型。造模成功后即给予强骨饮组生药灌胃量102 g·kg^-1,益生菌组灌胃量12.5 g·kg^-1,未给药组灌胃等量生理盐水,每日1次,连续6周。骨密度扫描仪测定骨密度,Micro-CT扫描股骨相关参数,酶联免疫法测定大鼠血清骨源性碱性磷酸酶（bone alkalinephosphatase,BALP）、骨钙素（osteocalcin,OC）、I型胶原C端肽（type I procollagen carboxy-terminal propeptide,CTX-I）,生化仪测定钙浓度（Ca）。HE观察骨折骨痂组织病理变化。结果 与骨质疏松性骨折组比较,强骨饮组或益生菌组均可促进骨折愈合（增加骨小梁数量和骨密度）,增加骨形成参数（BALP和OC）和降低吸收参数（CTX-I）,而强骨饮联合益生菌组作用更明显。结论 益生菌和强骨饮均有促进骨质疏松性骨折愈合的治疗作用,联合用药效果更佳。     

Oral delivery of mouse [d‐Leu‐4]‐OB3, a synthetic peptide amide with leptin‐like activity,in male C57BL/6J wild‐type and ob/ob mice: effects on energy balance,glycaemic control and serum osteocalcin levels

Background: We have recently shown that intranasal administration of mouse [d ‐Leu‐4]‐OB3 reconstituted in Intravail^® to male Swiss Webster mice resulted in significantly higher bioavailability than commonly used injections methods of delivery. The absorption profile associated with intranasal delivery of mouse [d ‐Leu‐4]‐OB3 showed an early peak representing absorption across the nasal mucosa, and a later peak suggesting a gastrointestinal site of uptake. Aim and Methods: In the present study, we examined the effects of orally administered (by gavage) mouse [d ‐Leu‐4]‐OB3 on energy balance, glycaemic control and serum osteocalcin levels in male C57BL/6J wild‐type and ob/ob mice allowed food and water ad libitum or calorie restricted by 40% of normal intake. Results: In wild‐type mice fed ad libitum, oral delivery of mouse [d ‐Leu‐4]‐OB3 reduced body weight gain, food intake and serum glucose, by 4.4, 6.8 and 28.2% respectively. Serum osteocalcin levels and water intake were essentially the same in control and treated wild‐type mice. In ob/ob mice fed ad libitum, mouse [d ‐Leu‐4]‐OB3 reduced body weight gain, food intake, water intake and serum glucose by 11.6, 16.5, 22.4 and 24.4% respectively. Serum osteocalcin in ob/ob mice treated with mouse [d ‐Leu‐4]‐OB3 was elevated by 62% over controls. Calorie restriction alone caused significant weight loss in both wild‐type (9.0%) and ob/ob (4.8%) mice, and mouse [d ‐Leu‐4]‐OB3 did not further enhance this weight loss. As expected, serum glucose levels in wild‐type and ob/ob mice were significantly reduced by calorie restriction alone. Mouse [d ‐Leu‐4]‐OB3 further reduced serum glucose in wild‐type mice and normalized levels in ob/ob mice. Calorie restriction alone reduced serum osteocalcin levels by 44.2% in wild‐type mice and by 19.1% in ob/ob mice. Mouse [d ‐Leu‐4]‐OB3 prevented this decrease in groups of mice. Conclusions: The results of this study suggest that oral delivery of mouse [d ‐Leu‐4]‐OB3 in Intravail^® is possible and may have potential not only as an alternative therapy in the treatment of human obesity and some of its associated metabolic dysfunctions, but also may help to prevent and/or reverse at least some of the bone loss which accompanies osteoporosis, anorexia nervosa and other wasting diseases.     

利塞膦酸钠治疗绝经后骨质疏松症骨转换标志物的变化

目的:通过利塞膦酸钠(RIS)治疗绝经后骨质疏松症,观察其对骨转换标志物的影响。方法:采用多中心、随机、双盲、安慰剂对照试验方法,RIS组118例,口服RIS5mg/d,安慰剂组119例,口服安慰剂1片/d。所有受试者均加凯思立D(含元素钙500mg和维生素D200IU)1片/d,治疗12个月。于治疗前、治疗后6个月和12个月测定血浆骨钙素(BGP)和空腹尿Ⅰ型胶原交联N端肽(NTX)。结果:RIS组血BGP治疗后6个月和12个月均下降(P<0.01),安慰剂组治疗后血BGP无明显变化(P>0.05),2组间治疗后比较差异有统计学意义(P<0.01)。RIS组尿NTX治疗后6个月和12个月均下降(P<0.01),安慰剂组尿NTX治疗后6个月无明显变化(P>0.05),12个月下降(P<0.05)。2组间治疗后比较差异有统计学意义(P<0.01)。结论:RIS能抑制骨吸收,降低骨转换。     

大鼠骨髓间充质干细胞的培养鉴定及向成骨细胞诱导分化研究

目的探讨大鼠骨髓间充质干细胞(BMSCs)的体外培养方法、生长规律及向成骨细胞分化的条件。方法采用全骨髓培养法分离成年大鼠BMSCs,经条件培养液诱导培养后,进行形态学观察及碱性磷酸酶活性、细胞矿化作用、骨钙素(OCN)等的测定。结果原代培养的BMSCs先形成细胞集落,14 d时集落融合;传代细胞体积变大,约5~7 d传代1次。诱导条件下,细胞碱性磷酸酶活性明显增高,并出现了矿化结节。经过体外成骨诱导的BMSCs表现出增殖、聚集、结节和矿化期的阶段性形态特征。在矿化期OCN表达呈阳性。结论BMSCs易于体外分离培养及扩增,体外成骨定向诱导的BMSCs具有典型的成骨细胞的形态和功能性特征,可以作为骨组织工程的种子细胞。     

骨金散对去卵巢大鼠骨钙素、降钙素的影响

目的:通过骨金散对去卵巢大鼠骨量及骨代谢的影响.方法:以6月龄雌性大鼠40只为研究对象,随机分为4组:对照组、假手术组、去卵巢组、实验组(骨金散治疗组),每组10只.观察骨金散对去卵巢大鼠股骨、腰椎骨、胫骨下端骨密度、血清雌激素(E2)、降钙素(CT)及骨钙素(BGP)水平的影响.结果:与假手术组比较,去卵巢大鼠股骨、腰椎骨、胫骨下端骨密度明显降低(P＜0.05),血浆BGP水平明显升高(P＜0.05),E2、CT水平明显降低(P＜0.05).骨金散治疗组可使去卵巢大鼠股骨、腰椎骨、胫骨下端骨密度增加(P＜0.05),升高血浆CT水平(P＜0.05),降低血清骨钙素水平(P＜0.05),但血清E2 水平无明显改变(P＞0.05).结论:骨金散可通过对CT分泌释放和对BGP的调节,减缓伴随去卵巢所致的骨量丢失,起到防治骨质疏松的作用.     

Intake of fermented soybean (natto) increases circulating vitamin K2 (menaquinone-7) and γ-carboxylated osteocalcin concentration in normal individuals

Changes in circulating vitamin K₂ (menaquinone-7, MK-7) and γ-carboxylated osteocalcin concentrations in normal individuals with the intake of fermented soybeans (natto) were investigated. Eight male volunteers were given sequentially fermented soybeans (natto) containing three different contents of MK-7 at an interval of 7 days as follows: regular natto including 775 μg/100 g (MK-7 × 1) or reinforced natto containing 1298 μg/100 g (MK-7 × 1.5) or 1765 μg/100 g (MK-7 × 2). Subsequently, it was found that serum MK-7 and γ-carboxylated osteocalcin concentrations were significantly elevated following the start of dietary intake of MK-7 (1298 or 1765 μg/100 g). Serum undercarboxylated osteocalcin concentrations were significantly decreased by dietary MK-7 (1765 μg/100 g) supplementation. Moreover, the changes in serum MK-7 level with the frequency of dietary natto intake were examined in 134 healthy adults (85 men and 39 women) without and with occasional (a few times per month), and frequent (a few times per week) dietary intake of regular natto including MK-7 (775 μg/100 g). Serum MK-7 and γ-carboxylated osteocalcin concentrations in men with the occasional or frequent dietary intake of natto were significantly higher than those without any intake. The present study suggests that intake of fermented soybean (natto) increases serum levels of MK-7 and γ-carboxylated osteocalcin in normal individuals. Received: Aug. 23, 1999 / Accepted: Nov. 24, 1999     

Porcine sinus mucosa holds cells that respond to bone morphogenetic protein (BMP)-6 and BMP-7 with increased osteogenic differentiation in vitro

The aim of this in vitro study was to determine whether the sinus mucosa holds cells with an osteogenic potential. Frozen sections of sinus mucosa from three adult pigs were investigated for the expression of STRO-1, a marker of mesenchymal progenitor cells, and alkaline phosphatase activity, an enzyme expressed by cells committed to the osteogenic lineage and by mature osteoblasts. To determine their osteogenic potential, mucosa-derived cells were incubated with bone morphogenetic protein (BMP)-6 and BMP-7, and alkaline phosphatase activity, osteocalcin expression, and mineralization of the extracellular matrix was measured. We found sinus mucosa cells staining positive for STRO-1 and alkaline phosphatase activity. When sinus mucosa tissue was placed in culture, alkaline phosphatase positive cells grew out from the explants and further increased alkaline phosphatase activity in response to BMP-6 and BMP-7. The expression level of the osteoblast-specific extracellular matrix protein osteocalcin, and the amount of calcium accumulation within the extracellular matrix was also increased in response to BMPs. We conclude that the sinus mucosa holds mesenchymal progenitor cells and cells committed to the osteogenic lineage that can respond to BMP-6 and BMP-7 by an increase of their osteogenic differentiation.     

Osteocalcin fragment in bone matrix enhances osteoclast maturation at a late stage of osteoclast differentiation

Although 14-day-old mouse embryonic calvarial cells cultured in plastic culture dishes in the presence of 1,25-dihydroxyvitamin D₃[1,25-(OH)₂D₃] for 7 days could barely resorb bone slices, the same calvarial cells cultured with an ethylenediaminetetraacetic acid (EDTA) extract from bovine bone powder under the same conditions stimulated pit formation on bone slices in a dose-dependent manner. Therefore, the present study was conducted to purify and characterize this osteoclast maturation-inducing factor(s) from the bone matrix. The protein having osteoclast maturation-inducing activity in the EDTA extract was purified by gel filtration over Superdex 75 preparation grade and chromatography on hydroxyapatite, Mono Q, and C8 reversed-phase HPLC by monitoring the ability of the eluted fractions to elicit pit formation on bone slices. The molecular weight of the purified protein estimated by high-resolution polyacrylamide gel electrophoresis was 5.7kDa and 6.8kDa in the respective absence and presence of 2-mercaptoethanol. The sequence of the 30-amino-acid purified protein corresponded to the 7th to 36th residues of bovine osteocalcin. The osteocalcin fragment, missing the initial 6 residues at the N-terminal region, exhibited higher osteoclast maturation-inducing ability than bovine intact osteocalcin on a per weight basis. The osteocalcin fragment had no effect on the expression of receptor activator of nuclear factor (NF)-B ligand (RANKL) and osteoprotegerin (OPG) genes in calvarial cells, nor did it enhance the bone-resorbing activity of mature osteoclasts. When the osteocalcin fragment was added to late-stage (days 4–7) or to early-stage (days 0–3) cultures of calvarial cells pretreated with 1,25-(OH)₂D₃, its stimulatory effect was observed in the late-stage cultures rather than in the early-stage ones. In addition, the osteocalcin fragment directly enhanced the formation of osteoclasts with bone-resorbing ability from Mac-1⁺ c-Fms⁺ cells in the presence of macrophage colony-stimulating factor (MCSF) and RANKL. These results suggest that the osteocalcin fragment in bone matrix is involved in osteoclast maturation, especially at a late stage of osteoclast differentiation.     

Vitamin D supplementation to healthy children does not affect serum osteocalcin or markers of type I collagen turnover

AIM: New serum markers have recently been introduced in the assessment of bone turnover. Such measures are osteocalcin, the C-terminal propeptide of type I procollagen (PICP), the N-terminal propeptide of type I procollagen (PINP) and the C-terminal pyridinoline cross-linked telopeptide of type I collagen (ICTP). This study aimed to determine whether supplementation with vitamin D3 to healthy children during the winter affects bone turnover in healthy children measured by serum osteocalcin, PICP, PINP or ICTP. METHODS: 12 girls and 8 boys aged 6.2-13.7 (mean 9.8) y, all proven healthy by medical examination and history, were enrolled in a double-blind, randomized, placebo-controlled, cross-over study with two 4 wk treatment periods and 2 wk washout. Vitamin D3 600 IU was given in one tablet of ABCDin daily. On the last day of the 4 wk periods blood was sampled for assessment of serum osteocalcin, PICP, PINP, ICTP, 25-OH-vitamin D, 1,25-diOH-vitamin D and parathyroid hormone (PTH). RESULTS: During supplementation and placebo periods serum osteocalcin (mean +/- SEM) was 53.9 +/- 5.7 and 54.4 +/- 3.8 microg l(-1) (p = 0.70), PICP was 437+/- 44 and 429 +/- 41 microg l(-1) (p = 0.73), PINP was 579 +/- 56 and 619 +/- 64 microg l(-1) (p = 0.33) and ICTP was 13.4 +/- 0.9 and 13.6 +/- 0.7 microg l(-1) (p = 0.52), respectively. Mean +/- SEM serum 25-OH-vitamin D was 47.0 +/- 2.3 and 33.0 +/- 3.0 nmol l(-1) during vitamin D3 supplementation and placebo (p < 0.001, t = 8.10, 95% CI = 10.3 to 17.6 nmol l(-1)), 1,25-diOH-vitamin D and PTH were 87.5 +/- 4.3 and 92.0 +/- 5.3 pmol l(-1) (p = 0.38), and 3.97 +/- 0.5 and 4.21 +/- 0.4 micromol l(-1) (p = 0.37), respectively. CONCLUSION: Supplementation with 600 IU vitamin D3 to healthy children in the winter does not affect bone turnover as measured by serum osteocalcin, PICP, PINP or ICTP. Vitamin D supplementation to healthy children may not be recommended on the ground of concern for bone turnover.