Mechanical Loading Stimulates Differentiation of Periodontal Osteoblasts in a Mouse Osteoinduction Model: Effect on Type I Collagen and Alkaline Phosphatase Genes

The effects of mechanical loading on the osteoblast phenotype remain unclear because of many variables inherent to the current experimental models. This study reports on utilization of a mouse tooth movement model and a semiquantitative video image analysis of in situ hybridization to determine the effect of mechanical loading on cell-specific expression of type I collagen (collagen I) and alkaline phosphatase (ALP) genes in periodontal osteoblasts, using nonosseous cells as an internal standard. The histomorphometric analysis showed intense osteoid deposition after 3 days of treatment, confirming the osteoinductive nature of the mechanical signal. The results of in situ hybridization showed that in control periodontal sites both collagen I and ALP mRNAs were expressed uniformly across the periodontium. Treatment for 24 hours enhanced the ALP mRNA level about twofold over controls and maintained that level of stimulation after 6 days. In contrast, collagen I mRNA level was not affected after 24 hours of treatment, but it was stimulated 2.8-fold at day 6. This increase reflected enhanced gene expression in individual osteoblasts, since the increase in osteoblast number was small. These results indicate that (1) the mouse model and a semiquantitative video image analysis are suitable for detecting osteoblast-specific gene regulation by mechanical loading; (2) osteogenic mechanical stress induces deposition of bone matrix primarily by stimulating differentiation of osteoblasts, and, to a lesser extent, by an increase in number of these cells; (3) ALP is an early marker of mechanically-induced differentiation of osteoblasts. (4) osteogenic mechanical stimulation in vivo produces a cell-specific 2.8-fold increase in collagen gene expression in mature, matrix-depositing osteoblasts located on the bone surface and within the osteoid layer. Received: 9 August 1999 / Accepted: 4 February 2000     

Serum Bone Alkaline Phosphatase and Calcaneus Bone Density Predict Fractures: A Prospective Study

The aim of this study was to assess the ability of serum bone-specific alkaline phosphatase (bone ALP), creatinine-corrected urinary collagen crosslinks (CTx) and calcaneus bone mineral density (BMD) to identify postmenopausal women who have an increased risk of osteoporotic fractures. Calcaneus BMD and biochemical markers of bone turnover (serum bone ALP and urinary CTx) were measured in 512 community-dwelling postmenopausal women (mean age at baseline 69 years) participating in the Hawaii Osteoporosis Study. New spine and nonspine fractures subsequent to the BMD and biochemical bone markers measurements were recorded over an average of 2.7 years. Lateral spinal radiographs were used to identify spine fractures. Nonspine fractures were identified by self-report at the time of each examination. During the 2.7-year follow-up, at least one osteoporotic fracture occurred in 55 (10.7%) of the 512 women. Mean baseline serum bone ALP and urinary CTx were significantly higher among women who experienced an osteoporotic fracture compared with those women who did not fracture. In separate age-adjusted logistic regression models, serum bone ALP, urinary CTx and calcaneus BMD were each significantly associated with new fractures (odds ratios of 1.53, 1.54 and 1.61 per SD, respectively). Multiple variable logistic regression analysis identified BMD and serum bone ALP as significant predictors of fracture (p = 0.002 and 0.017, respectively). The results from this investigation indicate that increased bone turnover is significantly associated with an increased risk of osteoporotic fracture in postmenopausal women. This association is similar in magnitude and independent of that observed for BMD. Received: 18 June 1999 / Accepted: 21 June 1999     

Investigation of Bone Disease Using Isomerized and Racemized Fragments of Type I Collagen

In the collagen type I C-telopeptide an aspartyl-glycine site within the sequence AHDGGR is susceptible to molecular rearrangement. In newly synthesized collagen this site is in the native form, denoted alpha L. During aging a spontaneous reaction occurs resulting in three age-modified forms: an isomerized form (beta L) a racemized form (alpha D), and an isomerized/racemized form (beta D). In this study, we measured the urinary excretion of the four forms of C-telopeptides (CTX) in healthy adults and in patients with bone diseases. Levels of all CTX forms were higher in healthy postmenopausal women (P<0.001) compared with premenopausal controls. Levels decreased within 3 days of bisphosphonate treatment indicating that all CTX forms reflect bone resorption. In hyperthyroidism, characterized by a generalized increased bone turnover, native (alpha L) and age-modified (beta L, alpha D and beta D) forms increased to a similar extent compared to controls, resulting in normal ratios between the alpha L and age-modified forms of CTX. Conversely, in Paget's disease and prostate cancer-induced bone metastases, conditions characterized by focal increased bone turnover, alpha L CTX levels were more elevated than those of age-related CTX forms, resulting in increased ratios between native and age-modified CTX. For example, the ratio alpha L/alpha D was increased 7-fold in Paget's disease (P<0.001) and 2-fold in prostate cancer-induced bone metastases (P<0.002). In conclusion, the study suggests that in conditions with a localized alteration in bone turnover the ratio between alpha L CTX and the age-modified forms is significantly elevated. This may provide a new diagnostic and monitoring tool for diseases such as metastatic bone cancer and Paget's disease.     

Posttranslational Modifications of Bone Collagen Type I are Related to the Function of Rat Femoral Regions

This study analyzes the relationship between the function of femoral regions in the rat and the extent of collagen type I posttranslational modifications, to assess whether the different functional roles, i.e., mechanical or metabolic, of the bone tissues are related to the molecular structure of the matrix. For this purpose, 18 female, 100-day-old Sprague-Dawley rats were sacrificed, under anesthesia, and their femurs were removed and dissected free of adhering tissue. The spongy bone of the proximal metaphysis and the diaphysis were then selected as regions exerting prevalently a mechanical function, and the spongy bone of the distal metaphysis was selected as mainly related to metabolic function. Bone prepared from these regions was used to extract and purify the major component of the matrix, type I collagen. The content of hydroxyproline, hydroxylysine, glycosylated hydroxylysine, and pyridinium crosslinks was evaluated and the amount of each compound was expressed as a molar ratio to hydroxyproline. The amount of glycosylated hydroxylysine and pyridinium crosslinks in the distal metaphysis are significantly different from the amounts measured both in the diaphysis and the proximal metaphysis. On the contrary, the amounts of the same compounds in the diaphysis and the proximal metaphysis are statistically the same. The amount of free hydroxylysine, however, appears to be different in the proximal metaphysis and in the diaphysis. The conclusion is that matrix composition differs among different skeletal regions according to the main function they exert. Received: 5 February 1999 / Accepted: 13 August 1999     

Lymphocyte Proliferation to Collagen Type I in Dogs

The objective of this study was to investigate if cellular reactivity to collagen type I exists in dogs with unilateral cranial cruciate ligament (CrCL) rupture and if it relates to disease progression. The patient group consisted of 10 dogs with unilateral CrCL rupture. The control dogs consisted of three healthy control dogs, and two healthy dogs with unilateral sham operations of the stifle joint. All dogs were assayed repeatedly every 6 months for 12–24 months. Peripheral blood mononuclear cells were isolated from whole blood and were cultured with human collagen type I at concentrations of 5, 20 and 40 μg/ml for 6 and 7 days. Lymphocyte reactivity to collagen type I occurred not only in dogs with CrCL rupture, but also in sham‐operated dogs and healthy dogs. Five of the eight assays (63%) performed at the time of operation or at the time of diagnosis of CrCL rupture had a stimulation index (SI) ≥3.0. This was not significantly different compared to healthy control dogs, not to the sham‐operated control dogs. The CrCL rupture was assessed intraoperatively in six cases. Three cases had partial rupture and three had complete rupture. Only one dog with partial rupture, and two dogs with complete rupture had a positive SI. An increase in proliferation to collagen type I was seen in dogs with CrCL rupture, whereas it either remained stable or decreased in the control dogs. No distinct pattern in lymphocyte reactivity to collagen type I could be established from the dogs that sustained a CrCL rupture in the contralateral stifle joint, although most dogs that did not sustain a CrCL rupture in the contralateral stifle joint remained negative during this study with exception of one dog. Further research is required to determine whether cellular reactivity to collagen type I may play an initiating role in cruciate degradation.     

Stimulated Type I Collagen Turnover in Patients With Giant Cell Tumor of Bone

The aim of this study was to determine type I collagen turnover in giant cell tumor of bone (GCT) by biochemical markers of type I procollagen aminoterminal propeptide (PINP) and type I collagen carboxyterminal telopeptide (ICTP) as synthesis and degradation markers, respectively. The serum concentrations of PINP and ICTP were measured in 11 patients with GCT using radioimmunoassay, and analyzed by the correlation to the grades of GCT progression described by Campanacci. Serum of the 11 healthy subjects was available for comparison. The serum concentration of PINP was significantly higher in patients with GCT (82.4 ± 46.2 ng/ml) than in controls (40.8 ± 12.1 ng/ml) (P < 0.01), and that of ICTP was also significantly higher in GCT (5.3 ± 2.0 ng/ml) than in controls (3.2 ± 0.8 ng/ml) (P < 0.01). In GCT, the PINP concentration of grade 3 (127.6 ± 38.8 ng/ml) was higher than that in grade 1 patients (46.9 ± 4.8 ng/ml) (P < 0.01). ICTP concentration of both grades 2 (7.1 ± 1.4 ng/ml) and 3 (5.8 ± 1.8 ng/ml) patients was significantly higher than that of grade 1 (3.5 ± 0.6 ng/ml) patients (P < 0.01, P < 0.05, respectively). Two cases of serum concentration of PINP and ICTP after resection of GCT demonstrated that these biomarkers decreased to the control levels in the absence of GCT. Our results indicated that type I collagen turnover evaluated by ICTP and PINP was stimulated in the presence of GCT. Moreover, this enhanced metabolic turnover reflects the grade of GCT.     

Serum Isoforms of Bone Alkaline Phosphatase Increase During Physical Exercise in Women

Physical activity is an important factor for maintaining and probably increasing bone mass in humans. However, the mechanism by which this takes place is not completely understood. The purpose of this study was to examine the influence of physical exercise on serum alkaline phosphatase (ALP) and in particular, the bone isoforms of ALP. Six ALP isoforms were quantified by high-performance liquid chromatography: three bone (B/I, B1, and B2), and three liver ALP isoforms. In addition, serum calcium, parathyroid hormone (PTH), and other markers of bone formation and degradation, as measured by osteocalcin and cross-linked carboxyterminal telopeptide of type I collagen (ICTP), were analyzed. The study groups comprised 15 women, 8 postmenopausal (range 51–62 years) and 7 near age of peak bone mass (range 21–27 years). When the postmenopausal women exercised on an ergometer cycle until exhaustion we found significant increases in serum of bone ALP isoforms B1 and B2, and phosphate, even considering the hemoconcentration that occurred during the exercise. When the young women jogged in a moderate tempo for 40–40 minutes the levels of serum B2 and PTH increased. All changes turned towards baseline within 20 minutes after exercise. In conclusion, exercise increased serum ALP bone isoforms B1 and B2, and their responses were differentiated. As B1 and B2 are known to represent specific bone compartments, cortical and trabecular bone, the present findings may indicate different effects on bone of weight- and nonweight-bearing exercise. Received: 10 February 1999 / Accepted: 10 December 1999     

Comparison of Bone and Total Alkaline Phosphatase and Bone Mineral Density in Postmenopausal Osteoporotic Women Treated with Alendronate

Alendronate therapy in osteoporotic women decreases bone turnover and increases bone mineral density (BMD). Optimal patient management should include verification that each patient is responding to therapy. Markers of bone turnover and BMD have both been proposed for this purpose. We have investigated changes resulting from alendronate therapy with an enzyme immunoassay for bone alkaline phosphatase (BAP) and compared it with total alkaline phosphatase (TAP) and BMD of the lumbar spine, hip, and total body. Subjects were drawn from a multicenter randomized, placebo-controlled trial of alendronate in postmenopausal women with osteoporosis. BAP and TAP levels were measured at baseline and following 3, 6 and 12 months of therapy with either placebo (n= 180) or alendronate 10 mg/day (n= 134). All subjects also received 500 mg/day supplemental calcium. BMD was measured at baseline and following 3, 6, 12, 18, 24 and 36 months of therapy. To compare BAP, TAP and BMD at each site for identifying women that experienced a skeletal effect of alendronate, we calculated least significant change (LSC) values from the long-term intraindividual variability in each placebo-treated woman. Median levels of BAP decreased by 34%, 44% and 43% at 3, 6 and 12 months, respectively, in alendronate-treated women (p<0.0001 compared with baseline and with placebo). These changes were significantly greater (p<0.0001) than changes observed for TAP. Following 6 months of alendronate therapy, 90% of the women had experienced a decrease in BAP exceeding the LSC compared with only 71% for TAP. The greatest number of women similarly identified with BMD at any site (i.e. a gain in BMD exceeding the LSC) was 81% for spinal BMD at 36 months. All other sites were less than 70% at 36 months. Short-term changes in BAP and TAP were modestly associated with subsequent changes in BMD at all sites (Spearman’s rho −0.22 to −0.52, p<0.05). Compared with TAP and BMD, BAP testing rapidly and sensitively identified skeletal effects of alendronate thus enabling appropriate drug monitoring of osteoporotic women. Though BAP and TAP changes were modestly predictive of BMD changes, the value of the bone marker tests is their ability to detect rapidly a skeletal effect of therapy. Received: 19 May 2000 / Accepted: 31 October 2000     

Collagen Modifications in Postmenopausal Osteoporosis: Advanced Glycation Endproducts May Affect Bone Volume,Structure and Quality

The classic model of postmenopausal osteoporosis (PM-OP) starts with the depletion of estrogen, which in turn stimulates imbalanced bone remodeling, resulting in loss of bone mass/volume. Clinically, this leads to fractures because of structural weakness. Recent work has begun to provide a more complete picture of the mechanisms of PM-OP involving oxidative stress and collagen modifications known as advanced glycation endproducts (AGEs). On one hand, AGEs may drive imbalanced bone remodeling through signaling mediated by the receptor for AGEs (RAGE), stimulating resorption and inhibiting formation. On the other hand, AGEs are associated with degraded bone material quality. Oxidative stress promotes the formation of AGEs, inhibits normal enzymatically derived crosslinking and can degrade collagen structure, thereby reducing fracture resistance. Notably, there are multiple positive feedback loops that can exacerbate the mechanisms of PM-OP associated with oxidative stress and AGEs. Anti-oxidant therapies may have the potential to inhibit the oxidative stress based mechanisms of this disease.     

Age-Related Changes in the Biochemical Properties of Human Cancellous Bone Collagen: Relationship to Bone Strength

The metabolism of bone collagen has received little attention in relation to age-related loss of bone mass and strength. The aim of the present study was to analyze bone collagen content and metabolism in human bone with respect to age. The material consisted of iliac crest bone biopsies from 94 individuals: 46 women (ages 18–96, mean age 60.8 years) and 48 men (ages 23–92, mean age 59.5 years). Excluded from the study were all individuals with known osteoporotic lumbar vertebral fractures and renal, hepatic, or malignant diseases. Prior to collagen analysis the biopsies were scanned in a pQCT scanner for density assessment and then tested biomechanically. The results showed a decline in apparent bone density with age (P < 0.0001), a decline in maximum stress, Young's modulus, and energy absorption with age (P < 0.001). Concomittantly, there was an age-related decline in the intrinsic collagen content with age (P < 0.001). However, there were no biochemical modifications of the bone collagen during aging. There were no significant differences between women and men in the slopes of the regressions-curves. When multiple regression analyses were performed, only apparent bone density came out as a significant contributor in the correlation to biomechanical properties. Nevertheless, the decrease in bone collagen content with age might indicate an increase in the mineralization degree (probably due to decreased bone turnover) and thereby a change in material properties of bone. In conclusion, the present study has shown that loss of bone mass plays the major role in loss of bone strength. However, there is also a change in bone composition during normal aging, leading to a decrease in collagen content and an increase in the degree of mineralization. At this skeletal site, in a normal population there was no change in the biochemical properties of bone collagen. Received: 3 November 1998 / Accepted: 12 March 1999     

Clinical Efficacy of Bone Alkaline Phosphatase and Prostate Specific Antigen in the Diagnosis of Bone Metastasis in Prostate Cancer

Purpose

We investigated the usefulness of bone alkaline phosphatase isoenzyme and prostate specific antigen (PSA) determined by radioimmunoassay to predict bone scan evidence of metastasis in newly diagnosed untreated and treated prostate cancer.

Materials and Methods

We analyzed bone alkaline phosphatase enzyme concentrations in 350 men, including 150 controls, 100 with benign prostatic hyperplasia and 100 with prostate cancer (52 with stage T1 to 4, M0 and 48 with stages T1 to 4, M1 to 4). We also analyzed bone alkaline phosphatase enzyme concentrations in 61 stages T1 to 4, M0 prostate cancer cases during followup after radical prostatectomy or hormonal therapy, and 17 had clinical progression (9 with local, 5 with lymph node and 3 with bone metastases). Simultaneously, we analyzed PSA concentrations.

Results

Average bone alkaline phophatase enzyme levels were 12, 11.1 and 10.0 ng./ml. in the control, benign prostatic hyperplasia and stage M0 prostate cancer groups, respectively (p not significant), and 83.2 ng./ml. in patients with stage M1 to 4 disease (p less than 0.001). Considering that to diagnose bone metastasis the cutoff for bone alkaline phosphatase enzyme and PSA is 30 ng./ml. and 100 ng./ml., respectively, clinical effectiveness was 93.7 percent and 81.8 percent, respectively. Finally, measurement of both substances at the same time increased clinical effectiveness to 97.9 percent. During followup a bone alkaline phosphatase enzyme level that becomes greater than 30 ng./ml. (0 percent in the local and lymphatic progression groups, and 100 percent in the bone metastasis group) indicates the need to perform a bone scan.

Conclusions

We recommend the clinical use of bone alkaline phosphatase enzyme determined by radioimmunoassay and PSA measurement for the diagnosis of bone metastases and progression of prostate cancer because of the good sensitivity and specificity.     

Glycolic Acid Treatment Increases Type I Collagen mRNA and Hyaluronic Acid Content of Human Skin

BACKGROUND: Chronic solar irradiation results in both morphologic and functional changes in affected skin. alpha-hydroxy acids, such as glycolic acid, have been shown to improve photodamaged skin. OBJECTIVE: To investigate alterations in collagen gene induction and epidermal and dermal hyaluronic acid production as a result of administered glycolic acid. METHODS: In this study we compared collagen gene expression from skin biopsy specimens, and epidermal and dermal hyaluronic acid immunohistochemical staining between glycolic acid-treated and vehicle-treated skin. Forearm skin was treated with 20% glycolic acid lotion or a lotion vehicle control twice a day for 3 months. RESULTS: Epidermal and dermal hyaluronic acid and collagen gene expression were all increased in glycolic acid-treated skin as compared to vehicle-treated controls. CONCLUSION: Our data suggest that epidermal and dermal remodeling of the extracellular matrix results from glycolic acid treatment. Longer treatment intervals may result in collagen deposition as suggested by the measured increase in mRNA.     

可注射型骨修复材料对兔MSC增殖及分化的影响

目的：观察以纤维蛋白胶为载体的骨修复材料对兔骨髓基质细胞（marrow stromal cell,MSC)增殖及分化的影响。方法；采用细胞培养及组织化学等方法对各材料组兔MSC的增殖、碱性磷酸酶（alkaline phosphase,ALP)的活性和染色、细胞贴壁率及I型胶原表达进行研究。结果：（1）各组材料对细胞贴壁率及促增殖作用的影响总体上由强到弱依次是：对照组2→实验组→对照组1[纤维蛋白胶（fibrin sealant,FS)]→对照组3→单纯对照组，差异有显著性（P＜0．05）。（2）各组细胞的I型胶原表达水平和ALP活性由强到弱依次是：实验组→对照组3→对照组1→对照组2→单纯对照组，差异有显著性（P＜0．05）。结论：以纤维蛋白胶为载体的注射型骨修复材料可显著保进MSC贴壁率和向成骨细胞方向的分化水平。     

bFGF增强rhBMP-2诱导成骨中Ⅰ、Ⅱ型胶原及碱性磷酸酶的表达

目的：对bFGF增强rhBMP-2诱导成骨中CollagenⅠ、Ⅱ及碱性磷酸酶（ALP）的表达进行研究。方法：110只BALB/c小鼠随机分为2组，每组55只，rhBMP-2/bFGF为实验组，rhBMP-2为对照组，分别于术后3-21d8个时间点取材，用免疫组化法对新生骨组织中的Ⅰ、Ⅱ胶原进行检测；对ALP活性进行定量分析，观察2组在诱导成骨中CollagenⅠ、Ⅱ及ALP的表达情况。结果：⑴Ⅱ型胶原和成软骨、软骨细胞的出现相伴随，但肥大、变性的软骨细胞并不表达Ⅱ型胶原。⑵作为成骨细胞成熟标志物的Ⅰ型胶原，ALP在骨形成期即有表达，但随着成骨细胞的出现，骨组织的形成Ⅰ型胶原表现为高表达，而ALP的表达则呈下降趋势。⑶实验组CollagenⅠ、Ⅱ及ALP的表达早于对照组。结论：bFGF增强了rhBMP-2诱导成骨中CollanenⅠ、Ⅱ及ALP的表达。     

Altered Collagen in Tartrate-Resistant Acid Phosphatase (TRAP)-Deficient Mice: A Role for TRAP in Bone Collagen Metabolism

Tartrate-resistant acid phosphatase (TRAP) is an iron-containing protein that is highly expressed by osteoclasts, macrophages, and dendritic cells. The enzyme is secreted by osteoclasts during bone resorption, and serum TRAP activity correlates with resorptive activity in disorders of bone metabolism. TRAP is essential for normal skeletal development. In knockout mice lacking TRAP, bone shape and modeling is altered with increased mineral density. Here, we report the effect of TRAP on the biochemical and biomechanical properties of collagen, the major protein constituting the bone matrix, using these mice. Femurs from TRAP^-/- and wild-type mice were used in these studies. The biomechanical properties were investigated using a three-point bending technique. Collagen synthesis was determined by measuring cross-link content using high-performance liquid chromatography and amino acid analysis. Collagen degradation was determined by measuring matrix metalloproteinase-2 (MMP-2) activity. The rates of collagen synthesis and degradation were significantly greater in bones from TRAP^-/- mice compared with wild type. At 8 weeks, there was an increase in the intermediate cross-links but no significant difference in animals aged 6 months. There was a significant increase in mature cross-links at both ages. A significant increase in MMP-2 production both pro and active was observed. A significant increase in ultimate stress and Young’s modulus of elasticity was needed to fracture the bones from mice deficient in TRAP. We conclude that both synthesis as well as degradation of collagen are increased when TRAP is absent in mice at 8 weeks and 6 months of age, showing that TRAP has an important role in the metabolism of collagen.     

rhBMP-2在体内诱导成骨中Ⅰ、Ⅱ型胶原mRNA及碱性磷酸酶的表达

目的：研究rhBMP-2在体内诱导成骨中CollagenⅠ、ⅡmRNA及碱性磷酸酶（ALP）的表达。方法：将含rhBMP-2 0.5mg的松质骨载体植入BALB/c小鼠的右股部肌袋内，于术后3-21d间8个时间点取材，用原位杂交法对新生骨组织中的Ⅰ、Ⅱ型胶原mRNA进行检测；定量分析ALP活性，观察rhBMP-2在诱导成骨中CollagenⅠ、ⅡmRNA及ALP的表达。结果：⑴Ⅱ型胶原mRNA的出现是和成软骨、软骨细胞的出现相伴随。肥大、变性的软骨细胞并不表达Ⅱ型胶原mRNA。⑵作为成骨细胞成熟标志物的Ⅰ型胶原mRNA、ALP在软骨形成期即有表达，随着成骨细胞的出现，骨组织的形成，Ⅰ型胶原mRNA仍表现为高表达，而ALP的表达则呈下降趋势。结论：在体内诱导成骨中rhBMP-2促进了CollagenⅠ、ⅡmRNA及ALP的表达。