The effect of long-term low-dose diphosphonate treatment on rat bone

Weaning, male rats were given ethane-1-hydroxy 1,1-diphosphonate (EHDP) or dichloromethylene diphosphonate (Cl2MDP) 0.5 mgP/kg/day for 140 days. Samples prepared after sacrifice were: (1) thin ground (30 micrometers) transverse tibial sections, (2) methacrylate-embedded 5 micrometers sections of the proximal tibial metaphysis, and (3) ultrathin calcified metaphyseal sections for electron microscopy. Cl2MDP reduced the diaphyseal medullary cavity, and EHDP increased the osteoid width. Both drugs increased endosteal bone apposition (possible secondarily to the impaired resorption) and, perhaps as a result, periosteal apposition was increased. Consequently, diaphyseal bone area was unchanged. Metaphyseal bone area was increased and osteoclast numbers reduced, in contrast to the increased osteoclast numbers reported previously in animal experiments with diphosphonates. Acid phosphatase activity within osteoclasts was markedly reduced with EDHP and unchanged with Cl2MDP. The ultrastructural changes in osteoclasts, i.e., a deficiency of ruffled borders, reduced numbers of cytoplasmic vacuoles and the presence of crystals between bone and the osteoclastic plasmalemma, were more marked with EHDP than Cl2MDP.     

Effect of pyrophosphate and two diphosphonates on 45Ca and 32Pi uptake and mineralization by matrix vesicle-enriched fractions and by hydroxyapatite

Inorganic pyrophosphate (PPi) and two diphosphonates, ethane-1-hydroxy-1, 1-diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl2MDP), were found to inhibit in vitro mineralization induced by matrix vesicle-enriched fractions from chicken epiphyseal cartilage. Inhibitor concentrations from 0.20 to 20 microM caused a dosage-dependent decrease in 45Ca and 32Pi uptake by the vesicle fraction. These inhibitors were also tested in a hydroxyapatite (HA)-seeded system to help distinguish between effects on the mineral vs nonmineral portions of the vesicle fraction. The order of inhibition of the HA-seeded system was EHDP greater than PPi greater than Cl2MDP, except for inhibitor concentrations of 0.20 microM where EHDP was the least inhibitory. This variation may be due to differences in the binding of the inhibitors to HA crystals. In general, inhibition of HA mineralization was greatest during later time periods, whereas vesicle ion uptake was affected more during early stages of incubation. The differential effects of the three inhibitors were most obvious at the 2.0 microM concentration. With PPi substantial inhibition of HA-seeded mineralization was observed even in late stages of the study; in contrast, with time the vesicle fraction overcame this inhibition. This suggests that alkaline phosphatase, an enzyme notably enriched in matrix vesicles, catalyzed the hydrolysis of PPi, reducing its concentration to a level where mineralization could proceed. Our findings show that matrix vesicle-induced mineralization differs significantly from apatite-induced mineralization. The data support the concept that vesicle alkaline phosphatase acts, at least in part, to remove physiological crystal growth inhibitors.     

Resorption is not essential for the stimulation of bone growth by hPTH-(1-34) in rats in vivo

Chronic low doses of hPTH-(1-34) stimulate bone growth in rats in vivo. The objective of these studies was to determine if the anabolic effect of hPTH-(1-34) on rat bone in vivo is dependent on an initial stimulation of resorption by blocking resorption with either salmon calcitonin (CT) or dichloromethylene diphosphonate (Cl2MDP). Male Sprague-Dawley rats, 70-100 g, were treated with daily subcutaneous (SC) injections of vehicle (V) or hPTH-(1-34), 8 micrograms per 100 g (PTH), for 12 days. In experiment 1, rats were given CT for 3 (CT3) or 12 (CT12) days, either alone or in combination with hPTH-(1-34) (CT3-PTH and CT12-PTH) or vehicle for 12 days. In experiment 2, rats were pretreated for 4 days with Cl2MDP or its vehicle before starting the daily PTH or vehicle injections. Rats were then killed. Sera, femora, tibiae, and kidneys were removed for chemical and histomorphometric analyses. PTH, PTH-CT3, and PTH-CT12 rats showed significant increases in total bone calcium (18-23%), dry weight (DW, 13-25%), and bone-forming surfaces compared with their respective controls. Eroded (resorption) surfaces were comparable between the groups. Although weight gain and serum calcium were normal in rats treated for 3 days with CT, rats treated for 12 days with CT gained 14% less weight than controls and were hypophosphatemic, with reduced serum calcium and urea nitrogen. Total bone mass increased both in Cl2MDP rats (Ca 21%, DW 2%), where resorption was presumably blocked, and in PTH rats (Ca 31%, DW 19%). The increase in bone mass was greater in PTH-Cl2MDP rats (Ca 48%, DW 29%) than in rats treated with Cl2MDP alone, suggesting that although Cl2MDP blocked resorption, the anabolic response to PTH was not altered. As neither short-term treatment with CT nor Cl2MDP blocked the anabolic response of bone to hPTH-(1-34), this response does not appear to depend on the early stimulation of resorption.     

The comparative effects of dichloromethylene diphosphonate (Cl2MDP) and ethane-1-hydroxy-1,1-diphosphonate (EHDP) on growth and modeling of the rat tibia

Summary Male rats weighing 100 g were assigned to groups and injected daily for 10 days with vehicle (control), 0.4, 2.0, 4.0, 10.0, or 20.0 mg/kg/day of ethane-1-hydroxy-1,1-diphosphonate (EHDP) or dichloromethylene diphosphonate (Cl₂MDP). The proximal tibial metaphysis and epiphysis were assayed for changes in percentage of hard tissue and bone formation parameters. From the data, information about hard tissue resorption was deduced.All doses of Cl₂MDP and doses of 2.0 mg EHDP/kg/day and greater caused significant increases in percentage of hard tissues with Cl₂MDP being more effective than similar doses of EHDP in decreasing bone resorption.Osteoclast population parameters were increased with all doses of both Cl₂MDP and EHDP with Cl₂MDP having a greater effect than similar doses of EHDP. Decreases in the proliferation of the osteoprogenitor pool parallel the decreases in osteoblasts and bone formation parameters. These decreases in osteoprogenitor pool proliferation do not account for the increases with diphosphonates in osteoclast population parameters.     

The effects of disodium ethane-1-hydroxy-1, 1-diphosphonate (EHDP) on mineral and organic components of rat bone

The purpose of this study was to determine the short-term effects of various systemic doses of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) on bone organic matrix and to relate these effects to the corresponding dose-related changes in bone mineral. EHDP was administered daily by subcutaneous injection at doses of 0.25, 2.5 and 40 mg/kg body weight for periods of one and two weeks. At both time intervals, rat tibiae were quantitatively analyzed for mineral content (ash, calcium and phosphorus) and for organic matrix content (matrix weight, nitrogen and certain amino acids). The latter data were correlated with semiquantitative histological analyses of the tibiae. Results of this study demonstrate that the short term effects of EHDP on bone chemistry and histology are variable and depend on the systemic dose and the duration of treatment. Systemic doses of 0.25 and 2.5 mg/kg EHDP following daily administration for one week resulted in transitory decreases in bone mineral content compared to controls. Following two weeks of treatment, both of these dose levels resulted in increased bone mineral content and, in addition, the 2.5 mg/kg dose resulted in tibiae which contained more organic matrix compared to control bones. In contrast to the low dose effects, a high systemic dose of EHDP—e.g. 40 mg/kg administered daily for 1 or 2 weeks—appears to act solely by inhibiting mineralization of newly-formed matrix.     

The effects of EHDP on regenerating trabecular bone usingin vivo microscopic,light and electron microscopic,and electron microprobe techniques

Metallic chambers were implanted into the proximal tibiae of rabbits to permit microscopic examination of living bonein situ. The bone repair process secondary to the injury produced during installation of the chamber, was visualized. Six to 8 weeks after implantation, osteoid and/or bone could be seen. The effects of various doses of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) on the repair and regeneration processes following chamber implantation were studied. Data from various techniques indicated that: (1) following low dose EHDP (0.25 mg/kg/day) chambers contained bone tissue morphologically and ultrastructurally indistinguishable from controls; and (2) with higher doses of EHDP (2.5 or 10 mg/kg/day) chamber contained spicules of normal osteoid, osteoblasts and osteocytes, but were devoid of osteoclasts. The effects of the various regimes of EHDP also were assessed on regenerated, trabecular bone contained within the tibia chambers three months after implantation of the chambers. Data from various methods of analysis supported the following conclusions: (1) the low dose of EHDP (0.25 mg/kg/day) had no toxic effects on the trabecular bone within the chambers but there appeared to be an increase in bone formation as compared to saline control; (2) higher doses of EHDP (2.5 or 10 mg/kg/day) were not toxic to bone cells but thick osteoid seams formed on the trabecular bone within the chambers. No osteoclasts were found associated with the bone apparently due to the coverage of bone surfaces by osteoid seams; and (3) osteoid which accumulated after EHDP treatment of 2.5 mg/kg/day for 2 months remained uncalcified for as long as 2 months following withdrawal of EHDP administration. The results showed the value of tibial chamber for examining microscopically living bonein situ and demonstrated the inhibitory effect of EHDP on mineralization of newly formed osteoid and a lack of effect on bone cells.     

应用牵拉成骨技术治疗股骨、胫骨肥大型骨不愈合合并畸形患者的疗效

目的探讨应用牵拉成骨技术治疗股骨、胫骨肥大型骨不愈合合并畸形患者的疗效。方法回顾性分析2016年11月至2019年11月在北京积水潭医院创伤骨科应用牵拉成骨技术治疗的3例股骨、胫骨肥大型骨不愈合合并畸形患者的病历资料。3例均为男性。所有骨折不愈合端均不切开。胫骨不愈合者均需经皮截断腓骨,使用Taylor Spatial Frame(施乐辉公司)固定,将13个参数输入计算机,设定牵开速度1 mm/d,生成电子处方。股骨不愈合者使用Orthofix外固定架固定。术后第2天按照1 mm/d的速度牵拉,在牵开恢复长度的过程中逐渐纠正畸形。随访期间,观察患者畸形纠正情况和骨愈合情况。结果病例A术后20 d畸形完全纠正,术后15个月获得骨性愈合。病例B术后25 d畸形完全纠正,术后15个月获得骨性愈合。病例C术后29 d成角畸形纠正,术后19个月获得骨性愈合。结论牵拉成骨技术可纠正肥大型骨不愈合的成角畸形及短缩,可作为肥大型骨不愈合的一种微创治疗方法。     

Boen remodelingin vitro: The effects of two diphosphonates on osteoid synthesis and bone resorption in mouse calvaria

Two Diphosphonates, ethane-1-hydroxy-1,1-diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl₂MDP) inhibitin vitro bone resorption, which is either stimulated by parathyroid hormone or intrinsic in a bone remodeling culture system. While Cl₂MDP is more effective than EHDP in inhibiting resorption, it also appears to result in a related diminution in osteoid formation. This effect causes a marked biochemical and morphological depression of bone remodelling with Cl₂MDP at a concentration equivalent to 10-μg-phosphorus/ml of culture medium. The difference in activity between EHDP and Cl₂MDP may be related to their relative affinities for the bone mineral surfaces and hence their effective free concentration in the bone extracellular fluid. It is hypothesized that diphosphonates may also affect bone formation indirectly if one assumes that the degree of mineralization of the matrix is important in the induction and regulation of osteoblastic activity in remodelling bone.     

Inhibition of bone resorption by difluoromethylene diphosphonate in organ culture

A newly synthesized diphosphonate, difluoromethylene diphosphonate (F₂MDP), was studied for its effects on bone resorption, as measured by the release of previously incorporated ⁴⁵Ca. F₂MDP (10 μM to 1000 μM) effectively inhibited both unstimulated and parathyroid hormonestimulated resorption, and the amount of ⁴⁵Ca release decreased with time. Dichloromethylene diphosphonate (Cl₂MDP) and ethane- 1-hydroxyl-1,1-diphosphonate (EHDP) inhibited resorption to similar extents with two exceptions: At concentrations of 10 μM and 100 μM F₂MDP was more effective than EHDP and less effective than Cl₂MDP. No greater inhibition was observed when bones had been stimulated with PTH prior to the addition of F₂MDP In addition, bones treated with F₂MDP only during the first half of the incubation period exhibited reductions in tha amount of ⁴⁵Ca released during the second half similar to that observed when F2MDP was continuously in the medium, indicating a prolonged effect. Morphologic alterations of osteoclasts suggestive of cell degeneration were observed in F₂MDP-treated bones, which were similar to those observed in bones treated with Cl₂MDP and EHDP. Due to the presence of fluorine, F₂MDP may be useful as an experimental tool to investigate the mode of action of all diphosphonates, in addition to its possible use as a therapeutic agent for diseases of increased bone resorption.     

Effect of diphosphonates and calcitonin on the chemistry and quantitative histology of rat bone

Young male rats were treated with one of three diphosphonates, disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP), disodium dichloromethylene-diphosphonate (Cl₂MDP), or disodium methane-1-cyclohexyl-1-hydroxy-1,1-diphosphonate (MCHDP), or with porcine calcitonin. Changes in the length weight, calcium, phosphorus, and ash of the femora and in plasma calcium, phosphate and alkaline phosphatase were measured. Bone formation rate was measured histologically in the tibial diaphysis using tetracycline markers. The lower dose (0.01–1mg P/kg/day s.c.) of the phosphonates, which block immobilization osteoporosis and reduce bone turnover measured by⁴⁵Ca kinetics, did not cause marked changes in the composition of the femora or in plasma values. This suggests that, at these doses in intact animals, the diphosphonates similarly reduce both resorption and mineralization rates. Higher doses of EHDP and MCHDP (10 or 30 mg P/kg/day s.c.) impaired bone growth both in length and width and inhibited mineralization of new cartilage and osteoid. The total calcium content of the bones decreased but the plasma values remained unchanged. Cl₂MDP did not impair mineralization at equivalent doses. EHDP, unlike Cl₂MDP, therefore seems to have two effects on bone. At lower doses it reduces bone turnover rate; but at higher doses it also directly prevents the full mineralization of new matrix. This difference between the effects of EHDP and Cl₂MDP may be important.     

Growth of embryonic chick tibiae in ovo and in vitro: A scanning electron microscope study

Summary The study describes the ultrastructure of the mineralized portion of chick tibiae from 10 days in ovo to 2 days post-hatch. At 10 days a single mineralized cylinder surrounds the diaphysis. On its outer surface columnar trabeculae join to form ridges parallel to the long axis of the bone. These ridges are covered by another cylinder and form the haversian canals. At 11 days vascular invasion of the marrow cavity occurs and resorption of the endosteal surface begins. This type of periosteal deposition and endosteal resorption is repeated during and subsequent to embryonic development. The mineralized portion of 10-day chick tibiae cultured for 2 days in modified BGJ medium was compared with 10-, 11-, and 12-day tibiae in ovo. Cultured tibiae were similar in length and calcium content to 11-day tibiae in ovo. The form of mineral deposited in ovo and in culture was the same, namely, aggregates of spherical mineral clusters. Differences in culture included the following: (a) few concentric cylinders were deposited as compared with tibiae in ovo; (b) trabeculae were not arranged in rows and ridges in culture; (c) osteocytic lacunae were restricted to bases of trabeculae rather than uniformly distributed as in ovo; and (d) the endosteal surface of tibiae in culture appeared etched.     

The effects of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) on bone and serum chemistry in rabbits

The effects of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) on bone and serum chemistry were investigated in adult rabbits. EHDP was administered by subcutaneous injection at doses of 0.25, 2.5 and 10 mg/kg body weight/day for of 28 days. Blood samples were obtained weekly from each rabbit and serum levels of total calcium, ionized calcium, inorganic phosphate and alkaline phosphatase were determined. At the end of the treatment period all rabbits were sacrificed and the tibiae removed for chemical analysis and histological evaluation. The effect of EHDP administration on serum chemistry was both dose- and time-related. The highest of the three doses, 10 mg/kg/day, resulted in a time-related decrease in total serum calcium. This dose also caused a rapid but transient reduction in serum ionized calcium. The effect of EHDP on serum inorganic phosphate was biphasic. Administration of 2.5 mg/kg/day resulted in a time-related elevation in this parameter, whereas the 10 mg/kg/day dose resulted in a time-related hypophosphatemic response. There were no significant drug-related changes in tibial fat-free dry weight, ash weight, total calcium or total phosphorus values. However, administration of 2.5 and 10 mg/kg/day EHDP resulted in increased osteoid tissue as measured histologically. These results are compared with data from other EHDP studies, and discussed in relation to the maturity and growth-state of the experimental animals.     

The binding of pyrophosphate and two diphosphonates by hydroxyapatite crystals

The binding on hydroxyapatite has been studied of inorganic pyrophosphate (PP_j) and two diphosphonates, disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) and disodium dichloromethylene diphosphonate (Cl₂MDP). The binding is greatest for PP_i, less for EHDP and least for Cl₂MDP. The binding of all three is accompanied by a release of orthophosphate into the solution and an uptake of calcium by the crystals. A competition exists between EHDP and PP_i, EHDP being more potent in displacing PP_i than the reverse. An analysis with Scatchard plots suggests that the binding is not restricted to one class of binding sites with equal affinity and that the affinity of the first site is in the order EHDP>PP_i>Cl₂MDP. The results correlate well with previous data on the effect of these compounds on apatite crystal formation and dissolutionin vitro and on bone formation and resorptionin vivo To Prof. F. Leuthardt to his 70th birthday.     

The kinetics of 99mTc labelled EHDP in paget's disease before and after dichloromethylene-diphosphonate treatment

The kinetics of ^99m labelled EHDP were studied before and during treatment with dichloromethylene diphosphonate (Cl2MDP) in 20 pagetic patients. The quantitative scintigraphic parameter analyzed was the ratio of activity per unit area over a pagetic bone localization to a symmetrical non-involved bone area evaluated at 10 minutes, 45 minutes, and 6 hours after Injection of the tracer. Urine hydroxyproline excretion (OHP) and serum alkaline phosphatase (SAP) were measured.Along with marked reductions of SAP and OHP the scintigraphic parameters decreased very significantly after 3 months of treatment and decreased even further after 6 months of treatment. These quantitative scintigraphic parameters seem very suitable for the assessment of therapy of specific pagetic lesions.     

Medial epiphysiolysis in severe infantile tibia vara

Recurrent varus deformity and shortening frequently result following valgus osteotomy alone for advanced stage infantile tibia vara (ITV), due to the proximal medial physeal dysfunction which resembles a complete growth arrest in Langenski?ld grade III or greater lesions. Epiphysiolysis is a possible treatment alternative which, if successful, can restore symmetric proximal tibial growth and obviate the need for additional complex treatments to address recurrent angular deformity and limb length inequality. We reviewed 24 patients, ages 5-10 years, with 27 affected tibiae, who underwent epiphysiolysis of the proximal medial tibia combined with valgus osteotomy. The procedure was >80% successful in restoring growth and avoiding varus recurrence in children <7 years old. Patients >age 7, patients with relative undercorrection of their mechanical axis at the time of surgery, and patients with previous failed surgical treatment were more likely not to benefit from epiphysiolysis, and consequently alternative methods may be more appropriate.     

Effect of ethane-1-hydroxy-1,1-diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl2MDP) on the calcification and resorption of cartilage and bone in the tibial epiphysis and metaphysis of rats

Young male rats (70–90 g) were treated for various periods with several doses of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) or disodium dichloromethylene diphosphonate (Cl₂MDP). Effects of treatment on the changes in the thickness, growth and mineralization of proximal growth plate and metaphysis of the tibia were assessed histologically and by micro-radiography. High doses (10 or 30 mg P/kg/day) of EHDP impaired mineralization of the growth cartilage, which became increased in thickness, and of the osteoid in the metaphysis and diaphysis. Matrix formation continued, although at a diminished rate. High doses (10 or 30 mg P/kg/day) of Cl₂MDP produced a different effect. There was no inhibition of mineralization, but there was a marked impairment of normal metaphyseal remodelling, with persistence of columns of calcified cartilage. Resorption at the periosteal surface in the metaphysis was also inhibited, so that the metaphysis became club-shaped. Osteoclasts were present in large numbers in the metaphysis, but their appearance was abnormal and similar to that seen in human osteopetrosis.     

胫骨延长后骨痂直径与新生骨预后关系的临床分析

目的探讨胫骨延长后骨痂直径与新生骨预后的关系.方法 1996年1月～2001年12月,对68例胫骨延长者测量达所需延长长度时的骨痂直径,计算骨痂直径率(callus diameter ratios,CDR);统计拆除外固定架后2～4周时新生骨发生骨折例数及畸形角度,比较骨折和成角畸形与CDR的关系.结果拆除外固定架前胫骨前屈角度5度以上畸形19例,其中11例发生骨折;拆除外固定架后胫骨出现5度以上畸形6例,其中2例骨折;其余43例胫骨延长骨痂正常愈合.计算CDR值:CDR值小于80% 23例,其中发生新生骨骨折13例,成角畸形5度以上者21例;CDR在81%～85%之间6例,成角畸形5度以上4例;CDR值超过86% 39例,无畸形或新生骨骨折.结论胫骨延长时CDR小于80%时,容易发生新生骨骨折和畸形;CDR大于85%时,无新生骨骨折和畸形发生;两组间具有统计学意义(P＜0.05).CDR是判断骨痂能否正常愈合较为理想的检测指标.     

A noninvasive, in vivo model for studying strain adaptive bone modeling

We present a noninvasive, in vivo model for strain application in the tibiae of rats. The hind limb of each animal was placed into a device that applied four point bending to the tibia. Bending was applied in the medial-lateral direction causing compression on the lateral surface of the tibia and tension on the anteromedial surface. The peak strain magnitudes were estimated to be between 1600 and 3500 mu strain. In this pilot work, data were collected from 12 rats. The rats received either one cycle per day, four cycles per day, 12 cycles per day, 36 cycles per day, or 108 cycles per day of bending. The experimental (right) tibiae from all of the rats showed new bone formation after 12 days. The control (left) tibiae showed no new bone formation over this period. A better organized, dense bony reaction occurred in regions of lesser strains than in regions of higher strains, where there was a large accumulation of bone easily identified as woven. The organization and density of the newly formed bone appeared to be inversely related to the peak strains in the region. After 40 days of daily loading, the new bone area appeared to be more compact and better mineralized. However, bone formation was still occurring after 40 days. The results of this study suggest that woven bone formation occurred due to the bending stimulus and not due to pathology.     

In vivo internalization of Staphylococcus aureus by embryonic chick osteoblasts

Staphlylococcus aureus is the primary pathogen associated with osteomyelitis, an acute and recurrent bone disease. Internalization of S. aureus by cultured embryonic chick calvarial osteoblasts has been observed. The purpose of this study was to demonstrate that internalization of bacteria by embryonic chick calvarial and tibial osteoblasts occurs in vivo. In initial experiments, 10(8) colony forming units (cfu) of S. aureus, strain UAMS-1 or Cowan 1, were injected subcutaneously under the scalp skin of 17 day chick embryos. After 45 min, calvariae were harvested and processed for transmission electron microscopy (TEM). In subsequent experiments, 10(9) cfu of UAMS-1 were injected into the allantoic sac of 17 day chick embryos via a small opening in the egg shell. After 48 h, calvariae and tibiae were harvested for TEM. S. aureus cells were found in approximately 14% of the calvarial osteoblasts after subcutaneous injection and in 11% of calvarial and tibial osteoblasts following intraallantoic injection. Endosomes were observed in some cells, but most bacteria internalized appeared to be free in the cytoplasm. Osteoblasts with as few as five bacteria had a greater loss of cytoplasmic integrity and a more heterochromatic nucleus than osteoblasts with fewer bacteria or than uninfected osteoblasts. S. aureus cells in calvariae and tibiae were also observed in the cytoplasm of approximately 4% of the osteocytes in mineralized bone matrix. Thus, internalization of S. aureus by osteoblasts in vivo augments the previous observation in vitro. This study has also shown that osteoblasts with few bacteria continue differentiating into osteocytes. Results of these experiments support the hypothesis that internalization of S. aureus by osteoblasts may play a role in the etiology of osteomyelitis.