Rolf Felix

Common herbs, essential oils, and monoterpenes potently modulate bone metabolism.

During our survey of herbs looking for activity on bone metabolism, we found that the dried leaves of sage strongly inhibit bone resorption. Therefore, we investigated several common herbs rich in essential oils (sage, rosemary, and thyme) and essential oils extracted from these herbs and other plants (oils of sage, rosemary, juniper, pine, dwarf pine, turpentine, and eucalyptus) as well as their monoterpene components (thujone, eucalyptol, camphor, borneol, thymol, alpha-pinene, beta-pinene, bornylacetate as well as menthol) and found that they inhibit bone resorption when added to the food of rats. Pine oil, used as a representative essential oil, protects an osteoporosis model, the aged ovariectomized rat, from bone loss. The monoterpenes borneol, thymol, and camphor are directly inhibitory in the osteoclast resorption pit assay. Nonpolar monoterpenes may require metabolism to be active in vitro, for example, cis-verbenol, a metabolite of alpha-pinene occurring in human urine, inhibits osteoclast activity in contrast to the parent compound. Within 30 min borneol inhibits the formation of actin rings, a characteristic of resorbing osteoclasts indicating cell polarization. Both the in vitro and the in vivo effects of borneol are reversible. Our study demonstrates for the first time that essential oils and monoterpenes are efficient inhibitors of bone resorption in the rat.

Murine osteoblastlike cells and the osteogenic cell MC3T3-E1 release a macrophage colony-stimulating activity in culture

SummaryThe osteoclast may be of hematopoietic lineage and as such its development could be regulated by colony-stimulating factors. Since there is much interest as to whether osteoblasts influence bone resorption, we examined whether bone cells produce colony-stimulating activity. Both cells isolated from neonatal calvaria and the osteogenic cell MC3T3-E1 were found to constitutively release a colony-stimulating activity possessing characteristics of a macrophage colony-stimulating factor, as determined by basic biochemical purification and by identity of colonies induced in cultures of bone marrow cells. Release could be increased by the presence of the bone-resorbing agents lipopolysaccharide and 1,25 dihydroxyvitamin D3. We conclude that the osteoblast may contribute to both the processes of osteoclast formation and of hematopoiesis through the secretion of colony-stimulating activity into the adjacent bone marrow.

Bone-resorbing cytokines enhance release of macrophage colony-stimulating activity by the osteoblastic cell MC3T3-E1.

SummaryIt has been observed that bone resorption in response to interleukin 1 (IL 1) or tumor necrosis factor (TNF) is accompanied by an increase in osteoclast number. Because the osteoclast is of hemopoietic lineage, recruitment could be regulated by colony-stimulating factors, one of which may be macrophage colony-stimulating factor (MCSF). In this study, we show that the constitutive release of M-CSF activity by the osteoblastic cell MC3T3-E1 is enhanced by the presence of recombinant IL 1α, recombinant TNFα, or by the concurrent presence of purified transforming growth factorβ (TGFβ) and epidermal growth factor (EGF). Increased release of CSF by the osteoblast in response to these agents may provide a signal for the growth and maturation of osteoclast precursors leading to subsequent bone resorption.

The effect of several diphosphonates on acid phosphohydrolases and other lysosomal enzymes.

Diphosphonates are known to inhibit bone resorption in tissue culture and in experimental animals. This effect may be due to their ability to inhibit the dissolution of hydroxyapatite crystals, but other mechanisms may be important. Since lysosomal enzymes have implicated in the process of bone resorption, we have examined the effect of several phosphonates and of a polyphosphate (P20,2) on lysosomal hydrolases derived from rat liver and rat bone. Dichloromethylene diphosphonate strongly inhibited acid beta-glycerophosphatase (EC 3.1.3.2) and acid p-nitrophenyl phosphatase (EC 3.1.3.2) and to a lesser degree (in descending order) acid pyrophosphatase (EC 3.1.3.-), arylsulfatase A (EC 3.1.6.1), deoxyribonuclease II(EC 3.1.4.6) and phosphoprotein phosphatase (EC 3.1.3.16) of rat liver. Inhibition of acid p-nitrophenyl phosphatase and arylsulfatase A was competitive. Ethane-1-hydroxy-1, 1-diphosphonate did not inhibit any of these enzymes, except at high concentrations. Neither dichloromethylene diphosphonate nor ethane-1-hydroxy-1, 1-diphosphonate had any effect on beta-glucuronidase (EC 3.2.1.31), arylesterase (EC 3.1.1.2) and cathepsin D (EC 3.4.23.5). Of several other phosphonates tested only undec-10-ene-1-hydroxy-1, 1-diphosphonic acid inhibited acid p-nitrophenyl phosphatase strongly, the polyphosphate (P20, I) had little effect. Acid p-nitrophenyl phosphatase in rat calvaria extract behaved in the same way as the liver enzyme and was also strongly inhibited by dichloromethylene diphosphonate, but not by ethane-1-hydroxy-1, 1-diphosphonate. It is suggested that the inhibition of bone resorption by dichloromethylene diphosphonate might be due in part to a direct effect of this diphosphonate on lysosomal hydrolases.

Aggregation of hydroxyapatite crystals.

A system has been developed to measure quantitatively the disaggregation of hydroxyapatite crystals. Disaggregation was induced by pyrophosphate, ethane-1-hydroxy-1,1-diphosphonate, dichloromethylene diphosphonate, haparin and citrate. Hyaluronic acid stimulated aggregation at low concentrations and disaggregation at high concentrations. Lactate had no effect. The possible role disaggregation might play in the resorption of calcified tissues in vivo id discussed.

Pyrophosphatase and ATPase of isolated cartilage matrix vesicles

Some of the characteristics of the pyrophosphatase and ATPase activities studied in isolated cartilage matrix vesicles were found to be similar to those already reported for the solubilized and purified bone alkaline phosphatase. Thus, the pH optimum of the pyrophosphatase activity responded similarly to changes in the concentration of Mg2+, Ca2+, and PPi. Further, the ATPase activity was not activated by Ca2+ in the presence of an optimal Mg2+ concentration. It is proposed that a function of the alkaline phosphatase of matrix vesicles in vivo is to hydrolyze the substrates PPi, ADP, and ATP, which are known inhibitors of calcium phosphate precipitation.

Effect of diphosphonates on the synthesis of prostaglandins in cultured calvaria cells.

SummaryIn calvaria cells cultured for 6 days in the presence of dichloromethanediphosphonate (Cl2MDP) (0.025 – 250 µM), the synthesis of prostaglandin E2 (PGE2) was inhibited by up to 90 %. Inhibition of PGE2 synthesis might be one mechanism whereby this diphosphonate inhibits bone resorption. This effect is not common to all diphosphonates since 25 µM 3-amino-1-hydroxypropane-1,1-diphosphonate (AHPDP) stimulated the synthesis of PGE2.

Transforming growth factor-β reduces the phenotypic expression of osteoblastic MP3T3-E1 cells in monolayer culture

Abstract Transforming growth factor beta (TGF-β) regulates cell growth and differentiation. Since it is abundant in bone, we have studied the effect of the polypeptide upon the growth and phenotypic expression of murine osteoblastic cells in monolayer culture. Its actions were compared to those of epidermal growth factor (EGF), another hormonally active polypeptide known to alter bone cell function. Picogram amounts of TGF-β were found to inhibit the growth and phenotype (alkaline phosphatase and cAMP response to parathyroid hormone) of the clonal nontransformed MC3T3-E1 osteoblastic cell line. EGF also inhibited phenotypic expression, although at higher (nanogram) concentrations, but stimulated cell growth. The low concentration of TGF-β required to inhibit growth and phenotype of osteoblastic cells together with its abundance in bone suggest that TGF-β may be an important regulator of bone cell function.

Colony-Stimulating Factor-1 Stimulates the Fusion Process in Osteoclasts

Colony‐stimulating factor‐1 (CSF‐1), also called macrophage colony‐stimulating factor, is required for growth, differentiation, activation, and survival of cells of the mononuclear phagocytic system. This cytokine has been shown to be essential for osteoclast development as well as for inducing both proliferation and differentiation of osteoclast progenitors. It also sustains survival of mature osteoclasts and stimulates spreading and migration of these cells. In the present in vitro study, the formation of large tartrate‐resistant acid phosphatase (TRAP)‐positive cells with a high number of nuclei was observed when osteoclasts isolated from rat long bones were incubated with CSF‐1. These large cells, cultured on plastic, bind calcitonin and form F‐actin along the edges of the cells. Fusion to such large TRAP‐positive multinucleated cells in the presence of CSF‐1 and the formation of pits were also observed on dentine slices. Quantitative data obtained from cultures on plastic demonstrated that the number of osteoclasts slightly increased in the course of 72 h in the presence of 250 pM CSF‐1, whereas it decreased rapidly after 24 h in the absence of CSF‐1, which confirms that this cytokine is required for the survival of osteoclasts. The number of nuclei per osteoclast was maximal after 16 h of incubation with CSF‐1, namely twice the value found in the absence of CSF‐1. The maximal effect of the cytokine on the fusion process was observed at a concentration of 250 pM. A calculation of the medians of the average frequency of nuclei distribution per osteoclast resulted in four nuclei per osteoclast in the absence and six in the presence of CSF‐1. Genistein and herbimycin A, inhibitors of tyrosine kinases, inhibited the fusion induced by CSF‐1. The data suggest that CSF‐1 induces osteoclast fusion and that tyrosine kinase(s) are involved in this process. The fusion process may continue throughout the entire life of an osteoclast.

Aggregation of calcium oxalate crystals: Effect of urine and various inhibitors

SummaryThe influence of various factors on aggregation of calcium oxalate crystals in vitro was determined. Aggregation was assessed by filtering the crystal suspension and measuring the flow rate through a filter. 10% urine completely inhibited aggregation. Orthophosphate and magnesium at concentrations occurring in urine had no effect. Citrate had no effect at 10-4 M, but did inhibit at 10-3 M. The latter effect is probably due to calcium binding. Pyrophosphate and disodium dichloromethylene diphosphonate (Cl2MDP) inhibited strongly at 10-4 M, disodium ethane-1-hydroxy-1, 1-diphosphonate (EHDP) at 10-5 M, whereas pentanemonophosphonate had no effect. Uromucoid also did not show any inhibitory activity. Studies by means of heat inactivation, ultrafiltration and fractionation on DEAE-cellulose and gel-filtration indicated that the inhibitory activity was heterogenous and that the major part was larger than 10000 daltons.