lates resorption and inhibits collagen synthesis in mouse calvaria bones in vitro. Addition of polymyxin B caused a dose-related inhibition ofLPS-stimulated bone ...
Br. J. exp. Path. (I986) 67, 699-705
In vitro inhibition of lipopolysaccharide-induced bone resorption by polymyxin B W. Harvey, M. Wilson* and S. Meghji Department of Oral and Maxillo-Facial Surgery and *Clinical Pathology and Immunology, Institute of Dental Surgery, Eastman Dental Hospital, Grays Inn Road, London Received for publication 3 April I986 Accepted for publication 6 June I986
Summary. Lipopolysaccharide (LPS) purified from Haemophilus actinomycetemcomitans stimulates resorption and inhibits collagen synthesis in mouse calvaria bones in vitro. Addition of polymyxin B caused a dose-related inhibition ofLPS-stimulated bone resorption and reversal of the inhibition of collagen synthesis. A polymyxin B to LPS ratio of 2: I prevented bone resorption and restored collagen synthesis to control levels. The activity of polymyxin B was specific for LPS as bone resorption induced by prostaglandin E2 or parathyroid hormone was unaffected at similar concentrations. These results indicate that polymyxin B and its analogues may have potential in the treatment of periodontal disease by reducing bone loss induced by lipopolysaccharides.
Keywords: lipopolysaccharide, bone resorption, polymyxin B, periodontal diseases
The subgingival growth and accumulation of Gram negative bacteria is considered to be central to the inflammatory and destructive changes in periodontal diseases. Many of these pathological changes have been attributed to the lipopolysaccharides (LPS) of Gram negative bacteria which exhibit a wide range of biological activities, both in vivo and in vitro, including complement activation, disseminated intravascular coagulation, pyrogenicity, bone resorbing ability, macrophage activation, and B-cell mitogenicity (Daly et al. I980). By virtue of such properties, lipopolysaccharides are considered to be responsible for the morbidity and high mortality resulting from infections with Gram negative bacteria (Drutz & Graybill I 9 78).
The discovery that some of the cyclic polypeptide antibiotics (e.g. polymyxin B) can bind specifically to LPS and neutralize some of its biological activities (Neter et al. I958) prompted some investigations into their possible use in the treatment of endotoxaemia. Polymyxin B (PB) was shown to reduce the lethality of LPS (Rifkind I967), prevent endotoxin-induced intravascular coagulation (Corrigan & Bell I 9 7I) and also to inhibit the Schwartzman reaction (Rifkind & Hill I967). However, despite the apparent importance of LPS in periodontal diseases, no attempts have been made specifically to neutralize or suppress its activity. Treatment of perindontal disease has consisted largely of attempts to remove bacteria and their
Correspondence: Dr W. Harvey, Institute of Dental Surgery, 2 5 6 Gray's Inn Road, London WCiX 8LD.
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W. Harvey et al. products by mechanical means or to reduce ml). The purified LPS was recovered by the number of bacteria present using a ultracentrifugation and then lyophilized. variety of antiseptics and antibiotics. Bone resorption. Bone resorption was meaThe purpose of this study was to determine sured by the release of calcium, assayed whether PB could inhibit the induction by colorimetrically, from 5 day old mouse calLPS of two pathological changes directly varia in vitro (Zanelli et al. I969). After relevant to periodontal disease, namely bone removal of any adherent connective tissue, resorption and the inhibition of bone colla-
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gen synthesis.
Materials and methods Bacterial cultivation. Haemophilus actinomycetemcomitans (NCTC 9710) used in this study was kindly supplied by Dr P. Marsh (P.H.L.S., Porton Down, UK). The organism was grown at 3 7°C under anaerobic conditions in a medium containing the following components (per litre of distilled water): io g tryptone, 2 g glucose, 5 g yeast extract, 5 g NaHCO3, 50 mg dithiothreitol, and i mg biotin. After 72 h incubation the cultures were checked for contamination by staining and cultural methods and centrifuged at 30000 g for 30 min at 40C. The harvested cells were washed with saline, recentrifuged and then lyophilized. Preparation of LPS. Capsular material was removed from the cells by saline extraction (Wilson et al. I985) and LPS opbtained by the hot phenol-water method of Westphal and Jann (I965). The crude LPS was then purified essentially as described by Kiley and Holt (I980). Briefly, the lyophilized cells were suspended in distilled water at 68°C and mixed with an equal volume of 90% phenol (w/v) at 68°C for I 5 min. The aqueous phase was then removed after cooling and centrifugation, and the phenol phase extracted twice more with equal volumes of water. The combined aqueous phases were dialysed against distilled water for 48 h at 40C and then lyophilized. The resulting crude LPS was ultracentrifuged at I00000 g for i h at 40C and the pellet treated sequentially with deoxyribonuclease (20 ,ug/ml), ribonuclease A (20 Mug/ml), and pronase (25 pg/
the calvaria were halved along the sagittal suture and each bone cultured on a I cm2 stainless steel grid in i.5 ml BGJ modified medium (Flow Laboratories) supplemented with antibiotics, ascorbate (I00 Mug/ml) and 5% complement-inactivated rabbit serum (Wellcome). After 24 h the media were removed and replaced with fresh media containing LPS (O.OI-50 ,ug/ml), prostaglandin E2 (PGE2) (i gM), or parathyroid hormone (PTH) (o.s u/ml) in groups of six cultures per concentration. For inhibition studies, PB (Sigma) was added to give final concentrations of I-I00og/ml in solutions of LPS at a concentration of I0 jug/ml (equivalent to approximately i08 bacteria/ ml), PGE2 (i gm) and PTH (o.5 u/ml). The cultures were incubated for a further 48 h and resorption expressed as the release of calcium into the medium over this period.
Collagen synthesis. Groups of six half calvaria, prepared as described above, were incubated for I8 h in somm culture dishes containing BGJ medium supplemented with 50 jug/ml vitamin C, 2 mM glutamine, 5% heat-inactivated rabbit serum, and 50 ,ug/ml fl-aminopropionitrile fumarate to inhibit collagen cross-linking. LPS (0-so jug/ml or io ,g/ml mixed with PB at I-50 ,ug/ml) was added to the culture medium before incubation. Tritiated proline (specific activity 31.5 Ci/ mmol, Amersham International, UK) was introduced into each culture to give a final concentration of o.5 !iCi/ml and incubation continued for a further 6 h. The accumulation of 3H-proline in labelled collagen was measured by an adaptation of the pepsin extraction method (Webster & Harvey I979). Collagen was extracted from each bone by limited pepsin digestion (o. s mg/ml
70I Polymyxin B and LPS-induced bone resorption nate (3.5 g/l) in an atmosphere of 5% CO2 in in 0. 5 M acetic acid for i 6 h at 40C). Insoluble
debris was removed by centrifugation, and the collagen was precipitated from solution by addition of o 9 M NaCl (w/v). The precipitate was centrifuged, redissolved in 0.5 M acetic acid, and re-precipitated with o 9 M NaCl. The final collagen pellet was dissolved in 0. 5 M acetic acid, mixed with 3 ml scintillant (Unisolve i, Koch-Light) and radioactivity measured on a LKB Rackbeta scintillation counter with external standardization. Fibroblast proliferation. Human gingival fibroblasts cultured from biopsies taken during routine surgical removal of third molar teeth were used between the fourth and sixth passage. Suspensions were prepared by detaching the cells with trypsin (0.25%) and inoculated into 96-well culture plates (Microtiter, Linbro) at 5000 cells per well in 200 pl Eagle's Minimal Essential Medium (MEM) supplemented with fetal calf serum (FCS) (io%), penicillin and streptomycin (ioo u/ml each) and buffered with bicarbo-
air. After overnight attachment, medium was replaced with MEM +2% FCS containing appropriate mixtures of LPS and PB in groups of six wells per concentration. The medium was removed after 5 days incubation and the cells fixed in methanol for 5 mins. Cell numbers were quantified by the methylene blue staining method of Currie (I98I): the cells were stained with I00 1l O.I% methylene blue in I0 mM borate buffer, pH 8.5, for 30 min and rinsed three times with 400 pui of the same buffer. The methylene blue was eluted from the cell layers with I00 ,l o.i M HCI containing 20% ethanol, and absorbance measured at 650 nm in an automated multichannel spectrophotometer (Titertek Multiskan, Flow Laboratories, UK) using the first column of wells which had contained culture medium alone as a reference blank. The linear relationship between methylene blue retention and the number of fibroblasts per well has been demonstrated elsewhere (Harvey et al. I985).
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Fig. i. The effects ofLPS from H. actinomycetemcomitans on bone resorption and bone collagen synthesis in vitro. Resorption of halved calvaria from 5-day old mice was measured colorimetrically by release of calcium into the culture medium over 3 days. Collagen synthesis was measured by incorporation of 3Hproline into pepsin-resistant native collagen during the last 6 h of a 24 h incubation. Points represent mean values and vertical lines the standard error of the mean. *Indicates significant difference from control values, P< o.os.
W. Harvey et al.
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Inhibition of LPS-induced bone resorption by polymyxin B (PB). Different concentrations of PB Mg/ml LPS before incubation with calvaria (six per group) for 3 days. PGE2 (i ,UM) and parathyroid hormone (PTH, 0.5 u/ml) were also used to stimulate resorption and demonstrate the specificity ofPB for the inhibition ofLPS-induced resorption. *, Spontaneous release of calcium in control calvaria incubated with culture medium alone; PGE2; 0, PTH; 0, LPS. Each point represents the mean value of six cultures. *Significantly reduced resorption compared with LPS, PGE2 or PTH alone, P < 0.0 5.
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Fig. 3. Reversal by PB of LPS-inhibited bone collagen synthesis. PB at the concentrations indicated was mixed with LPS (io ig/ml) before culture with calvaria for 24 h; collagen synthesis was measured as described in Fig. and in the text. *Significantly different from cultures incubated with pg/ml LPS alone, P
