bacterial calcification: friend or foe? - j-stage

Sammons et al., Nano Biomedicine 2(2), 71-80, 2010

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Biomineralisation Biomineralisation is the process whereby living organisms harden organic matter within them-selves by the incorporation of minerals, as in the formation of the skeleton and teeth of verte-brates. Whilst such processes are clearly essen-tial to life, pathogenic calcifications also occur in a great number of human disorders including atherosclerosis, gall stones, nephrolithiasis, den-tal calculus and pulp stones, scleroderma, tym-panosclerosis, calcinosis cutis, calcific tenditis, synovitis and arthritis [1]. Calcification readily occurs in body fluids because calcium and phosphate ions are normally present in metasta-ble solution at concentrations too low for spon-taneous precipitation but high enough to cause calcium phosphate formation once nucleation has begun. If the solubility product of 75 mg/dl of CaHPO4 (i.e.[Ca2+] x [HPO4

2-]) is exceeded in physiological fluids, precipitation may occur if

inhibitors are absent and nucleation sites, such as calcium binding proteins and lipids are avail-able [2]. Several types of bacteria can provoke calcification by a variety of different mecha-nisms. These will be described briefly in the first part of this review, with reference to some well studied examples (Fig. 1). In the second part the mechanism of calcification by a species of Serratia is described and potential applications are discussed. Pathogenic calcifications involving bacteria Formation of bladder and kidney stones and urinary catheter encrustation. Certain types of bladder and kidney stones are formed due to the action of bacterial urease en-zyme. In urinary tract infections these include Proteus spp., especially P. mirabilis, Staphylo-coccus aureus, Klebsiella spp., Providencia spp., Pseudomonas spp. and the mycoplasma, Urea-

Bacterial Calcification: Friend or Foe?

Rachel SAMMONS1, Anqi WANG2, Ania THACKRAY1, Ping YONG 3, Yoshinori KUBOKI4, Akihiro AMETANI5,

and Lynne MACASKIE3

1 University of Birmingham School of Dentistry, 2 University of Birmingham School of Metallurgy and Materials,

3 University of Birmingham School of Biosciences, Birmingham, UK 4 Professor Emeritus, University of Hokkaido, Sapporo, Japan

5 HI-LEX Corporation Inc., Hyogo, Japan Synopsis Biomineralisation occurs in nature in many forms, some of which are beneficial to humans andsome detrimental. Biomineralisation leads to the formation of the skeleton but also to pathogeniccalcification in arteries, on heart valves, kidney stones and medical devices such as urinary cathe-ters. Several pathogenic calcifications are associated with bacterial activity. Can we exploit thiscapacity for our benefit? Serratia sp. NCIMB 40259 is a non-pathogenic Gram-negative bacteriumwhich is capable of growing as a biofilm on almost all surfaces. A bacterial cell-wall located acidphosphatase enzyme liberates phosphate ions from organic phosphates and these combine withCa2+ ions to form hydroxyapatite (HA) crystals. We discuss the potential use of Serratia HA formedical and other applications. Key words: biomineralisation, hydroxyapatite, Serratia, biofilm, biomaterials, water purification

REVIEW

Sammons et al., Bacterial Calcification: Friend or Foe? Nano Biomedicine 2(2), 71-80, 2010

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plasma urealyticum [3, 4, 5]. Urease breaks down urea into ammonium ions, CO2 and water, resulting in an increase in urine pH from 4.6-7.4 to 8.3-8.6, which favours the precipitation of carbonate-substituted hydroxyapatite (Ca 10 (PO4 , CO3) 6 (OH, CO3) 2 ) and ammonium-containing salts such as struvite (Mg (NH4) (PO4).6H2O) [3]. Associated with proteins and lipids, the deposits (Fig. 1a) accumulate and may obstruct the blad-der and kidneys, often causing severe pain [3, 4, 5]. The same process leads to encrustation of urinary catheters (Fig. 1b), a problem which has been the object of intensive research over the last 40 years. Some patients may respond to an-tibiotics targeting P. mirabilis but others are prone to rapid and chronic catheter encrustation where the only preventative measure is to in-crease citrate-containing fluid intake (reviewed by Hamil et al., 2007 [6] and Stickler and Feneley, 2010 [7]).

Dental calculus Dental calculus is essentially calcified plaque that adheres strongly to teeth and dental appli-ances [8] (Fig. 1c). It is composed of a hetero-geneous organic matrix consisting of carbohy-drates, small amounts of lipids and proteins in-cluding calcium-binding proteins such as osteo-pontin, and bone sialoprotein that are also asso-ciated with bone matrix and calprotectin [9-11]. The mineral phase consists of calcium carbonate and various phases of calcium phosphate [12]. Many different types of oral bacteria have been identified in calculus, but only a few can calcify in vitro. The most well-studied of these is Cory-nebacterium matruchotii, a filamentous Gram-positive organism commonly found in supragingival plaque where it may associate with certain species of streptococci forming ag-gregations known as “corn-cobs” [13]. The cal-cification process (Fig. 2a) occurs extracellularly and, after cell death, intracellularly (Fig. 2b,c). Acidic Ca-binding phospholipids in the cell membrane associate with specific proteolipids that are present only in calcifying bacteria to form a Ca-phospholipid phosphate complex (CPLX), which acts as a nucleation site for hy-droxyapatite formation (Fig. 2a) [14, 15, 16]. Ca2+ may be taken up by an ion exchange mechanism mediated by the same proteolipids which therefore have two functions: to partici-pate in the formation of CPLX and in pH regu-lating ion translocation, exchanging H+ (extru-sion) for Ca2+ (uptake) [14].

Calcification of dental plaque is initiated by nucleation on bacterial phospholipid bilayer vesicles released into the biofilm (analogous to matrix vesicles in calcifying cartilage in bone formation in vertebrates) and it is also promoted by certain elements in food, for example, silica, presumably because of its Ca-binding potential [17]. Calcification within dental plaque may also occur due to the development of alkaline condi-tions in microcosms as a result of the action of urease-producing bacteria (as described above) [18, 19]. In addition, calcified nanoparticles (CPN) that have been identified in human gin-gival fluid and proposed to be a contributory factor in periodontal disease [20] may play a role. Still controversial, CPN are reported to be bacteria-like, infectious, self-replicating particles

Fig. 1. Examples of pathological calcifications: a) kidneystones; b) encrustation of a urinary catheter; c) dentalcalculus at the base of the teeth.


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that contain 16S rRNA but lack DNA and are sensitive to some antibiotics. They have been isolated from deposits associated with a variety of non-skeletal calcification disorders including kidney stones (reviewed by Ciftiçioğlu and McKay, 2010) [1].

Calculus is not generally considered to be a serious clinical problem although subgingival plaque may contribute to periodontitis by inhib-iting epithelial reattachment [8]. However it has been suggested that calcifying oral bacteria could be involved in calcifications elsewhere in the body with more serious consequences: For example, Cohen et al. (2004) [21] demonstrated that rabbits infected with a very low dose of Streptococcus sanguinis and a high dose of C. matruchotii developed large calcifications on the heart valves. This did not occur when the two organisms were injected separately and it was proposed that the streptococcus attached to the valves first and then C.matruchotii attached to them. In so doing oral bacteria may play a role in calcifying aortic stenosis, a disorder af-fecting approximately 3% of the US population over 75 years of age [22].

Biomineralisation by a species of Serratia Serratia sp. NCIMB 40259 is a non-pathogenic Gram-negative bacterium that was originally isolated from heavy-metal contaminated soil in Cheshire, England. Scientists were seeking bac-teria that were resistant to cadmium (Cd), with a view to obtaining strains suitable for removing this highly toxic element from industrial wastes. In laboratory tests the bacterium which proved most efficient at removal was Citrobacter N14 [23]. This bacterium was subsequently reclassi-fied, following 16S rRNA sequence analysis and biochemical studies, as a species of Serratia and is now known as Serratia NCIMB 40259 [24].

The mechanism by which metals are accu-mulated by Serratia 40259 involves an acid phosphatase enzyme located within the cell wall periplasmic space and attached to the fimbriae and extracellular polymeric material (EPM) that surround the cell [25]. The enzyme cleaves or-ganic phosphates, such as glycerol 2-phosphate (G2P), liberating inorganic phosphate, which combines with metal ions to produce metal phosphates [26, 27, 28]. These precipitate ex-tracellularly within the EPM, that provides the

Fig. 2 Calcification in Corynebacterium matruchotii. a) Proposed mechanism of calcification, adapted from[14], with reference to [15] and [16]. Calcium ions are taken up by the cell in exchange for H+ ions andtranslocated by ion-transport proteolipids to the inner side of the membrane where the proteolipids forma complex with Ca-phospholipids ; Ca2+ ions associate with phosphate groups to form HA. b) SEMimage showing HA crystals forming on C. matruchotii grown in vitro; c) TEM image of cells from thesame culture showing crystal formation throughout the cells after death.


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nucleation sites for crystal formation and re-stricts crystal growth [28]. Interestingly, the ini-tial event involves coordination of incoming metal ion to phosphate groups within the lipopolysaccharide [28] in an analogous way to the biomedically-important biomineralization described above but without Ca2+ transporters having been directly linked to biomineralisation. This Serratia strain can be used to remove heavy metals from contaminated water either by the production of metal phosphates, as described above, as in the case of uranium phosphate [28, 29] or by ion-exchange intercalation into the pre-formed biogenic uranium phosphates, as in the case of nickel [30, 31] and radionuclides such as 90Sr, 137Cs and 60Co [32]. Formation of hydroxyapatite by Serratia Calcium phosphates, especially HA and deriva-tives, are used extensively as biomaterials in dental and orthopaedic applications as bone-graft substitutes and as coatings for metal-lic prostheses such as hip and knee replacements, to promote osseointegration without the need for cement. The more soluble β-tricalcium phos-

phate (β- TCP) is often used in combination with HA to promote osseointegration whilst HA pro-vides stability during healing. When pre-grown Serratia bacteria are presented with calcium ions from calcium chloride and a source of organic phosphate (G2P), crystals are produced (Fig. 3) that have been shown by X-ray diffraction, se-lected area diffraction and FTIR to consist of Ca-deficient HA with a Ca/P ratio of 1.65 + 0.09, or lower, dependent on the mineralisation condi-tions: Mineralisation at pH 8.6 yields calcium deficient HA which may either sinter (at 1,100-1,200°C) to β-TCP [33], or remain as Ca-deficient HA [34], depending on the initial Ca/P ratio, determined primarily by the concen-tration of G2P and CaCl2 in the mineralisation medium. Crystals made at pH 9.2 in the pres-ence of citrate sinter to HA [35]. As in many biological apatites, the absence of the typical HA O-H stretching peak in the FTIR spectrum sug-gests that the HA crystal lattice is defective [34]. Na+ and Cl- ions are present at about 1% in the original crystals and remain as impurities in the final sintered product [35, 36].

Fig. 3 HA crystal production by Serratia NCIMB 40259. Crystals are produced in the presence of anorganic phosphate (G2P) and calcium chloride. A phosphatase enzyme in the bacterial cell wallcleaves the phosphate, liberating HPO42- ions that then combine with Ca2+ ions to form ex-tracellular HA crystals. b) higher magnification of the needle shaped crystals; c) selected areadiffraction pattern from b) showing rings corresponding to HA. The partial rings indicate thatthere may be a preferred crystal orientation.


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Bio-fabrication of coatings and scaffold mate-rials To manufacture HA, Serratia cells are first grown as a biofilm on a support material in an air-lift fermenter under lactose-limiting condi-tions, when the acid phosphatase enzyme activ-ity is up-regulated and the production of fim-briae is induced [37]. These hair-like cell wall appendages enable bacteria to attach to solid surfaces and, in the case of Serratia, this in-cludes glass, plastics, ceramics and metals [36]. They also have numerous flagellae that enable them to swim into pores and internal cavities and hence to form a biofilm on substrates with complex shapes [33, 35, 36]. After a few days (depending on the desired thickness of the sub-sequent mineralised coating) the biofilm-coated supports are transferred to buffered mineralisa-tion medium, containing calcium chloride and G2P, where HA crystals are produced by the ac-tion of the phosphatase enzyme. As the enzyme is stable for at least a year biofilm can be pre-grown and stored for future use. Because of the ability of the bacteria to form a biofilm on convoluted surfaces and subsequently mineralise,

the method is a potentially useful “non-line-of site” method for coating 3D structures with HA, unlike, for example plasma spraying, in which only surfaces in the direct line of fire are coated [38]. Using a micromanipulation technique, the adhesion and cohesion strength of HA-coated biofilm was found to be up to 40 times higher than native or dried biofilms on polypropylene [39]. Subsequent sintering at 1,100-1,200C to consolidate the HA crystals and form the ce-ramic destroys the bacteria and any potential pyrogens. Thermogravimetric analysis showed that all organic material was removed by ap-proximately 500º C [33].

If the bacteria are grown and mineralised on a combustible porous support such as polyure-thane foam, the sintered product is a porous “skeleton” of the original mineralised biofilm-coated foam with interconnected chan-nels (Fig. 4) and grains ranging in size from 0.5 – 10 μm. The surface replicates the original biofilm structure and its uniquely convoluted architecture presents a large surface area for cell attachment, supporting cell growth in vitro [33, 40].

Fig. 4 SEM images of sintered HA scaffold. a) 1cm3 scaffold produced by growing a Serratia biofilmon polyurethane foam, mineralising and then sintering. All organic material is burnt leaving thecalcium phosphate foam replica. b) higher magnification of one of the struts shows biofilmstructures preserved in the sintered material; the bacteria formed aligned chains wrapping aroundthe polymer. c) higher magnification of b) showing grain structure and porous surface.


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Potential Applications of Serratia HA The chemical composition of Serratia HA closely resembles that of bone [34] and the po-rous scaffolds are bone-like in appearance with inter-connecting channels and what appears to be an ideal surface for cell attachment. However, cubic scaffold specimens (10mm3) had a maxi-mum load at failure of only 12.12N (~0.12MPa) [36], which is too friable for clinical use. It may be possible to increase the strength by infiltra-tion with an organic matrix such as collagen. Alternatively the method could be used to coat a stronger, reinforcing support material. As an example, it has been used to coat the wires of a

three-dimensional titanium wire mesh (Ti web [41]; Fig. 5; Wang, unpublished). The bacteria penetrate into the structure, bridging gaps be-tween the wires and filling up much of the space within it. After sintering the construct in argon, to avoid growth of titanium oxide crystals and at 550° C for 3 hours and 800° C for 5 hours, to avoid exceeding the titanium beta transus tem-perature (882 °C), the calcium phosphate coating was retained on the wires (Fig. 6), as confirmed by energy dispersive Xray spectroscopy (EDX) (Fig. 6d-f). Further optimisation of the drying/ sintering conditions may be required to avoid the formation of cracks in the coating.

Fig. 5SEM images of Serratia HA on a titanium web(HI-LEX Corporation, Inc. Hyugo, Japan). Smallimage: low magnification view of uncoated wiremesh. The main image shows the wires completelycovered with mineralised bacterial biofilm. Occa-sional “naked” bacteria can be seen (arrows) butmost are covered in a thick crystalline coating. Sizebar = 20μm.

Fig. 6 SEM images of titanium web coated with Serratia HA after sintering in an Argon atmosphere. b) and c) show thatthe coating is retained but is cracked; d) is a back scattered electron image of resin-embedded web cross sectionedfor EDX mapping. The presence of Ca and P around the titanium wires shown by EDX e) and f) confirms thepresence of the coating on wires throughout the web.


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Serratia HA crystals are initially nanophase: Yong et al. [42] reported crystal dimensions of 15-25nm, from XRD results, smallest crystals being produced when citrate was added to in-hibit crystal growth by restricting the amount of free Ca2+ ions in solution. Once crystal forma-tion has commenced it continues without deple-tion of the enzyme and following “priming” with a small amount of organic phosphate, it has been used to prepare HA from inorganic phosphates in waste-water, depleting the phosphate levels and thus coupling the manufacture of a valuable product to an environmentally beneficial process [43-45]. As in other aqueous precipitation methods for the manufacture of HA, the Serratia biominer-alisation process readily permits the substitution of other ions into the HA crystal lattice. For example, by mixing SrCl and CaCl2 in the min-eralisation solution in various combinations, Ca-deficient HA crystals with corresponding amounts of strontium substitution are produced (Fig. 7). Strontium-substituted HA is potentially

beneficial to promote bone healing in patients suffering from osteoporosis [46,47].

As already mentioned, biogenic Serratia hydrogen uranyl phosphate has been promoted for use in water purification as an ion exchanger for remediation of water contaminated with ra-dionuclides. However the use of uranium in treatment of potable water is undesirable and HA may also be useful when used in this way [48]. In the case of biogenic HA this was ~ 7 times more efficient at removing Sr2+ from synthetic groundwater than commercial HA (attributed to the small nanoparticle size) and, furthermore, the biomaterial was more stable in terms of its dis-solution [49]. Further potential applications in-clude drug delivery, especially since some pro-teins show a greater avidity for poorly crystal-line calcium-deficient HA than for well-crystallised stoichiometric apatites [50]. In all of these applications the very large surface area and convoluted surface structure provided by the sintered Serratia mineralised biofilm (Figs 4 and 5) could be highly beneficial.

Fig. 7 Bar chart showing the composition of Sr-substituted hydroxyapatite formed by mineralisationin medium containing G2P and various mixtures of SrCl and CaCl2. XRD results (notshown here) confirmed the substitution of Sr into the crystal lattice. Substitution occurred inthe presence and absence of citrate; slightly highly levels of substitution are achieved in itsabsence.


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Summary Bacteria may induce the precipitation of calcium salts in the body by a variety of mechanisms and such calcifications are generally harmful. On the other hand, Serratia NCIMB 40259 biominer-alisation can be exploited, not only for recovery of toxic and precious metals and bioremediation of water supplies but to build three-dimensional hydroxyapatite structures with complex biofilm-generated architecture that can act as scaffolds for cell growth and could be developed for use as biomaterials to deliver drugs and cells for tissue repair. References 1) Ciftiçioğlu N, McKay DS, Pathological calci-

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Corresponding author: Dr. Rachel Sammons University of Birmingham School of Dentistry St Chad’s Queensway, Birmingham B4 6NN, UK E-mail: [email protected]

bacterial calcification: friend or foe? - j-stage

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