In vitro-activity of oily calcium hydroxide suspension on microorganisms as well as on human alveolar osteoblasts and periodontal ligament fibroblasts

Periodontitis is a bacterially induced chronic inflammatory disease, and an imbalance of innate immune-defence system markedly contributes to the destruction of the periodontium [1]. A small group of predominantly gram-negative anaerobic or microaerophilic bacteria within plaque is associated with initiation and progression of periodontitis. Organisms strongly implicated as etiologic agents of periodontitis include Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola[2]. Moreover, other species such as Campylobacter rectus, Eubacterium nodatum, Fusobacterium nucleatum, Prevotella intermedia, Parvimonas micra, Streptococcus constellatus support pathogenesis of disease [2, 3]. Bacteria interact with host cells resulting in expression of inflammatory mediators, transition of polymorphonuclear neutrophils to the gingival crevice [1, 4]. Host response contributes to tissue destruction and bone resorption with the main mechanism of the ratio of RANKL (receptor-activator of nuclear-factor-κB ligand) to osteoprotegerin [1].

It has been well documented that resolution of inflammation and stop of disease progression can be predictably obtained with nonsurgical and conventional surgical periodontal therapy [5]. The ultimate goal of periodontal therapy is however the regeneration of the tooth’s supporting structures lost due to periodontal disease and should result in formation of new root cementum, periodontal ligament and bone [6]. Treatment with barrier membranes alone or in combination with different grafting materials, the use of biologic active substances such as enamel matrix proteins or growth factors have been shown to promote periodontal regeneration and to significantly improve the clinical outcomes evidenced by probing depth reduction, clinical attachment gain and defect fill [7]. A few of the materials have been described to act antimicrobial, e.g., enamel matrix derivatives inhibit the growth of P. gingivalis[8]. A study analysing the influence of five different biomaterials used for regenerative periodontal surgery against two A. actinomycetemcomitans showed antimicrobial activity of two materials against one of the two tested strains, most inhibitory was an oily calcium hydroxide suspension [9].

This oily calcium hydroxide containing paste (OCHS) (Osteora®, previously Osteoinductal®, DFS-Diamon GmbH Riedenburg, Germany) has been suggested to possess properties which may positively affect periodontal wound healing/regeneration [10‐14]. It is based on calcium hydroxide (Ca(OH)₂) and uses a carrier substance consisting of synthetically produced porcine oleum pedum and vaselinum album. Calcium hydroxide, a white odourless powder with a low solubility in water, has antibacterial properties by the release of highly reactive hydroxyl ions in aqueous fluids which damages cytoplasmatic membranes, proteins and DNA [15]. In endodontic treatment it is used as a pulp-capping agent [16], as a disinfectant for root canal treatment [17] and for apexification after pulp death [18]. In the pulp, a superficial necrosis induced by the high pH occurs with a mild inflammatory response and hard tissue formation in the environment [19].

Several animal studies which have evaluated the effects of OCHS on bone regeneration in various types of defects yielded different outcomes [10, 11, 20, 21]. In a guided bone regeneration model using minipig calvaria, OCHS failed to exert osteoinductive properties and hampered bone healing when used in conjunction with guided bone regeneration [22]. Furthermore, the healing of endosseous implants was not improved when those were inserted together with OCHS [21]. On the other hand, application of OCHS during the osteotomy phase of distraction osteogenesis improved formation of new bone [10] while in experimentally created intrabony periodontal defects, the application of OCHS in conjunction with access flap surgery promoted periodontal regeneration [11].

Varying outcomes related to wound healing and regeneration were also found in the few clinical studies. In one study, OCHS improved early wound healing when used in conjunction with non-surgical therapy [12]. In another controlled clinical trial evaluating the healing of intrabony defects treated with access flap surgery with and without OCHS, significantly higher pocket depths reductions and clinical attachment level gains were found in the defects treated filled with OCHS compared to the controls (i.e. access flap surgery alone) [13]. On the contrary, another recent controlled clinical study using a similar design has failed to demonstrate any superior outcomes following the application of OCHS when compared with access flap surgery alone [23].

Although much investigation has been obtained in animal and clinical models, knowledge about its mode of action and effects on periodontal ligament (PDL) cells, bone-forming osteoblasts as well as oral microbes is still limited. The aim of the present study was two-fold; 1) To determine a potential antimicrobial activity of OCHS including its components against bacterial species involved in pathogenesis of periodontitis and 2) to determine the effect on attachment and proliferation of host cells (periodontal ligament fibroblasts and osteoblasts).

OCHS is an oily suspension which contains Ca(OH)₂ as the active constituent. Other components are the synthetically produced oleum pedum tauri, a carrier material containing triglycerides including oil acid, palmitin acid, hexadecen acid and vaselinum album.

An antibacterial effect by OCHS was not observed even in extremely high concentrations by performing a modified agar diffusion test. Only one previous study reported on the effects of OCHS on periodontopathogens. In that study, OCHS acted inhibitory on A. actinomycetemcomitans strain ATCC 33384 (serotype c), however no antibacterial effect was observed following culture of A. actinomycetemcomitans strain ATCC 43718 (serotype b) with OCHS [9]. We also included in our study one clinical isolate belonging to serotype c. In contrast to the results described previously [9], we did not observe any antibacterial effect following application with OCHS in the present study on either tested strains of A. actinomycetemcomitans.

Due to the insolubility of the material, we were only able to determine the minimal inhibitory concentrations of individual components of OCHS by a micro-broth dilution technique. It was observed that although oleum pedum did not have any antibacterial property, the use of calcium hydroxide in high concentrations such as those present in OCHS acted as growth-inhibitory. Calcium hydroxide is used widely in endodontic treatment and in vitro evidence demonstrates it suppresses the growth of Candida albicans[28] and certain other anaerobically growing bacteria [29] although limited antimicrobial efficacy has translated in vivo following the analysis of root canals post-treatment [30, 31]. In our in vitro assays, in contrast to calcium hydroxide, OCHS did not have any antibacterial effect which may suggest a counteracting effect of the other components of OCHS to calcium hydroxide. Due to the nature of periodontal therapy, bacteria associated with periodontitis should be eliminated or reduced in any treatment modality. The missing antimicrobial activity of OCHS suggests the additional use of an antimicrobial, such as chlorhexidine. For these reasons, addition of 0.4% CHX to a calcium hydroxide paste did not affect osteoblastic cell biology in vivo [32].

In the present study, we investigated the effects of OCHS on HAO on cell attachment and proliferation. Our results demonstrated increased cell behavior following culture with OCHS and demonstrated concentration sensitivity. Low to moderate concentrations of OCHS acted clearly stimulatory, whereas the application with a concentration of 3U (7.5 mg) demonstrated in part detrimental results. To retard the release of Ca(OH)₂, OCHS contains a high percentage of oleum pedum. We investigated HAO counts cultured in the presence of oleum pedum and found a reduction in cell behavior follwing its application. Oleum pedum seems to counteract the stimulating activity of Ca(OH)_2. In an animal model using open Teflon capsules, OCHS inhibited bone formation and an active resorption of OCHS was not observed [20]. It can be suggested that the missing degradable properties are certainly from the oleum pedum and a more pronounced effect appears to be observed with increasing concentrations. A second logical explanation might be due to the cytotoxicity of OCHS. Although oleum pedum prevents the cytotoxicity of Ca(OH)₂, toxic effects to a certain degree were seen when OCHS was used at high concentrations. This obvious sensitivity of the used concentration might partly explain the reported different outcomes in animal and clinical studies. Accordingly, following our first results sets of experiments, a moderate concentration was chosen for ongoing experiments based on its positive results. Using this concentration, an increase in osteoblast mineralization was found 1 – 2 weeks post seeding with culture media containing OCHS. Recently it was demonstrated that Ca(OH)₂ is able to stimulate mRNA expression of bone sialoprotein and Runx2 [33]. Runx2 is an essential transcription factor in osteoblast differentiation [34] and bone sialoprotein is a late marker for osteoblast differentiation found during the mineralization process of osteoblasts [35].

Despite the stimulatory effects of OCHS on osteoblasts, OCHS did not exert any positive effect on attachment or proliferation of PDL fibroblasts. Similarly to our HAO experiments, the highest used concentration of OCHS also reduced proliferation of PDL fibroblasts after 48 h. Proliferation of PDL fibroblasts was induced by Ca(OH)₂ as described recently; but in contrast to our results, OCHS also had a stimulatory effect on proliferation in that study [36]. In a similar study [14] these authors found high attachment of PDL fibroblasts on OCHS treated root surfaces. Future research to determine the mechanisms influencing the attachment of cells via integrin binding and its downstream molecular mechanisms in host cells (e.g. cellular pathways) should investigated.

Following experimental testing of OCHS with cells from the periodontium, a co-culture system was designed to determine the influence of OCHS on cells exposed to periodontal pathogens simultaneously to simulate a clinical setting. Exposure of HAO cells and PDL fibroblasts to periodontopathogenic bacteria did not negatively influence the promoting effect of OCHS on attachment and proliferation of these cells. Therefore, the results from this experiment demonstrate the potential use of OCHS even in the presence of periodontal pathogens without affecting the potential benefit from OCHS. Surprisingly, the addition of bacteria enhanced the number of attached HAO cells. This result should be taken with caution and might be associated with the in vitro culture conditions. The effect was especially pronounced when A. actinomycetemcomitans was used. The cytotoxic potential of periodontopathogens is well known. Gingipains are responsible for the majority of the proteolytic activity of P. gingivalis[37] and are able to inhibit proliferation of osteoblasts by causing early G1 arrest in cell cycle [38]. A capsular-like polysaccharide antigen of A. actinomycetemcomitans induces apoptotic cell-death in osteoblastic cells [39]. But recently it was shown that a low concentration of A. actinomycetemcomitans LPS in contrast to high concentrations was able to increase bone sialoprotein gene transcription [40]. A topic of future research might be the interfering and interaction of periodontopathogens with host cells and regenerative materials.

Although, our in vitro studies did not consider the complexity of an in vivo system, the results from the present study may partly explain the different outcomes observed in animal and clinical studies following use with OCHS. In conclusion, OCHS promotes attachment and proliferation of osteoblasts as well as mineralization of their tissue. The effects seem to be concentration-dependent and too high a concentration acts negatively, thus suggesting an application of a moderate concentration. Infection of PDL fibroblasts and osteoblasts with certain periodontopathogens did not negatively interfere with the attachment and early proliferation of osteoblasts and PDL fibroblasts. Nevertheless, the missing antimicrobial activity of OCHS suggests an additional use of an antimicrobial for clinical application. Further in vitro studies should be aimed to investigate the cellular pathways and mechanisms by which these interactions take place.