Medizinische Fakultät Zahnmedizinische Kliniken Klinik für Parodontologie

Klinik für Parodontologie, Freiburgstrasse 7, CH-3010 Bern

Prof. Dr. Anton Sculean

Klinikdirektor

Freiburgstrasse 7 CH-3010 Bern

Tel.: +41 (0)31 632 25 89

Fax: +41 (0)31 632 49 15

www.zmk.unibe.ch

In vitro-efficacy of oily calcium hydroxide suspension on human

alveolar osteoblasts, periodontal ligament fibroblasts and

microorganisms

S. Eick, T. Strugar, S. Ruggiero, R. J. Miron, A. Sculean

Department of Periodontology, Dental School, University of Bern, Bern, Switzerland

Running title: OCHS and host cells and bacteria

Sigrun Eick, Department of Periodontology, Laboratory of Oral Microbiology, Dental

School, University of Bern, Freiburgstrasse 7, CH-3010 Bern, Switzerland

Tel.: +41 31 632 2542

Fax: +41 31 632 8608

e-mail: sigrun.eick@zmk.unibe.ch

Key words: oily calcium hydroxide suspension, human alveolar osteoblasts,

periodontal ligament fibroblasts, periodontopathogens

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Abstract

Background and Objective: Nowadays, several methods have been established for

the complete reconstruction of the periodontium for periodontitis treatment. Recently

an oily calcium hydroxide suspension has been introduced to favour the tissue

regeneration. The purpose of this in vitro study was to investigate the effect of an

oily calcium hydroxide suspension (OCHS) on attachment and proliferation of

osteoblasts and periodontal ligament fibroblasts as well as periodontopathogenic

bacteria.

Methods: Human alveolar osteoblasts (HAO) and periodontal ligament (PDL)

fibroblasts were cultured on OCHS. Adhesion and proliferation were counted up to 48

h and mineralization was assayed after 1 and 2 weeks. Furthermore antimicrobial

efficacy as well as the influence of infected PDL cells and OCHS on the HAO counts

were determined.

Results: More than a 2-fold increase in adherent HAO cells was observed at 4 h

following application of OCHS when compared to the control group. Proliferation of

HAO cells at 48 h was stimulated by moderate concentrations of OCHS, whereas a

high concentration of OCHS was inhibitory. Mineralization was observed only for

HAO cells treated with OCHS. For PDL fibroblasts, OCHS did not exert any positive

effect on attachment or proliferation. PDL fibroblasts infected with

periodontopathogens negatively influenced the proliferation of osteoblasts

independent of OCHS application.

Conclusions: The data suggest that OCHS promotes osteoblast attachment,

proliferation and mineralization in a concentration-dependent basis. OCHS does not

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have any significant influence on attachment of PDL fibroblasts.

In addition periodontopathogens may negatively influence the

proliferation of osteoblasts.

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Pathogenesis of periodontitis is thought to be a response of the host to microbial

plaque. 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 (1). Moreover, other species such as

Campylobacter rectus, Eubacterium nodatum, Fusobacterium nucleatum, Prevotella

intermedia, Parvimonas micra, Streptococcus constellatus support pathogenesis of

disease (1, 2). Bacteria interact with host cells resulting in expression of inflammatory

mediators which are capable of stimulating bone resorption and soft tissue

destruction via activation of matrix-metallo-proteases (3).

Resolution of inflammation and arresting disease progression is the minimal goal of

periodontal and periimplant treatment. Nowadays, the complete reconstruction of the

periodontium is the desired outcome. Several methods have previously been

introduced including guided tissue regeneration using non-resorbable and resorbable

membranes (4, 5). In combination with guided tissue regeneration or alone, several

compounds were proven to promote new bone formation. The application of enamel

matrix proteins to intrabony defects has been an established treatment regimen for

many years (6-8). Furthermore, bone grafts (9, 10) bone substitute materials (11, 12)

and growth factors (13-15) have also been successfully utilized.

Recently an oily calcium hydroxide containing paste (Osteora®, before

Osteoinductal®, DFS-Diamon GmbH Riedenburg, Germany) was introduced.

Calcium hydroxide (Ca(OH)2) which is a white odourless powder with a low solubility

in water has antibacterial properties by the release of highly reactive hydroxyl ions in

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aqueous fluids which damages cytoplasmatic membranes,

proteins and DNA (16). In endodontic treatment it is used as a

pulp-capping agent (17), as a disinfectant during the root canal treatment (18) and for

apexification after pulp death (19). In the pulp, a superficial necrosis induced by the

high pH occurs with a mild inflammatory response and hard tissue formation in the

environment (20).

The calcium hydroxide containing paste uses as a carrier substance synthetically

produced porcine oleum pedum and vaselinum album. In several animal models,

studies of this oily calcium hydroxide suspension (OCHS) resulted in different

outcomes. In a guided tissue regeneration model in the calvaria of minipigs, OCHS

failed to exert osteoinductive properties (21). Application during the osteotomy phase

of distraction osteogenesis in a rabbit mandible increased regeneration and new

bone formation (22). In dogs, OCHS favoured periodontal regeneration when used

after access flap surgery of intrabony periodontal defects (23). In rats, it hampered

bone healing when applied with guided bone regeneration (24). Similarly, in a dog

model the healing process of endosseous implants was retarded after application of

OCHS (25).

In the few clinical studies, varying outcomes were also reported. OCHS improved

early wound healing after non-surgical therapy (26). In a study on access flap

surgery, higher pocket depths reductions and clinical attachment level gains were

found (27), whereas in a similar study OCHS failed to demonstrate any superior

outcome when compared with open flap surgery alone (28).

Despite the clinical use of OCHS, knowledge about the mode of action remains

limited. The purpose of the scheduled study was to determine the effect of OCHS on

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osteoblasts and periodontal ligament fibroblasts cell behavior as

well as on periodontopathogenic bacteria.

Material and methods

Test substances

OCHS (Osteora®, DFS-DIAMON GmbH, Riedenburg, Germany) was used.

According to manufacturer’s information it is composed of 20% w/w Ca(OH)2, 40%

oleum pedum and 40% vaselinum album. Three different concentrations (1 U, 2 U, 3

U) were chosen. 1 U represents 2.5 mg of total material. As controls, calcium

hydroxide in aqueous solution (1.5 mg corresponding to 3 U of OCHS) and oleum

pedum substance (3 mg corresponding to 3 U of OCHS) were used. OCHS was also

tested at higher concentrations (50 mg, 100 mg) to determine a possible

antimicrobial effect.

Cells

Human alveolar osteoblasts (HAO) were obtained from three periodontally healthy

patients during surgery (extraction of teeth for orthodontic reasons). Following colla-

genase digestion, cells were plated in T-75 flasks containing cell cultivation medium

(DMEM, Invitrogen, Carlsbad, CA) supplemented with 10% of fetal bovine serum

(FBS, Invitrogen).

Human periodontal ligament (PDL) fibroblasts were collected from three periodontally

healthy patients during surgery (extraction of teeth for orthodontic reasons). PDL

cells were harvested from the middle third portion of tooth and placed in T-25 cell

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culture flasks till cell confluency. The cultivation medium was

DMEM supplemented with 10% FBS.

Using the tissue for research purposes was approved by the Ethical commission of

the Canton Bern. All patients gave their consent.

Microorganisms

The following species have been tested for antimicrobial assays: F. nucleatum ATCC

25586, P. intermedia ATCC 25611, P. gingivalis (ATCC 33277 and three clinical iso-

lates), T. forsythia ATCC 43037, A. actinomycetemcomitans (Y4 and three clinical

isolates), C. rectus ATCC 33238, Eikenella corrodens ATCC 23834, E. nodatum

ATCC 33099, P. micra ATCC 33270, and Capnocytophaga gingivalis ATCC 33624.

All strains were precultivated 42 h before experiments. Modified tryptic soy agar (29)

was used as cultivation media.

A. actinomycetemcomitans Y4 as well as P. gingivalis ATCC 33277, T. forsythia

ATCC 43037 and T. denticola ATCC 35405 in combination were used in the experi-

ments which analyzed the effect of OCHS on microorganisms in interaction with host

cells.

Determination of effect of oily calcium hydroxide suspension on osteoblasts

Slides were placed into 24-well plates and covered with the test substances (1-3 U

OCHS, 1.5 mg Ca(OH)2 and 3 mg oleum pedum). Immediately thereafter, HAO cells

were added at a density of 10,000 cells / well and the wells were incubated at 37°C

with 5% CO2. Cells were fixed and stained for adhesion and proliferation experiments

at 2 h, 4 h, 24 and 48 h using DAPI staining. Cell differentiation was analyzed by

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determination of alkaline phosphatase activity and

mineralization by using 2% alizarin red S staining 1 and 2

weeks after starting experiments. Each 10 fields of 1 mm2 were counted. A counting

grid was used and each field (50 µm × 50 µm) with positive staining was counted; the

mean was used as a single value for analysis.

Further, supernatants were collected at 24 h and 48 h and the levels of released

active TGF-β1 were determined by using ELISA-kits (R&D Systems Europa Ltd.,

Abingdon, UK) according to the manufacturer’s description. The detection level of the

kit was 50 pg/mL.

Determination of effect of oily calcium hydroxide suspension on PDL

fibroblasts

Similarly to HAO cells, effects on PDL fibroblasts were determined. Slides which

have been placed into 24-well plates were covered with the test substances. PDL

fibroblasts were added at a density of 10,000 cells / well. Cells were fixed and

stained for adhesion and for proliferation experiments at 2 h, 4 h, 24 h and 48 h using

DAPI staining as described for HAO cells.

Supernatants were collected 4, 24 and 48 h after starting the experiments. In the

supernatants, the levels of released active transforming growth factor (TGF)-β1 and

matrix metalloproteinase (MMP)-1 were determined by using ELISA-kits (R&D

Systems Europa Ltd.) according to the manufacturer’s description. The detection

level of the MMP-1 kit was 5 pg/mL.

Determination of antimicrobial efficacy of oily calcium hydroxide suspension

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A modified agar diffusion method was used due to the

insolubility of OCHS. One hundred µl of bacterial suspension

(MacFarland 0.5) were spread on agar plates (Wilkins Chalgren agar supplemented

with 5% blood). Then, each two gaps were prepared using a cork borer (diameter 7

mm). After that, the gap was filled with 100 µL of agar followed by the test substance

(50 mg and 100 mg of OCHS). After incubation at 37°C in the anaerobic atmosphere

for 42 h, the inhibition zones were measured. To exclude a growth-promoting effect

of OCHS, suspensions of selected bacterial strains (P. gingivalis ATCC 33277, P.

gingivalis M5-1-2, A. actinomycetemcomitans Y4 and F. nucleatum ATCC 255866)

were added to 200 µL nutrient broth added with 50 mg of OCHS (20%w/v). Tubes

had been incubated for 24 h anaerobically. Immediately before removing 25 µl of

mixture, suspensions were mixed by vortexing and short centrifugation at 400 g. The

removed 25 µl were serially diluted and each 100 µl were plated on agar plates. The

numbers of viable bacteria were determined by counting the colony forming units

(cfu). All experiments were performed in duplicates.

Determination of the effect of oily calcium hydroxide suspension on

osteoblasts and PDL fibroblasts interaction with microorganisms

The concentration of 2 U (5 mg) OCHS was selected and placed on each well of a

24-well plate for experiments focusing on the interaction of host cells with bacterial

strains.

HAO cells and PDL fibroblasts respectively were seeded on the slides with and

without coverage of 2 U of OCHS. A. actinomycetemcomitans Y4 as well as the

combination of P. gingivalis ATCC 33277, T. forsythia ATCC 43037 and T. denticola

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ATCC 35405 were added. The bacterial load was always 106

per well. The HAO cells and PDL fibroblasts respectively were

fixed and stained at 4 h.

Additionally HAO cells were exposed to supernatants of PDL fibroblasts on or without

OCHS and infected with bacteria. For that purpose, 2 U of OCHS were added to

each one well of a 24-well-plate. Then PDL fibroblasts were placed to that and

another plate without OCHS. In part PDL fibroblasts were infected with A.

actinomycetemcomitans Y4 and the combination of P. gingivalis ATCC 33277, T.

forsythia ATCC 43037 and T. denticola ATCC 35405 as described before. The cell

cultivation medium was DMEM without FBS. After 4 h incubation at 37°C,

supernatants were collected and sterile filtered. HAO cells were seeded on plates

and treated with the filtered supernatants of PDL cells mixed 1 : 1 with DMEM and

10% FBS. As made before, cells were fixed and stained for adhesion experiments at

4 h and for proliferation experiments at 48 h using DAPI staining.

Statistical analysis

Except for the antimicrobial assays at least six independent experiments were made

per group. Statistical analysis was made by using Student’s t-test for independent

samples and one way ANOVA test followed by Post Hoc LSD analysis.

Results

Effect of oily calcium hydroxide suspension on osteoblasts

Addition of OCHS promoted the attachment of HAO cells. After 2 h, 11.00 ± 4.90

cells / mm2 adhered to a surface covered with 2 U of OCHS. Using 3 U the value was

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11.67 ± 2.89 cells / mm2. These differences were significant in

comparison to the control, where 6.57 ± 3.55 HAO cells were

found per mm2 on the surface. After 4 h, 20.33 ± 14.13 HAO cells / mm2 were

counted after coverage with 1 U of OCHS and 18.11 ± 6.66 HAO cells / mm2 when 3

U of OCHS were used being significantly different from the controls (8.67 ± 3.50 HAO

cells / mm2). Stimulated proliferation was found 48 h after starting the experiments

and coverage with 1 U and 2 U of OCHS (1 U: 46.00 ± 13.11 HAO cells / mm2, 2 U:

46.67 ± 18.23 HAO cells / mm2 in comparison to control: 26.89 ± 7.25 HAO cells /

mm2). In contrast to the 1 U and 2 U of OCHS, the high concentration of 3 U

significantly inhibited the proliferation of HAO cells (13.44 ± 5.20 HAO cells / mm2).

An influence of the used Ca(OH)2 concentration was not registered. Coverage with

oleum pedum was followed by a decreased cell number 24 h after addition of the

cells. The results are presented in Figure 1.

Twenty-four hours after applying HAO cells to OCHS (all three used concentrations)

the amount of released TGF-β1 was significantly reduced, whereas after 48 h the

level of TGFβ-1 tended to increase in comparison to the control (Figure 2).

The differentiation and mineralization of the HAO cells was also analyzed. In all

experiments only HAO cells positively stained for alkaline phosphatase were found.

The mineralization was measured by counting the calcium noduli (stained area in %

of the whole area) 1 week and 2 weeks after beginning the experiments. To ensure

that only mineralization of cells was counted, OCHS with no cells was used as a

negative control. Without addition of OCHS, no mineralization was present on the

surface. When the surface was covered with OCHS, extracellular mineralization of

HAO was detectable after one week (5.57±1.97% stained area; Figure 3). The

quantity did not significantly change after two weeks (4.12±1.59%).

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Effect of oily calcium hydroxide suspension on PDL fibroblasts

Addition of OCHS did not have any significant influence on attachment of PDL

fibroblasts. Reduced proliferation was found 48 h after starting the experiments and

coverage with 3 U of OCHS (13.22 ± 4.12 PDL fibroblasts / mm2) in comparison with

the controls (33.89 ± 17.48 PDL fibroblasts / mm2). In contrast, Ca(OH)2 stimulated

the proliferation of PDL fibroblasts (53.44 ± 15.22 PDL fibroblasts / mm2 compared to

control values at the 48 h time-point (Figure 4).

Four hours after applying PDL fibroblasts to 2 U of OCHS the amount of released

TGF-β1 was significantly increased, whereas after 48 h the level of TGFβ-1 was

reduced in comparison with the control (all three used concentrations of OCHS;

Figure 5). MMP-1 was not detectable 4 h after beginning the experiments. After 24 h,

all used concentrations of OCHS elevated significantly the levels of MMP-1 in the

supernatants. This effect was still visible after 48 h, but only for the two higher

concentrations of OCHS (Figure 5).

Antimicrobial efficacy of oily calcium hydroxide suspension

Experiments using modified agar diffusion technique did not reveal any inhibition

zone by OCHS in the two tested concentrations. Final experiments using selected

species underline that OCHS does not influence growth of periodontopathogens.

Neither growth-suppressing nor growth-promoting effects were visible; differences to

controls were always below 0.2 log10-stages (data not shown). It has to be noticed

that due to the insolubility of OCHS the tubes contained two layers, one of OCHS

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and one of broth; thus only at the interface compounds released

from OCHS might interfere with bacteria.

Effect of oily calcium hydroxide suspension on osteoblasts and PDL

fibroblasts in interaction with microorganisms

Surprisingly, the addition of bacteria enhanced the numbers of attached HAO cells

(A. actinomycetemcomitans Y4: 16.00 ± 4.44 HAO cells / mm2, P. gingivalis, T.

forsythia, T. denticola in mixture: 14.56 ± 4.07 HAO cells / mm2) being significantly

higher than the controls (10.87 ± 8.43 HAO cells / mm2). Otherwise, the addition of

bacteria did not significantly change the numbers of adhered HAO cells when the

slides were covered with 2 U of OCHS. If A. actinomycetemcomitans Y4 was present,

the difference of numbers of HAO cells with 2 U of OCHS (25.08 ± 10.04 HAO cells /

mm2) was still significant in comparison with those without OCHS (controls). Contact

with bacteria did not significantly influence the numbers of attached PDL fibroblasts

(Figure 6).

Most supernatants of PDL fibroblasts cultured on OCHS tended to promote

attachment and proliferation of HAO cells, the difference was significant for all tests

(p=0.015) and for A. actinomycetemcomitans at the 4 h time-point. Infection of PDL

fibroblasts with A. actinomycetemcomitans did not influence the numbers of attached

HAO cells. In contrast, the infection of PDL fibroblasts with the combination of P.

gingivalis, T. forsythia and T. denticola remarkably decreased the HAO cell counts at

the 48 h time-point (Figure 7).

Discussion

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OCHS is an oily suspension which contains Ca(OH)2 as the

active constituent. Other components are synthetically produced

oleum pedum tauri as the carrier material which contains triglycerides including

oilacid, palmitin acid, hexadecen acid and vaselinum album.

Seeding of HAO cells on OCHS affected their attachment and proliferation. Our

results indicate concentration sensitivity. Low to moderate concentrations of OCHS

acted clearly stimulatory, whereas after application of the highest used concentration,

in part detrimental results were seen. To retard release of Ca(OH)2, OCHS contains a

high percentage of oleum pedum. The HAO counts were reduced after placement on

oleum pedum. 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 (24). It can be suggested that the

missing degradable properties are certainly based on the oleum pedum. This seems

to be of more importance with increasing concentrations. This obvious sensitivity of

the used concentration might explain the reported different outcomes in animal and

clinical studies. According to our first results, a moderate concentration was chosen

for ongoing experiments. Using this concentration, mineralization was found 1 – 2

weeks after exposing osteoblasts to OCHS. Recently it was demonstrated that

Ca(OH)2 is able to stimulate mRNA expression of bone sialoprotein and Runx2 (30).

Runx2 is an essential transcription factor in osteoblast differentiation (31). Bone

sialoprotein is known to mediate cell attachment and promote bone formation and

mineralization (32).

OCHS did not exert any positive effect on attachment and proliferation of PDL

fibroblasts. Similarly to osteoblasts, the highest used concentration reduced

proliferation of PDL fibroblasts after 48 h. This was opposite to calcium hydroxide in

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aqueous solution. Proliferation of PDL fibroblasts induced by

Ca(OH)2 was described recently; but in contrast to us, OCHS

also had a stimulatory effect on proliferation in that study (33).

TGF-β1which belongs to the TGF-β family is secreted in an inactive form; it is

activated by plasmin, thrombospondin, thrombin and MMPs (34, 35). TGF-β1 is a

multifunctional cytokine, it promotes biosynthesis of extracellular matrix in fibroblasts,

controls physiological processes during wound and bone healing and promotes

survival of myofibroblasts (34, 35). HAO cells released significantly less active TGF-

β1 after attachment when pretreated with OCHS, by trend this effect was weakened

48 h after the beginning of the experiments. Our first determination was at 4 h after

exposure to OCHS which may influence the results. In another study, 5 minutes after

treatment of bone surfaces with calcium hydroxide, bone cells secreted more TGF-

β1, but already after 15 min the levels of TGF-β1 returned to those without treatment

(36). In contrast, PDL fibroblasts released more TGF-β1 at the beginning, the effect

became opposite after 48 h in our study. MMP-1 was not detected initially. 24 h and

48 h after beginning, the levels of MMP-1 were increased when PDL fibroblasts were

cultured on CHOS. This was contradictory to the TGF-β1 levels and confirms findings

about reduced expression of MMP-1 in fibroblasts characterized by elevated TGF-β1

expression (37). MMP-1 is able to degrade fibrillar collagens and other matrix

molecules (38). The high initial amount of MMP-1 may support tissue remodeling.

Supernatants of PDL fibroblasts pretreated with OCHS promoted the attachment and

proliferation of HAO cells. This might be caused in part by the released calcium

hydroxide together with high amount of TGF-β1 in the environment of PDL fibroblasts

exposed to OCHS. Calcium hydroxide in aqueous solution was found to provide a

favorable environment for the anabolic effect of TGFβ1 on osteoblasts (39).

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An antibacterial effect by OCHS even in extremely high

concentration was not found by using a modified agar diffusion

test. The result was confirmed by using liquid medium for selected species. Calcium

hydroxide is widely used in endodontic treatment. Also there, a limited antimicrobial

efficacy was described analyzing root canals after treatment in vivo (40, 41). Only

one other study reported the effect of OCHS on periodontopathogens. Two A.

actinomycetemcomitans strains were selected; OCHS did not exert an antibacterial

effect against the ATCC 43718 strain (serotype b) (42). This strain is the Y4 strain

which was included in our study. In our study also one clinical isolate belonged to

serotype c. In contrast to the results described before (42), we did not find any

antibacterial effect. Surprisingly, the addition of bacteria enhanced the number of

attached HAO cells. The cell cultivation medium contained serum. Serum may

provide receptors for attachment of bacteria and the bacteria themselves may serve

as a receptor for HAO cells. Coverage with OCHS neglected that effect. Interestingly,

even in the presence of A. actinomycetemcomitans OHCS stimulated the attachment

of HAO cells. Also infection of PDL fibroblasts with that species did not affect the

later attachment and proliferation of HAO cells independently if the PDL fibroblasts

were exposed to OCHS or not. A. actinomycetemcomitans stimulates the mRNA

expression of inflammatory cytokines differently, it induces mRNA expression of IL-6

and IL-8 but not of IL-1β (43), moreover capsular polysaccharide of A.

actinomycetemcomitans inhibits the release of interleukin (IL)-6 and IL-8 (44). On the

other hand capsular-like polysaccharide antigen of A. actinomycetemcomitans

induces apoptotic cell-death in osteoblastic cells (45).

Otherwise, P. gingivalis, T. forsythia and T. denticola in combination were selected.

These three species represent the so called “red complex”; they are highly

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proteolytic, occur together in the late plaque and therefore in

closest contact with the adjacent tissue (46). Treatment of

osteoblasts with supernatants of infected PDL fibroblasts did not influence

attachment of HAO cells but it inhibited remarkably proliferation of HAO cells

independently if the PDL fibroblasts were exposed to OCHS or not. Sterile filtered

supernatants in the environment of infected PDL fibroblasts might contain secreted

virulence factors of bacteria. E.g., secreted proteases may influence proliferation of

HAO cells. Gingipains responsible for the majority of the proteolytic activity of P.

gingivalis (47), are able to destroy osteoprotegerin (48) and can inhibit proliferation of

osteoblasts by causing early G1 arrest in cell cycle (49). A T. forsythia protease has

been characterized recently (50), no data about a direct influence on osteoblasts are

available up to now. An outer membrane protein of T. denticola was shown to inhibit

osteoprotegerin expression (51). Further, periodontopathogens stimulate the release

of inflammatory cytokines from gingival fibroblasts which may affect proliferation of

osteoblasts. Viable P. gingivalis induce a strong mRNA expression of IL-1β, IL-6 etc.

(52). Thus, elimination of proteolytic pathogens is essential in treatment of

periodontitis. The missing antimicrobial activity of OCHS suggests the additional use

of an antimicrobial, such as chlorhexidine. In combination with a calcium hydroxide

paste 0.4% chlorhexidine did not affect osteoblastic cell biology in vivo (53).

Although, our in vitro studies did not consider the complexity in vivo, they may

explain in part the different outcomes observed in animal and clinical studies. In

conclusion, OCHS promotes attachment, proliferation of osteoblasts and

mineralization of their tissue. The effect seems to be concentration-dependent and

suggests an application of a moderate concentration. OCHS did not have significant

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influence on attachment of PDL fibroblasts.

Periodontopathogens may negatively influence the proliferation

of osteoblasts.

Acknowledgement

We are grateful to Regula Hirschi and Marianne Weibel (Department of Periodontology, University of

Bern) for excellent assistance in performing in vitro assays. This study was funded by DFS-DIAMON

GmbH, Riedenburg, Germany. The authors declare that they have no conflicts of interest.

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Legends of figures

Fig. 1 - Attachment and proliferation of HAO cells (mean and SD) after coverage with

different amounts of oily calcium hydroxide suspension (OCHS) as well as Ca(OH)2

and oleum pedum

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Fig. 2 - Released TGF-β1 (mean and SD) in the supernatants of

osteoblasts after coverage the surface with different amounts of

oily calcium hydroxide suspension (OCHS)

Fig. 3 - Cell mineralization stained with alizarin red one week after seeding HAO

cells. The mineralization is visible by the pink color (), left untreated HAO cells,

right HAO cells seeded on OCHS

Fig. 4 - Attachment and proliferation of PDL fibroblasts (mean and SD) after

coverage with different amounts of oily calcium hydroxide suspension (OCHS) as

well as Ca(OH)2 and oleum pedum

Fig. 5 - Released TGF-β1 and MMP-1 (mean and SD) in the supernatants of PDL

fibroblasts after coverage with different amounts of oily calcium hydroxide

suspension (OCHS)

Fig. 6 - Attachment of HAO and PDL fibroblasts cells (mean and SD) 4 h after

coverage with and without oily calcium hydroxide suspension (OCHS) and addition of

A. actinomycetemcomitans Y4 as well as the combination of P. gingivalis ATCC

33277, T. forsythia ATCC 43037, T. denticola ATCC 35405

Fig. 7 - Attachment and proliferation of HAO cells (mean and SD) 4 h and 48 h after

exposure to supernatants of PDL fibroblasts cultured 8 h on 2 U of oily calcium

hydroxide suspension (OCHS) and infected with addition of A.

actinomycetemcomitans Y4 as well as the combination of P. gingivalis ATCC 33277,

T. forsythia ATCC 43037, T. denticola ATCC 35405

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0

10

20

30

40

50

60

2 h 4 h 24 h 48 h

Att

ach

ed H

AO

ce

lls /

mm

2

1 U OCHS 2 U OCHS 3 U OCHS Ca(OH)2 Oleum pedum control

p=0.015p=0.004

p=0.007p=0.032

p=0.016

p<0.001

p<0.001

p=0.009

Figure 1

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p=0.024

p=0.013

p=0.016

Figure 2

Figure 3

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0

10

20

30

40

50

60

70

2 h 4 h 24 h 48 h

Att

ach

edP

DL

fib

rob

last

s/

mm

2

1 U OCHS 2 U OCHS 3 U OCHS Ca(OH)2 Oleum pedum control

p=0.001

p=0.002

Figure 4

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600

650

700

750

800

850

900

950

1000

4 h 24 h 48 h

pg/

mL

TGF-β

1

0

100

200

300

400

500

4 h 24 h 48 h

pg/

mL

MM

P-1

1 U OCHS 2 U OCHS 3 U OCHS control

p=0.047

p=0.002p=0.010

p=0.003

p=0.001

p=0.023

p<0.001p<0.001

p<0.001

Figure 5

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0

10

20

30

40

50

controls A. actinomycetem. P.g., T.f., T.d.

Att

ache

d PD

L fi

bro

blas

ts /

mm

2

no OCHS 2 U OCHS

0

5

10

15

20

25

30

35

controls A. actinomycetem. P.g., T.f., T.d.

Att

ache

d H

AO

cel

ls /

mm

2

p=0.013p=0.003 p=0.017

p=0.015

Figure 6

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0

5

10

15

20

25

30

35

control A. a. P.g. + T. f. + T. d. control A. a. P.g. + T. f. + T. d.

4 h 48 h

Att

ach

ed H

AO

cel

ls /

mm

2

p=0.013

p=0.001

p=0.001

Figure 7