529
A
Quantitative Analysis of the Migration, Attachment, and Orientation of Human Gingival
Fibroblasts to Human Dental Root Surfaces in vitro 0. Fardai* and B.F.
Lowenbergf
The purpose of this study was to assess the migration, attachment, and orientation of human gingival fibroblasts to human dental roots over a period of 21 days in vitro. The fibroblasts were incubated with a total of 120 periodontally diseased and non-diseased root slices (200 µ thickness) which had been treated in the following manner: 1) Root planed diseased root (DT); 2) Root planed and citric acid demineralized diseased root (DTD); 3) Non-treated diseased root (DNT); 4) Citric acid demineralized diseased root (DNTD); 5) Non-diseased control (ND); 6) Citric acid demineralized non-diseased root (CA); 7) Citric acid and collagenase digested non-diseased root (CAC); 8) EDTA demineralized non-diseased root (E); and 9) EDTA-demineralized and collagenase-digested non-diseased root (EC). The results showed that the most active phase of cell attachment and orientation occurred during the first 10 days of the experiment. Statistical differences were observed between the variables, and, in terms of cell attachment and orientation to the root slices, it was concluded that: 1) Root planing improves diseased roots; 2) Acid
demineralization subsequent to root planing improved diseased roots to such an extent as to render them comparable to non-diseased roots; 3) Citric acid demineralization alone improved diseased roots to the same extent as root planed diseased roots; 4) The exposure of collagen fibrils resulting from acid demineralization of the roots is not the sole reason for the improvement of the root surface, but rather a combination of the exposed collagen fibrils with the creation of a more hospitable environment was found to be responsible. JPeriodontol 1990;61:529-535.
Key Words: Tooth root; fibroblasts; cell attachment.
periodontal therapy is to gain a full regeneration of the periodontium destroyed by Periodontitis. From experiments in vitro,1'2 fibroblast migration, attachment, and orientation seem to be important steps in the The ultimate
goal
of
formation of a new connective tissue attachment to the root surfaces. It is not unreasonable to assume, therefore, that following periodontal therapy similar behavior of fibroblasts is important in the regenerative process. Different root surface qualities are reported to influence fibroblast functions; e.g., endotoxins from periodontal pathogens may penetrate the root surface and have been implicated in inhibiting fibroblast proliferation, synthesis, and attachment.3-11 However, Fardai et al.12 reported that periodontally
'University of Toronto, Faculty of Dentistry, 124 Edward ON M5G 1G6. Current address, Egersund, Norway. 'University of Toronto.
Street, Toronto,
diseased roots did not inhibit initial attachment in vitro and Adelson et al.13 could not find any difference in fibroblast attachment, pattern of cell growth, cell migration, or cytotoxic reaction in vitro on curetted versus non-curetted periodontally diseased root surfaces. The use of acid as an adjunct to scaling and calculus removal is reported to have been used as early as in 1846.14 Partial surface demineralization by citric acid at pH 1 for 3 minutes15 may influence new periodontal attachment attempts by one or more of the following mechanisms:16 1) Accelerated cementogenesis; 2) Widening of the dentinal tubules to allow for connective tissue ingress; 3) Extracting endotoxin and other toxic plaque products; 4) Inducing mesenchymal cell differentiation to osteoblasts or possibly cementoblasts; or 5) Exposing root dentinal collagen. Garrett et al.17 suggested that newly synthesized collagen may
J Periodontol 530
FIBROBLAST ATTACHMENT AND ORIENTATION TO ROOT SURFACES
splice to the exposed root dentinal collagen fibrils, while Boyko et al.18 proposed that demineralized root surfaces with the exposed fibrils offer a more "hospitable" surface
for fibroblast attachment to tooth surfaces. More recent in vitro studies1-2 have shown an enhanced effect on the migration, attachment, and orientation of fibroblasts by treating the root surface with 18% EDTA (pH 7.4) for 30 minutes. This suggests that there is a greater chance of new connective tissue attachment to root surfaces which have been demineralized. A number of in vivo studies however, report no additional advantage of surface demineralization over root planing at least in terms of flap healing.19"21 The inconsistency of these studies may be due to differences in experimental systems and techniques. For example, in vivo studies are frequently compared with in vitro studies; surface demineralization of periodontally diseased roots has been compared with surface demineralization of non-diseased roots, often utilizing roots from different species; as well as different demineralizing agents, and finally different criteria for the assessment of wound healing have been used. Pitaru et al.2 described an assay in which the migration, attachment, and orientation of fibroblasts to root surfaces could be studied over time. However, only healthy porcine teeth were used. The aims of the present study were first to establish and compare the effects of a variety of surface treatments of human periodontally diseased roots on fibroblast migration, attachment and orientation in vitro, with time; second, to compare cell attachment and orientation to healthy human dental root surfaces conditioned with either citric acid or EDTA; and third to determine what effect collagenase digestion would have on the cell attachment and orientation to these root surfaces when the digestive process was carried out subsequent to both types of demineralization. MATERIALS AND METHODS This study was carried out in two sets of experiments: Experiment 1: Assessing the fibroblast response to different root treatments of periodontally diseased roots; Experiment 2: Comparing the effects of citric acid/EDTA/collagenase digestion of healthy roots on fibroblast response.
Preparation
of Root Slices
Experiment 1: Eleven extracted human teeth were obtained from
healthy
individuals
attending the oral surgery departsingle teeth. In eight of these,
ment for the extraction of
the teeth had a loss of attachment of more than 6 mm around the circumference of the root resulting from adult periodontal disease, and none of the patients had a history of periodontal therapy for at least 6 months previously. The teeth from the remaining 3 patients showed no periodontal disease. The remains of the periodontal ligament were gently re-
August 1990
moved from the healthy teeth using a scalpel with the aid of a dissecting microscope. In the 8 periodontally diseased teeth, the level of attachment was identified with the aid of a dissecting microscope and this was marked by drilling a circumferential groove with a water-cooled diamond bur. Four of these roots were then cleaned using a Gracey curette and an ultrasonic sealer until the surface was clean, hard, and smooth. The remaining 4 teeth were not cleaned. Portions of the roots affected by periodontal disease and the corresponding portions on the non-diseased roots were then cut transversely into slices of similar thickness (200 µ ) using a rotary saw with water coolant. A total of 60 slices were prepared, 12 non-diseased, 24 from treated periodontally diseased teeth, and 24 from non-treated periodontally diseased teeth. The slices were checked individually with a dissecting microscope and any remaining projections of soft and hard tissue were removed to ensure that the slices were horizontally flat. All the slices were passed through 3 washes of antibiotics (1.0 mg/ml penicillin G, 0.05 mg/ ml gentamicin, and 3.0 µg/ml fungizone) and left overnight in a fourth wash of antibiotics at 37°C. Half of the diseased and half of the treated diseased root slices were then partially demineralized using citric acid (pH 1) for 3 minutes. All the slices were then washed with several washes of -minimal essential medium (a-MEM). For control purposes, all the non-demineralized slices were also washed with -MEM. Finally, the slices were conditioned using 2 changes of a-MEM, 15% fetal bovine serum, and antibiotics 1:10 of the above described concentration over a 24-hour period before being plated in the culture dishes.
Experiment 2: Seven teeth extracted from healthy individ-
uals were used. The teeth were not extracted due to periodontal disease, and the gingival sulci did not have a probing depth of more than 4 mm. The roots were then cut transversely into slices with similar thickness (200 µ ) using a rotary saw with water coolant; 60 slices were prepared in total. The most coronal and apical portions of the roots were not used. The root slices were then checked individually under a dissecting microscope and a scalpel used to remove the outermost layer of cementum and thus the remnants of the periodontal ligament. All the slices were passed through 3 washes of an antibiotic solution (1.0 mg/ml penicillin G, 0.05 mg/ml gentamicin and 3.0 µ / fungizone). Twenty-four root slices were partly demineralized using citric acid (pH 1) for 3 minutes. The acid was prepared by adding citric acid crystals to 10 ml of distilled water until the pH-meter indicated 1. A further 24 root slices were partly demineralized for 30 minutes in 18% ethylenediamine tetra-acetic acid (EDTA) pH 7.2. All root slices, demineralized and non-demineralized, were washed in several washes of -MEM. Half of the demineralized root slices from each of the above 2 categories were then incubated in .25 ml of 50 mM CaCl2 (pH 7.4 to 7.6) and 50
Volume 61 Number 8
FARDAL, LOWENBERG
531
µg of purified bacterial collagenase for 2 hours at 37°C and finally passed through several washes of PBS. Cell Culture Human gingival fibroblasts (HGF) (Gin 1, American Type Culture Collection) were used at the tenth subculture. The cells were cultured in -MEM supplemented with 15% fetal bovine serum and antibiotics at '/loth of the concentration used above in a humidified atmosphere of 95% air and 5% CO at 37°C. The HGF were plated in each of twelve 35 mm culture dishes at a concentration of 4.5 x 10 cells per dish. The medium was changed daily. On the sixth day of the experiment, when the cells were confluent in all 12 dishes, 5 root slices were placed gently onto the confluent cell layer in each dish as descibed below.
Diseased treated by mechanical means (DT); 2) Diseased treated and demineralized (DTD); 3) Diseased and not treated (DNT); 4) Diseased and not treated, but demineralized (DNTD); and 5) Non-diseased (ND).
Experiment
1:
1)
Experiment 2: 1) Citric acid demineralized (CA); 2) Citric
acid demineralized and subsequently collagenase digested (CAC); 3) EDTA demineralized (E); 4) EDTA demineralized and collagenase digested (EC); and 5) Untreated control root slice (C). The day the root slices were placed onto the HGF cell layer is referred to as day 0 of the experiment. The medium was supplemented with 50 µg/ml of ascorbic acid from day 0 until the end of the experiment. The method of measurement was based on the observation of Pitaru and Melcher1 that sheets of orientated cells in vitro exhibit orientated refractile material when viewed by phase contrast microscopy in which the iris diaphragm has been partially closed. A planachromatic objective (1.0/ 0.04 Zeiss) was used on an inverted Leitz microscope with phase contrast optics to photograph each root slice in its entirety at a final magnification of 5. Photographic films (Kodak technical pan 2415) were developed to produce high contrast and printed on medium contrast paper (Kodak paper 2) in order to achieve optimal delineation of the refractile material. Three parameters (the attachment index, the orientation index, and the modified orientation index), were calculated at days 3, 7, 10, 14, and 21. In addition, the attachment index was assessed at day 1 in experiment 1. In experiment 2, only the attachment index and the orientation index at days 3, 7, 10, and 14 were calculated. An example of this methodology is given in Figure 1, with explanatory text. The attachment index (AI), which describes the extent of the periphery of the root slices to which HGF are attached, was calculated by determining the proportion of the circumference of the root slice (TCL) occupied by a rim of refractile material (LA) (AI LA/TCL). The orientation index (OI), describing the extent of development of orientated sheets of HGF extending from the =
Figure 1: Sample showing photograph of DT (diseased treated) root at day 7 of the experimental period. Top: Root slice with the refractile material recorded using a phase contrast microscope in which the irisdiaphragm has been partially closed. Bottom: Showing how the various parameters were traced; solid line, total circumferential length (TCL) of the root slice, broken line, length of HGF attachment (LA); stippled area, area of orientated sheet of HGF (AO). of the root slice, was calculated by determining each orientated sheet of HGF (AO) relative to of area of length the arc to which it was attached (LA) (OI AO/ LA). The modified orientation index (MOI) also describes the extent of development of orientated sheets of HGF, but is expressed by determining the area of each orientated sheet of HGF (AO) relative to the total periphery of the root slice (TCL) (i.e., MOI AO/TCL). The required parameters (TCL, AO, and LA) were quantified using a Digital Equipment Corporation LSI-11/23 computer equipped with a digitizing tablet for area measurements and using an opisometer for linear measurements. The means, standard deviations, and standard errors were calculated for each of the 3 parameters (AI, Ol, and MOI). (Experiment 2 only utilized AI and OI). Each of the variables was compared with all the other variables at all the recorded time intervals using paired i-tests. Repeated r-tests may inflate the number of significant comparisons. This was overcome by treating differences as significant only
periphery the the
=
=
J Periodontol
532
FIBROBLAST ATTACHMENT AND ORIENTATION TO ROOT SURFACES
when P
A
Quantitative Analysis of the Migration, Attachment, and Orientation of Human Gingival
Fibroblasts to Human Dental Root Surfaces in vitro 0. Fardai* and B.F.
Lowenbergf
The purpose of this study was to assess the migration, attachment, and orientation of human gingival fibroblasts to human dental roots over a period of 21 days in vitro. The fibroblasts were incubated with a total of 120 periodontally diseased and non-diseased root slices (200 µ thickness) which had been treated in the following manner: 1) Root planed diseased root (DT); 2) Root planed and citric acid demineralized diseased root (DTD); 3) Non-treated diseased root (DNT); 4) Citric acid demineralized diseased root (DNTD); 5) Non-diseased control (ND); 6) Citric acid demineralized non-diseased root (CA); 7) Citric acid and collagenase digested non-diseased root (CAC); 8) EDTA demineralized non-diseased root (E); and 9) EDTA-demineralized and collagenase-digested non-diseased root (EC). The results showed that the most active phase of cell attachment and orientation occurred during the first 10 days of the experiment. Statistical differences were observed between the variables, and, in terms of cell attachment and orientation to the root slices, it was concluded that: 1) Root planing improves diseased roots; 2) Acid
demineralization subsequent to root planing improved diseased roots to such an extent as to render them comparable to non-diseased roots; 3) Citric acid demineralization alone improved diseased roots to the same extent as root planed diseased roots; 4) The exposure of collagen fibrils resulting from acid demineralization of the roots is not the sole reason for the improvement of the root surface, but rather a combination of the exposed collagen fibrils with the creation of a more hospitable environment was found to be responsible. JPeriodontol 1990;61:529-535.
Key Words: Tooth root; fibroblasts; cell attachment.
periodontal therapy is to gain a full regeneration of the periodontium destroyed by Periodontitis. From experiments in vitro,1'2 fibroblast migration, attachment, and orientation seem to be important steps in the The ultimate
goal
of
formation of a new connective tissue attachment to the root surfaces. It is not unreasonable to assume, therefore, that following periodontal therapy similar behavior of fibroblasts is important in the regenerative process. Different root surface qualities are reported to influence fibroblast functions; e.g., endotoxins from periodontal pathogens may penetrate the root surface and have been implicated in inhibiting fibroblast proliferation, synthesis, and attachment.3-11 However, Fardai et al.12 reported that periodontally
'University of Toronto, Faculty of Dentistry, 124 Edward ON M5G 1G6. Current address, Egersund, Norway. 'University of Toronto.
Street, Toronto,
diseased roots did not inhibit initial attachment in vitro and Adelson et al.13 could not find any difference in fibroblast attachment, pattern of cell growth, cell migration, or cytotoxic reaction in vitro on curetted versus non-curetted periodontally diseased root surfaces. The use of acid as an adjunct to scaling and calculus removal is reported to have been used as early as in 1846.14 Partial surface demineralization by citric acid at pH 1 for 3 minutes15 may influence new periodontal attachment attempts by one or more of the following mechanisms:16 1) Accelerated cementogenesis; 2) Widening of the dentinal tubules to allow for connective tissue ingress; 3) Extracting endotoxin and other toxic plaque products; 4) Inducing mesenchymal cell differentiation to osteoblasts or possibly cementoblasts; or 5) Exposing root dentinal collagen. Garrett et al.17 suggested that newly synthesized collagen may
J Periodontol 530
FIBROBLAST ATTACHMENT AND ORIENTATION TO ROOT SURFACES
splice to the exposed root dentinal collagen fibrils, while Boyko et al.18 proposed that demineralized root surfaces with the exposed fibrils offer a more "hospitable" surface
for fibroblast attachment to tooth surfaces. More recent in vitro studies1-2 have shown an enhanced effect on the migration, attachment, and orientation of fibroblasts by treating the root surface with 18% EDTA (pH 7.4) for 30 minutes. This suggests that there is a greater chance of new connective tissue attachment to root surfaces which have been demineralized. A number of in vivo studies however, report no additional advantage of surface demineralization over root planing at least in terms of flap healing.19"21 The inconsistency of these studies may be due to differences in experimental systems and techniques. For example, in vivo studies are frequently compared with in vitro studies; surface demineralization of periodontally diseased roots has been compared with surface demineralization of non-diseased roots, often utilizing roots from different species; as well as different demineralizing agents, and finally different criteria for the assessment of wound healing have been used. Pitaru et al.2 described an assay in which the migration, attachment, and orientation of fibroblasts to root surfaces could be studied over time. However, only healthy porcine teeth were used. The aims of the present study were first to establish and compare the effects of a variety of surface treatments of human periodontally diseased roots on fibroblast migration, attachment and orientation in vitro, with time; second, to compare cell attachment and orientation to healthy human dental root surfaces conditioned with either citric acid or EDTA; and third to determine what effect collagenase digestion would have on the cell attachment and orientation to these root surfaces when the digestive process was carried out subsequent to both types of demineralization. MATERIALS AND METHODS This study was carried out in two sets of experiments: Experiment 1: Assessing the fibroblast response to different root treatments of periodontally diseased roots; Experiment 2: Comparing the effects of citric acid/EDTA/collagenase digestion of healthy roots on fibroblast response.
Preparation
of Root Slices
Experiment 1: Eleven extracted human teeth were obtained from
healthy
individuals
attending the oral surgery departsingle teeth. In eight of these,
ment for the extraction of
the teeth had a loss of attachment of more than 6 mm around the circumference of the root resulting from adult periodontal disease, and none of the patients had a history of periodontal therapy for at least 6 months previously. The teeth from the remaining 3 patients showed no periodontal disease. The remains of the periodontal ligament were gently re-
August 1990
moved from the healthy teeth using a scalpel with the aid of a dissecting microscope. In the 8 periodontally diseased teeth, the level of attachment was identified with the aid of a dissecting microscope and this was marked by drilling a circumferential groove with a water-cooled diamond bur. Four of these roots were then cleaned using a Gracey curette and an ultrasonic sealer until the surface was clean, hard, and smooth. The remaining 4 teeth were not cleaned. Portions of the roots affected by periodontal disease and the corresponding portions on the non-diseased roots were then cut transversely into slices of similar thickness (200 µ ) using a rotary saw with water coolant. A total of 60 slices were prepared, 12 non-diseased, 24 from treated periodontally diseased teeth, and 24 from non-treated periodontally diseased teeth. The slices were checked individually with a dissecting microscope and any remaining projections of soft and hard tissue were removed to ensure that the slices were horizontally flat. All the slices were passed through 3 washes of antibiotics (1.0 mg/ml penicillin G, 0.05 mg/ ml gentamicin, and 3.0 µg/ml fungizone) and left overnight in a fourth wash of antibiotics at 37°C. Half of the diseased and half of the treated diseased root slices were then partially demineralized using citric acid (pH 1) for 3 minutes. All the slices were then washed with several washes of -minimal essential medium (a-MEM). For control purposes, all the non-demineralized slices were also washed with -MEM. Finally, the slices were conditioned using 2 changes of a-MEM, 15% fetal bovine serum, and antibiotics 1:10 of the above described concentration over a 24-hour period before being plated in the culture dishes.
Experiment 2: Seven teeth extracted from healthy individ-
uals were used. The teeth were not extracted due to periodontal disease, and the gingival sulci did not have a probing depth of more than 4 mm. The roots were then cut transversely into slices with similar thickness (200 µ ) using a rotary saw with water coolant; 60 slices were prepared in total. The most coronal and apical portions of the roots were not used. The root slices were then checked individually under a dissecting microscope and a scalpel used to remove the outermost layer of cementum and thus the remnants of the periodontal ligament. All the slices were passed through 3 washes of an antibiotic solution (1.0 mg/ml penicillin G, 0.05 mg/ml gentamicin and 3.0 µ / fungizone). Twenty-four root slices were partly demineralized using citric acid (pH 1) for 3 minutes. The acid was prepared by adding citric acid crystals to 10 ml of distilled water until the pH-meter indicated 1. A further 24 root slices were partly demineralized for 30 minutes in 18% ethylenediamine tetra-acetic acid (EDTA) pH 7.2. All root slices, demineralized and non-demineralized, were washed in several washes of -MEM. Half of the demineralized root slices from each of the above 2 categories were then incubated in .25 ml of 50 mM CaCl2 (pH 7.4 to 7.6) and 50
Volume 61 Number 8
FARDAL, LOWENBERG
531
µg of purified bacterial collagenase for 2 hours at 37°C and finally passed through several washes of PBS. Cell Culture Human gingival fibroblasts (HGF) (Gin 1, American Type Culture Collection) were used at the tenth subculture. The cells were cultured in -MEM supplemented with 15% fetal bovine serum and antibiotics at '/loth of the concentration used above in a humidified atmosphere of 95% air and 5% CO at 37°C. The HGF were plated in each of twelve 35 mm culture dishes at a concentration of 4.5 x 10 cells per dish. The medium was changed daily. On the sixth day of the experiment, when the cells were confluent in all 12 dishes, 5 root slices were placed gently onto the confluent cell layer in each dish as descibed below.
Diseased treated by mechanical means (DT); 2) Diseased treated and demineralized (DTD); 3) Diseased and not treated (DNT); 4) Diseased and not treated, but demineralized (DNTD); and 5) Non-diseased (ND).
Experiment
1:
1)
Experiment 2: 1) Citric acid demineralized (CA); 2) Citric
acid demineralized and subsequently collagenase digested (CAC); 3) EDTA demineralized (E); 4) EDTA demineralized and collagenase digested (EC); and 5) Untreated control root slice (C). The day the root slices were placed onto the HGF cell layer is referred to as day 0 of the experiment. The medium was supplemented with 50 µg/ml of ascorbic acid from day 0 until the end of the experiment. The method of measurement was based on the observation of Pitaru and Melcher1 that sheets of orientated cells in vitro exhibit orientated refractile material when viewed by phase contrast microscopy in which the iris diaphragm has been partially closed. A planachromatic objective (1.0/ 0.04 Zeiss) was used on an inverted Leitz microscope with phase contrast optics to photograph each root slice in its entirety at a final magnification of 5. Photographic films (Kodak technical pan 2415) were developed to produce high contrast and printed on medium contrast paper (Kodak paper 2) in order to achieve optimal delineation of the refractile material. Three parameters (the attachment index, the orientation index, and the modified orientation index), were calculated at days 3, 7, 10, 14, and 21. In addition, the attachment index was assessed at day 1 in experiment 1. In experiment 2, only the attachment index and the orientation index at days 3, 7, 10, and 14 were calculated. An example of this methodology is given in Figure 1, with explanatory text. The attachment index (AI), which describes the extent of the periphery of the root slices to which HGF are attached, was calculated by determining the proportion of the circumference of the root slice (TCL) occupied by a rim of refractile material (LA) (AI LA/TCL). The orientation index (OI), describing the extent of development of orientated sheets of HGF extending from the =
Figure 1: Sample showing photograph of DT (diseased treated) root at day 7 of the experimental period. Top: Root slice with the refractile material recorded using a phase contrast microscope in which the irisdiaphragm has been partially closed. Bottom: Showing how the various parameters were traced; solid line, total circumferential length (TCL) of the root slice, broken line, length of HGF attachment (LA); stippled area, area of orientated sheet of HGF (AO). of the root slice, was calculated by determining each orientated sheet of HGF (AO) relative to of area of length the arc to which it was attached (LA) (OI AO/ LA). The modified orientation index (MOI) also describes the extent of development of orientated sheets of HGF, but is expressed by determining the area of each orientated sheet of HGF (AO) relative to the total periphery of the root slice (TCL) (i.e., MOI AO/TCL). The required parameters (TCL, AO, and LA) were quantified using a Digital Equipment Corporation LSI-11/23 computer equipped with a digitizing tablet for area measurements and using an opisometer for linear measurements. The means, standard deviations, and standard errors were calculated for each of the 3 parameters (AI, Ol, and MOI). (Experiment 2 only utilized AI and OI). Each of the variables was compared with all the other variables at all the recorded time intervals using paired i-tests. Repeated r-tests may inflate the number of significant comparisons. This was overcome by treating differences as significant only
periphery the the
=
=
J Periodontol
532
FIBROBLAST ATTACHMENT AND ORIENTATION TO ROOT SURFACES
when P
