626
The Effects of the Nd:YAG Laser in Vitro Fibroblast Attachment to Endotoxin-Treated Root Surfaces David J. Trylovich, * Charles M. William J. Killoy*
on
Cobb,f David J. Pippin, * Paulette Spencer, * and
The
purpose of this study was to evaluate the effects of the NdrYAG laser on in vitro fibroblast attachment to endotoxin-treated root surfaces and to describe any laser-induced 4 mm cementum segments were obtained cementum surface alterations. Thirty 4 mm from unerupted third molars. The treatment groups were as follows: 1) control, healthy root segment; 2) non-lased, endotoxin treated; and 3) lased, endotoxin treated. The endotoxin treated roots were soaked in E. coli 055 :B5 lipopolysaccharide (556 EU/ml) for 72 hours. The lased, endotoxin-treated root segments were treated with a Nd:YAG laser using a 320 µ contact optic fiber handpiece with an energy setting of 80 mJ at 10 pulses per second for one minute. The root segments were subsequently placed in fibroblast culture dishes for 40 hours and then prepared for scanning electron microscopy (SEM) observation. SEM examination revealed two different types of attachment: flat and round. Flat cells represented firmly attached cells due to well-defined points of attachment and numerous lamellapodia. Round cells possessed few attachment processes and were, therefore, considered poorly attached. The lased, endotoxin-treated root segments had significantly decreased numbers of flat fibroblasts versus the control and nonlased, endotoxin-treated root segments. The absence of flat fibroblasts in the laser treated root segments was a consistent finding. The non-lased, endotoxin-treated root segments had significantly increased numbers of round fibroblasts versus the control and lased, endotoxin treated groups. The lased root segments exhibited surface alterations which included charring, crater formation, cementum meltdown, and tracking. The organic matrix appeared to have been burned off leaving behind a resolidified substance with a lava-like appearance. The results of this in vitro study suggest that the laser alters the biocompatibility of the cementum surface so as to make it unfavorable for fibroblast attachment. Healing studies are necessary to determine if these effects observed in vitro are of clinical importance. / Periodontol 1992; 63:626-632.
Key Words: Fibroblasts; lasers; tooth root; cementum; endotoxins.
The cementum of roots exposed to plaque-infected periodontal pockets undergoes several changes that decrease biocompatibility; i.e., hypermineralization and adsorption of endotoxin, antigen-antibody complexes, and products of microbial metabolism.1,2 The reattachment of gingival tissues to previously diseased root surfaces is a major goal of periodontal therapy. However, this requires that the root
'Department of Periodontics, R.L. Thompson Strategic Hospital, Carswell AFB,
TX.
department of Periodontics, School of Dentistry, University of MissouriKansas City, Kansas City, MO. department of Pediatrie Dentistry. The opinions or conclusions contained in this article are those of the authors and are not to be construed as official or reflecting the views of the United States Air Force or the Department of Defense.
surface be rendered biologically compatible to the adjacent tissues. The classic method of treating root surfaces to achieve biocompatibility has been scaling and root planing with hand and ultrasonic instrumentation.3 Recently, the NdrYAG (neodymium: yttrium, aluminum, and garnet) laser has been proposed as an instrument with great potential for effective root preparation.4 Use of the Nd:YAG laser on contaminated root surfaces as an adjunct to hand and ultrasonic instrumentation may have a role in both surgical and nonsurgical periodontal therapy. In theory, the laser's ability to sterilize, vaporize, and ablate appears to offer an effective means of removing or altering adsorbed endotoxin, calculus, plaque, and other root surface contaminants. Myers5 used a Nd:YAG laser on enamel and dentin.
Volume 63 Number 7
TRYLOVICH, COBB, PIPPIN, SPENCER,
Scanning electron microscopy (SEM) showed enamel and dentin cratering with resolidified spheres of hydroxyapatite. Cratering of the dentin surface was noted and ranged in depth from 40 to 60 µ . No subsurface cracking or fissuring was observed, indicating the absence of thermal damage. However, changes in dentinal tubule structures in depth below a carbonized were observed up to 40 µ layer. The tubular structure was apparently normal beyond the 40 micron level. Hess6 examined morphologic changes
in enamel after Nd:YAG laser exposure. SEM evaluation revealed a pock-marked surface with indistinct crater formation at low energy levels and increasing severity of cratering as energy levels were increased. Globules of resolidified enamel were observed and tended to accumulate at the periphery of craters. The surface of lased areas had a porous, lava-like appearance. To date, there are no studies evaluating the biocompatibility of lased cementum surfaces, nor are there any descriptive reports of laser-induced cementum surface alterations. Thus, the purpose of this study was to evaluate the effects of the Nd:YAG laser on in vitro fibroblast attachment to endotoxin-treated root surfaces and to describe any laserinduced cementum surface alterations. MATERIALS AND METHODS Collection third molars were collected from the offices of local oral surgeons. A siliconized bottle filled with 50 ml of triple distilled water and 1 ml of a solution containing 10,000 units penicillin, 10 mg streptomycin, and 25 µg amphotericin per ml in 0.9% sodium chloride was provided for specimen collection. Thirty root segment squares, approximately 4 mm x 4 mm x 1 to 2 mm, were cut with a water-cooled, high-speed handpiece using a #557 carbide bur. The segments were cut from the mesial or distal root surface from an area beginning 2 mm below the cemento-enamel junction (CEJ).
Sample
Thirty unerupted
Treatment
Groups
The segments
were
randomly assigned to 1 of 3 groups: 1)
control, healthy root segment; 2) non-lased, endotoxintreated; and 3) lased, endotoxin-treated. The root segments coded on the pulpal side indicate their treatment group.
were
using
a
1/4 round bur
to
Endotoxin Preparation A commercially available endotoxin8 (Escherichia coli 055 :B5 lipopolysaccharide) was prepared by adding 16,700 endotoxin units (EU) to 30 ml of endotoxin-free water. The solution was vortex mixed for 5 minutes as directed. This yielded a concentration of approximately 556 EU/ml. 'Endotoxin Standard, 210-SE,
Sigma Chemical Company, St. Louis, MO.
KILLOY
627
Sample Preparation
Twenty-five of the root segments were embedded in baseplate wax so that only the cementai surfaces were exposed.
Five of these were control segments which were embedded to determine if the wax had any detrimental effects on fibroblast attachment. The remaining 5 controls were not embedded in wax. All of the root segments were subsequently exposed to an abrasive sodium bicarbonate aerosol spray11 for 10 seconds followed by a 10-second water rinse. The aerosol spray ensured the removal of any residual root surface contaminants. The 20 segments assigned to the endotoxin-treated group were placed in 2 Petri dishes each containing 10 ml of E. coli endotoxin solution. The 10 segments in the control group were placed in a similar Petri dish with 10 ml of the specimen collection solution. Specimens were allowed to remain in the Petri dishes for approximately 72 hours. All root segments were then removed from their respective solutions and allowed to air dry under a vacuum hood for approximately 1 hour. After air drying, the baseplate wax was removed from the 25 root segments. Laser Treatment Ten root segments
comprising the lased, endotoxin-treated lased with an Nd:YAG laser1 using a 320 µ group were fiber with an average energy reading of 80 contact optic second for 1 minute each. The average 10 mJ at pulses per obtained was prior to lasing each root segenergy reading ment using a Model EM22 Energy Meter.* The fiber was held perpendicular to the root segment and moved in a back and forth motion in an attempt to expose the entire root segment equally.
Fibroblast Culture Preparation and Incubation Human gingival fibroblasts were grown from expiants of normal tissue obtained during surgical reduction of retromolar tissue. The cell culture media consisted of Dulbecco's Modified Eagle's Medium with the following formulation of additives: 2 mmol L-glutamine, 100 units/mL penicillin, 100 ^g/mL streptomycin, 50 µg/mL gentamicin, 2.5 µg/ mL amphotericin (Fungizone), and 15% (by volume) fetal bovine serum. Cells were cultured in a humidified atmosphere of 5% C02 in air at 37°C until confluent monolayers were obtained. The attachment assay was performed with sixth-generation fibroblasts. All 30 root segments were placed in 3 30 mL Petri dishes keeping the 3 treatment groups separate. The root segments were incubated for 40 hours at 37°C in a humidified atmosphere of 5% C02 in air with fibroblasts at a concentration on 2.5 x 10s cells/mL of medium. The incubation period was terminated for all specimens by gentle rinsing in Hank's balanced salt solution to remove nonadherent cells followed by immersion fixation in ice-
uProphy-Jet 30, Dentsply International, York, PA. 'American Dental Laser Inc., Birmingham, MI. 'Sunrise Technologies, Inc., Fremont, CA.
628
J Periodontol July 1992
EFFECTS OF Nd:YAG LASER ON FIBROBLAST ATTACHMENT
cold 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer at pH 7.4 for 2 hours. After fixation, the segments were prepared for SEM examination by dehydration in a series of graded ethyl alcohol solutions (50% to 100%). The final dehydration step was accomplished in hexamethyldisilazane. Specimens were then sputter-coated with 20 nm of gold-palladium and subsequently examined in a Philips 515 Scanning Electron Microscope.** SEM Observation All photographs used for cell counting were taken at a magnification of 170 at 15 kV and a positive angle of 15°. The examiner (CMC) operating the scanning electron microscope and counting the fibroblasts was blind as to the treatment group of the segment. During SEM observation, an imaginary diagonal line was drawn from the upper left corner through the center to the lower right corner of the root segments. One photomicrograph was then randomly taken in each of the 3 areas (upper left, center, and lower right). This methodology aided in consistency of sampling areas between root segments. Data Collection and Analysis The number of attached flat fibroblasts and the number of attached round fibroblasts observed in each SEM micrograph was recorded separately. When specimens showed proliferative attachment of fibroblasts, resulting in a confluent monolayer, the fibroblasts were counted in a small subset area and calculated for the entire area of the micrograph. A maximum number was set at 80 fibroblasts. The micrographs were re-examined in 10 days when the fibroblasts were recounted. Intra-examiner correlation (Pearson) was determined. The data were analyzed using a one-factor analysis of variance (ANOVA) test for both round and flat fibroblasts and the Tukey studentized range method.
RESULTS Intra-examiner reliability between the first and second counting exhibited a very high correlation (r 0.99). Attached fibroblasts were observed to be either flat (Fig. 1) or round (Fig. 2) in appearance. Flat cells represented firmly-attached cells due to well-defined points of attachment and numerous lamellapodia. Round cells possessed few attachment processes and were, therefore, considered poorly-attached. The fibroblasts occasionally coalesced to form a confluent monolayer (Fig. 3). There was a low correlation (r 0.30) between the number of round and flat cells among the 30 specimens; this allowed them to be examined as separate dependent variables. The control root segments were originally separated into 5 wax-mounted and 5 non-wax-mounted root segments, to determine if the wax-mounting process had any effect upon the attachment of fibroblasts to the cementum. T-tests revealed that there was no significant difference in the number
Photomicrograph shows a high-power view of a flat fibroblast considered healthy and firmly attached due to the presence of well-developed lamellapodia (arrows) (original magnification 2500; bar 10 µ/ ). Figure which
1.
was
=
58jiml5.1kU 3.26E2 0601^81
LftSER-S
2.
Figure Left-side photomicrograph shows both round (wide arrow) and flat (narrow arrow) fibroblasts (bar 50 µ/ ; original magnification x 326). At higher magnifications (right side) rounded cells exhibited damaged cell membranes (arrow) and few lamellapodia (bar 10 µ/ ; original magnification 1863). =
=
=
=
"'Philips Electronic Instruments, Mahwah,
NJ.
Figure blasts
3. Control
specimen
covered
(original magnification
by
a
confluent monolayer of fibra 0.1
170; bai -
nun).
Volume 63 Number 7
TRYLOVICH, COBB, PIPPIN, SPENCER,
Table 1. Mean (Standard Control Root Segments
Deviation) of Attached Fibroblasts
Treatment
Round
Wax embedded
629
to
Flat
4.00
30.80
(2.55) Non-wax embedded
KILLOY
The Effects of the Nd:YAG Laser in Vitro Fibroblast Attachment to Endotoxin-Treated Root Surfaces David J. Trylovich, * Charles M. William J. Killoy*
on
Cobb,f David J. Pippin, * Paulette Spencer, * and
The
purpose of this study was to evaluate the effects of the NdrYAG laser on in vitro fibroblast attachment to endotoxin-treated root surfaces and to describe any laser-induced 4 mm cementum segments were obtained cementum surface alterations. Thirty 4 mm from unerupted third molars. The treatment groups were as follows: 1) control, healthy root segment; 2) non-lased, endotoxin treated; and 3) lased, endotoxin treated. The endotoxin treated roots were soaked in E. coli 055 :B5 lipopolysaccharide (556 EU/ml) for 72 hours. The lased, endotoxin-treated root segments were treated with a Nd:YAG laser using a 320 µ contact optic fiber handpiece with an energy setting of 80 mJ at 10 pulses per second for one minute. The root segments were subsequently placed in fibroblast culture dishes for 40 hours and then prepared for scanning electron microscopy (SEM) observation. SEM examination revealed two different types of attachment: flat and round. Flat cells represented firmly attached cells due to well-defined points of attachment and numerous lamellapodia. Round cells possessed few attachment processes and were, therefore, considered poorly attached. The lased, endotoxin-treated root segments had significantly decreased numbers of flat fibroblasts versus the control and nonlased, endotoxin-treated root segments. The absence of flat fibroblasts in the laser treated root segments was a consistent finding. The non-lased, endotoxin-treated root segments had significantly increased numbers of round fibroblasts versus the control and lased, endotoxin treated groups. The lased root segments exhibited surface alterations which included charring, crater formation, cementum meltdown, and tracking. The organic matrix appeared to have been burned off leaving behind a resolidified substance with a lava-like appearance. The results of this in vitro study suggest that the laser alters the biocompatibility of the cementum surface so as to make it unfavorable for fibroblast attachment. Healing studies are necessary to determine if these effects observed in vitro are of clinical importance. / Periodontol 1992; 63:626-632.
Key Words: Fibroblasts; lasers; tooth root; cementum; endotoxins.
The cementum of roots exposed to plaque-infected periodontal pockets undergoes several changes that decrease biocompatibility; i.e., hypermineralization and adsorption of endotoxin, antigen-antibody complexes, and products of microbial metabolism.1,2 The reattachment of gingival tissues to previously diseased root surfaces is a major goal of periodontal therapy. However, this requires that the root
'Department of Periodontics, R.L. Thompson Strategic Hospital, Carswell AFB,
TX.
department of Periodontics, School of Dentistry, University of MissouriKansas City, Kansas City, MO. department of Pediatrie Dentistry. The opinions or conclusions contained in this article are those of the authors and are not to be construed as official or reflecting the views of the United States Air Force or the Department of Defense.
surface be rendered biologically compatible to the adjacent tissues. The classic method of treating root surfaces to achieve biocompatibility has been scaling and root planing with hand and ultrasonic instrumentation.3 Recently, the NdrYAG (neodymium: yttrium, aluminum, and garnet) laser has been proposed as an instrument with great potential for effective root preparation.4 Use of the Nd:YAG laser on contaminated root surfaces as an adjunct to hand and ultrasonic instrumentation may have a role in both surgical and nonsurgical periodontal therapy. In theory, the laser's ability to sterilize, vaporize, and ablate appears to offer an effective means of removing or altering adsorbed endotoxin, calculus, plaque, and other root surface contaminants. Myers5 used a Nd:YAG laser on enamel and dentin.
Volume 63 Number 7
TRYLOVICH, COBB, PIPPIN, SPENCER,
Scanning electron microscopy (SEM) showed enamel and dentin cratering with resolidified spheres of hydroxyapatite. Cratering of the dentin surface was noted and ranged in depth from 40 to 60 µ . No subsurface cracking or fissuring was observed, indicating the absence of thermal damage. However, changes in dentinal tubule structures in depth below a carbonized were observed up to 40 µ layer. The tubular structure was apparently normal beyond the 40 micron level. Hess6 examined morphologic changes
in enamel after Nd:YAG laser exposure. SEM evaluation revealed a pock-marked surface with indistinct crater formation at low energy levels and increasing severity of cratering as energy levels were increased. Globules of resolidified enamel were observed and tended to accumulate at the periphery of craters. The surface of lased areas had a porous, lava-like appearance. To date, there are no studies evaluating the biocompatibility of lased cementum surfaces, nor are there any descriptive reports of laser-induced cementum surface alterations. Thus, the purpose of this study was to evaluate the effects of the Nd:YAG laser on in vitro fibroblast attachment to endotoxin-treated root surfaces and to describe any laserinduced cementum surface alterations. MATERIALS AND METHODS Collection third molars were collected from the offices of local oral surgeons. A siliconized bottle filled with 50 ml of triple distilled water and 1 ml of a solution containing 10,000 units penicillin, 10 mg streptomycin, and 25 µg amphotericin per ml in 0.9% sodium chloride was provided for specimen collection. Thirty root segment squares, approximately 4 mm x 4 mm x 1 to 2 mm, were cut with a water-cooled, high-speed handpiece using a #557 carbide bur. The segments were cut from the mesial or distal root surface from an area beginning 2 mm below the cemento-enamel junction (CEJ).
Sample
Thirty unerupted
Treatment
Groups
The segments
were
randomly assigned to 1 of 3 groups: 1)
control, healthy root segment; 2) non-lased, endotoxintreated; and 3) lased, endotoxin-treated. The root segments coded on the pulpal side indicate their treatment group.
were
using
a
1/4 round bur
to
Endotoxin Preparation A commercially available endotoxin8 (Escherichia coli 055 :B5 lipopolysaccharide) was prepared by adding 16,700 endotoxin units (EU) to 30 ml of endotoxin-free water. The solution was vortex mixed for 5 minutes as directed. This yielded a concentration of approximately 556 EU/ml. 'Endotoxin Standard, 210-SE,
Sigma Chemical Company, St. Louis, MO.
KILLOY
627
Sample Preparation
Twenty-five of the root segments were embedded in baseplate wax so that only the cementai surfaces were exposed.
Five of these were control segments which were embedded to determine if the wax had any detrimental effects on fibroblast attachment. The remaining 5 controls were not embedded in wax. All of the root segments were subsequently exposed to an abrasive sodium bicarbonate aerosol spray11 for 10 seconds followed by a 10-second water rinse. The aerosol spray ensured the removal of any residual root surface contaminants. The 20 segments assigned to the endotoxin-treated group were placed in 2 Petri dishes each containing 10 ml of E. coli endotoxin solution. The 10 segments in the control group were placed in a similar Petri dish with 10 ml of the specimen collection solution. Specimens were allowed to remain in the Petri dishes for approximately 72 hours. All root segments were then removed from their respective solutions and allowed to air dry under a vacuum hood for approximately 1 hour. After air drying, the baseplate wax was removed from the 25 root segments. Laser Treatment Ten root segments
comprising the lased, endotoxin-treated lased with an Nd:YAG laser1 using a 320 µ group were fiber with an average energy reading of 80 contact optic second for 1 minute each. The average 10 mJ at pulses per obtained was prior to lasing each root segenergy reading ment using a Model EM22 Energy Meter.* The fiber was held perpendicular to the root segment and moved in a back and forth motion in an attempt to expose the entire root segment equally.
Fibroblast Culture Preparation and Incubation Human gingival fibroblasts were grown from expiants of normal tissue obtained during surgical reduction of retromolar tissue. The cell culture media consisted of Dulbecco's Modified Eagle's Medium with the following formulation of additives: 2 mmol L-glutamine, 100 units/mL penicillin, 100 ^g/mL streptomycin, 50 µg/mL gentamicin, 2.5 µg/ mL amphotericin (Fungizone), and 15% (by volume) fetal bovine serum. Cells were cultured in a humidified atmosphere of 5% C02 in air at 37°C until confluent monolayers were obtained. The attachment assay was performed with sixth-generation fibroblasts. All 30 root segments were placed in 3 30 mL Petri dishes keeping the 3 treatment groups separate. The root segments were incubated for 40 hours at 37°C in a humidified atmosphere of 5% C02 in air with fibroblasts at a concentration on 2.5 x 10s cells/mL of medium. The incubation period was terminated for all specimens by gentle rinsing in Hank's balanced salt solution to remove nonadherent cells followed by immersion fixation in ice-
uProphy-Jet 30, Dentsply International, York, PA. 'American Dental Laser Inc., Birmingham, MI. 'Sunrise Technologies, Inc., Fremont, CA.
628
J Periodontol July 1992
EFFECTS OF Nd:YAG LASER ON FIBROBLAST ATTACHMENT
cold 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer at pH 7.4 for 2 hours. After fixation, the segments were prepared for SEM examination by dehydration in a series of graded ethyl alcohol solutions (50% to 100%). The final dehydration step was accomplished in hexamethyldisilazane. Specimens were then sputter-coated with 20 nm of gold-palladium and subsequently examined in a Philips 515 Scanning Electron Microscope.** SEM Observation All photographs used for cell counting were taken at a magnification of 170 at 15 kV and a positive angle of 15°. The examiner (CMC) operating the scanning electron microscope and counting the fibroblasts was blind as to the treatment group of the segment. During SEM observation, an imaginary diagonal line was drawn from the upper left corner through the center to the lower right corner of the root segments. One photomicrograph was then randomly taken in each of the 3 areas (upper left, center, and lower right). This methodology aided in consistency of sampling areas between root segments. Data Collection and Analysis The number of attached flat fibroblasts and the number of attached round fibroblasts observed in each SEM micrograph was recorded separately. When specimens showed proliferative attachment of fibroblasts, resulting in a confluent monolayer, the fibroblasts were counted in a small subset area and calculated for the entire area of the micrograph. A maximum number was set at 80 fibroblasts. The micrographs were re-examined in 10 days when the fibroblasts were recounted. Intra-examiner correlation (Pearson) was determined. The data were analyzed using a one-factor analysis of variance (ANOVA) test for both round and flat fibroblasts and the Tukey studentized range method.
RESULTS Intra-examiner reliability between the first and second counting exhibited a very high correlation (r 0.99). Attached fibroblasts were observed to be either flat (Fig. 1) or round (Fig. 2) in appearance. Flat cells represented firmly-attached cells due to well-defined points of attachment and numerous lamellapodia. Round cells possessed few attachment processes and were, therefore, considered poorly-attached. The fibroblasts occasionally coalesced to form a confluent monolayer (Fig. 3). There was a low correlation (r 0.30) between the number of round and flat cells among the 30 specimens; this allowed them to be examined as separate dependent variables. The control root segments were originally separated into 5 wax-mounted and 5 non-wax-mounted root segments, to determine if the wax-mounting process had any effect upon the attachment of fibroblasts to the cementum. T-tests revealed that there was no significant difference in the number
Photomicrograph shows a high-power view of a flat fibroblast considered healthy and firmly attached due to the presence of well-developed lamellapodia (arrows) (original magnification 2500; bar 10 µ/ ). Figure which
1.
was
=
58jiml5.1kU 3.26E2 0601^81
LftSER-S
2.
Figure Left-side photomicrograph shows both round (wide arrow) and flat (narrow arrow) fibroblasts (bar 50 µ/ ; original magnification x 326). At higher magnifications (right side) rounded cells exhibited damaged cell membranes (arrow) and few lamellapodia (bar 10 µ/ ; original magnification 1863). =
=
=
=
"'Philips Electronic Instruments, Mahwah,
NJ.
Figure blasts
3. Control
specimen
covered
(original magnification
by
a
confluent monolayer of fibra 0.1
170; bai -
nun).
Volume 63 Number 7
TRYLOVICH, COBB, PIPPIN, SPENCER,
Table 1. Mean (Standard Control Root Segments
Deviation) of Attached Fibroblasts
Treatment
Round
Wax embedded
629
to
Flat
4.00
30.80
(2.55) Non-wax embedded
KILLOY
