990
In-Vivo Comparisons of Clot Formation on Titanium and HydroxyapatiteCoated Titanium Arnold D.
Steinberg, * Ruth Willey,T and James L. Drummond*
We investigated in vivo clot formation on the surface of hydroxyapatite (HA)coated titanium (Ti) implants and on non-coated Ti implants. Immediately after tooth extraction implant samples were inserted into the blood clot, in the same extraction site, for 1, 30, 60, or 120 seconds. Samples were processed for scanning electron microscopy (SEM). Qualitative observations of clotting topography were made by direct SEM viewing. Neither of the implant surfaces appeared to differ markedly in the degree of clotting during the 120 seconds of implantation; they revealed very early clot formation and limited clot attachment. These results were compared to the findings obtained in a previous study using identical methods with an intact periodontal ligament (PDL), root planed roots, and roots planed and treated with pH 1 citric acid. The PDL surface had the most rapid clot formation at all time periods. By 120 seconds, all root surfaces had completed clot formation. J Periodontol 1992; 63:990-994.
Key Words: Dental implants; titanium; hydroxyapatite; blood coagulation.
Titanium (Ti) dental implants are designed to enhance rapid bone growth around the implant and provide mechanical retention by osseointegration.1 Hydroxyapatite (HA) coated Ti is also currently used as a dental implant material because its direct and rapid surface interaction with adjacent bone2*4 results in bioactive retention or biointegration.1 The first interaction of an implant surface placed in intimate contact with freshly-prepared bone is with clotting blood. This initial interaction results in a coagulation cascade which, if undisturbed, will undergo organization, incorporation of a variety of cellular elements, and finally, bone formation resulting in osseointegration of the implant. Deviation from this biological order of events may result in fibrous tissue formation around the implant. Thus, we believe it is essential that we understand the initial blood interaction with the implant surface to determine if differences in blood clot formation exist between those surfaces leading to fibrous tissue formation and those surfaces leading to osseointegration. Additionally, such findings must be compared to our human tooth replantations (under similar conditions) having a variety of root surface treatments.5,6 van Steenberghe,7 in a review, suggests that rapid formation of a blood clot favors rapid bone apposition and osseointegration of the dental implant rather than fibrous scar formation. d'Hoedt,8 using scanning electron micros-
*Departments of Periodontics and Biochemistry, University of Illinois Chicago, Chicago, IL. Department of Biological Science. •Department of Operative Dentistry.
at
and freshly drawn human blood, observed in clot formation at 30, 90, and 240 seconds after that vitro, aluminum and Ti oxide surfaces was more on application stainless steel. than on He, too, suggested the imrapid formation for enhanced bone formation. of clot portance rapid This study was designed to evaluate and compare, in vivo, the rapidity of clot formation and maturation on HAcoated Ti implants and on non-coated Ti implants in humans. It was also designed to compare these results with a recent investigation, performed under identical conditions, where intact periodontal ligament (PDL) and periodontally diseased root surfaces with variety of root treatments were studied.6 Evaluation of the clot forming potential of these 2 types of implant surfaces is intended to serve as a measure of potential biocompatibility and provide information about the initial events leading to osseointegration and copy
(SEM)
biointegration. This protocol for human investigation was approved by the University of Illinois Institutional Review Board. The procedure and purpose of the project was described to each patient prior to signing the consent form. MATERIALS AND METHODS Dental Implant Materials The dental implant samples were prepared by the manufacturer5 of material identical to that used in their commer-
§Calcitek, Inc., Carlsbad, CA.
Volume 63 Number 12
STEINBERG, WILLEY,
daily available HA-coated implants. The HA was plasma sprayed onto 2 mm thick Ti (TÌ-6A1-4V) plates and sectioned into pieces 5 mm x 10 mm. The samples had HA (coated) on one surface and only Ti (uncoated) on the other surface. They were then sterilized by gamma irradiation and packaged in individually sealed containers for storage. Patient Selection and Extraction Site Selection
Healthy volunteers undergoing uncomplicated tooth extraction at the Oral Surgery Clinic at the University of Illinois at Chicago Dental School were used in this study. Informed consent for the short-term implantation of the dental implant materials into the sockets of freshly extracted teeth was obtained from all patients. Only patients who had not taken any medication for 10 days were selected. The extraction sites chosen were from an area of the mandible where complete anesthesia could be achieved with block anesthetic. This prevented infusion of local anesthetic and vasoconstrictor drugs into the clotting extraction site. Placement of Implant Into Extraction Site
Immediately after extraction, one sample at a time was inserted into the blood clot in the extraction site for 1, 30, 60, and 120 second time periods. These time periods were selected for comparison with human root replantations in a previous study.6 The "1" second time was the time required to insert the sample into the clot in the extraction site and remove it immediately. The samples were held in the extraction site by locking forceps. One extraction site was used for 4 implant samples for each of the 4 time periods. A blood clot was always present in the extraction socket into which the samples were inserted. Five different patients were used, thus 5 samples were obtained for each time period. Preparation
of
Samples
for SEM
Immediately upon removal of the implant material from the extraction site, the samples were rinsed in sterile saline and fixed initially in 0.1% glutaraldehyde (GA) in 0.1 M phosphate buffer, pH 7.2 for 30 minutes at room temperature. This was followed by immersion into 2% GA in 0.1 M phosphate buffer and refrigerated at 4° C. for 24 hours.6 Stereoscopic microscope observations (20 x ) of the amount of blood clot attachment to the implant surface were made immediately after fixation. Samples were washed in distilled water, mounted on stubs, frozen in liquid nitrogen, and dried for 12 hours in a Pearse freeze drier. After coating with 120 A gold, the samples were examined with an ISI DS-130 SEM at 10 kV. SEM Observations
Qualitative observations were made by direct SEM viewing and
appropriate photographs of clotting topography. These included observations of film deposits, initial platelet attachment and aggregation, the presence of erythrocytes or any other blood cells and fibrin formation, and lysis. It was
DRUMMOND
991
Table 1. Blood Clot Formation on Titanium (Ti) and Hydroxyapatite-Coated Titanium (HA) Implant Surfaces
Time
Material/
Sample
Number
(seconds)
30
60
120
1.5 1.0 1.0 2.5 1.0
1.0 1.5 1.0 1.5 1.0
1.0 1.0 1.0 1.0 1.0
1.0
1.0 1.0 1.5 3.0 1.0
1.0 1.5 2.5 2.0 2.0
1.5 1.0 1.0 1.0 2.5
Ti 1 2 3 4 5 HA 1 2 3
The numbers
2.0 1.5 2.0 1.0
4.0 2.5
(1 through 5) represent an arbitrary rank classification.
assumed that the film deposits observed on some of the surfaces after blood contact were plasma proteins. The observations were made by dividing each implant sample into quadrants and evaluating one arbitrarily chosen site from each quadrant. A mean was then obtained from the numerical evaluation of the 4 observations for each of the implant samples (Table 1). The following rank classification of the degree of clot formation and prepared for assessing the degree of clotting at the observed sites:6 1. Film may or may not cover surface. No blood elements or fibrin observed. 2. Initial clot formation: plasma protein layer may be visible, fibrin strands with or without the presence of scattered and aggregating platelets. 3. Organized clot: surface layer not visible, organized mesh-work consisting of aggregating platelets, fibrin, and
erythrocytes. 4. Retracting clot: dense fibrin mesh, platelets not visible, few erythrocytes. 5. Lysis of clot: dense fibrin mesh disappearing, eryth-
to be present outside the remnants of the fibrin mesh. No statistical evaluations were made of these observations due to the limited number of samples.
rocytes appear
RESULTS The photomicrographs in Figures 1 through 5 show the control surfaces and examples of clotting at the different time periods that Ti and HA were exposed to clotting blood in the extraction sites. On most of the implant surfaces, initial blood clotting was observed and included fibrin formation, along with aggregating platelets or individual platelets. On all Ti surfaces the implant surface was visible, while on the HA surfaces, the implant surface usually was completely or partially obscured by fibrin and/or a film assumed to be plasma protein. The data in Table 1 show that the Ti surfaces had clot grading from 1.0 to 2.5 while the HA surface grading ranged from 1.0 to 4.0. On the HA surface only 2 of the surfaces
992
IN VTVO CLOT FORMATION ON IMPLANTS
Figure
1. Control specimen
showing
Ti
surface (bar
=
10
J Periodontol December 1992
µ/ ).
Figure
4. Control
specimen showing HA-coated
surface (bar
Ti
=
10
-m.
Figure 2. One-second implantation of Ti sample into an extraction site showing limited fibrin formation with scattered and aggregating platelets. 10 µ/ ). The underlying Ti surface is visible (bar =
•¿ Ai extraction site Figure Thirty-second implantation sample showing fibrin formation along with some aggregating platelets. The underlying HA surface appears obscured (bar 10 µ/ ). ·.
5.
of HA
'
into
an
=
had a grade of 3.0 or higher. Thus, no marked differences in the degree of clot formation appeared to be present between the Ti and the HA surfaces at the various time periods. Stereoscopic microscope observations were made to estimate the degree of blood clot covering the implant surfaces. These observations suggested limited but similar degrees of blood clot covering both the Ti or HA coated implant surfaces.
Figure 3. One hundred-twenty second implantation of Ti sample into an extraction site showing fibrin formation and some aggregating platelets. 10 µ/ ). The underlying Ti surface is visible (bar =
DISCUSSION Blood clot formation is the first event in all wound healing. Intravascular blood studies show that initial clot formation begins with plasma protein adsorption at the injured site, rapidly followed by platelet attachment, aggregation, and activation. These platelet events are usually accompanied by the specific extrusion of granule-bound substances.9'10 Platelets are an important initial component of early clot formation at the wound site.9-11 Concomitantly, fibrin strands
Volume 63 Number 12
appear along with an influx of erythrocytes. This rapidly leads to an organized meshwork consisting of platelets, fibrin, and erythrocytes. Clot retraction and lysis rapidly follows.6 Within several hours polymorphonuclear leukocytes accumulate at the wound site and their numbers peak within 24 to 48 hours. They are followed by macrophages which appear to reach their peak at about 3 days.12 The very early events in extravascular blood clotting at the interface of the root surface and supporting connective tissues during healing are poorly understood.5-6,13 However, events similar to those described for other wound sites occur."12 Most periodontal wound healing studies focus on interpretation of observations one day or longer after the initiation of wound healing.14 Wikesjö et al.,13 using dogs having small dentin blocks surgically implanted in bone cavities in edentulous alveolar ridges, observed healing at the dentin-connective tissue interface as early as 10 minutes after wound initiation. This study is focused upon in vivo time periods in humans at the initiation of wound healing and at time periods up to 2 minutes. We suggest that the initiating factors in wound healing, namely clot formation, are responsible for inducing the cascade of events which will finally result in the union of the periodontium with the root surface. We have shown that during the first several minutes of the healing process on root surfaces, platelets are the initial cellular elements to become involved at the site of injury and appear to disappear within 2 minutes as fibrinolysis appears. Very limited numbers of polymorphonuclear leukocytes or other blood cells were observed during this time period.6 Neither the Ti or HA-coated implant surfaces in this study appeared to differ markedly from each other in the degree of clotting during the 120 seconds of implantation into the extraction sites (Table 1). At nearly all time periods only very early clot formation was observed (Figs. 2, 3, and 5) and a limited number of surface sites showed frank, macroscopic clot attachment. On all Ti observations, there were no visible plasma protein deposits to obscure the implant surface (Figs. 2 and 3). On HA-coated surfaces (Fig. 5) variability was found in our ability to observe the underlying implant surface. This suggests that in the Ti samples there either were no such deposits or the deposits were present in such limited amounts that the surface of the implant was still visible. If the implant clotting on Ti and HA coated surfaces in Table 1 is compared to identical experiments with root segments, it can be observed that, on root surfaces with intact PDL, a more advanced clot formation occurred for all time periods and clotting was completed before 120 seconds. Periodontally-diseased root surfaces, treated by root planing or root planing followed by 3 minute pH 1 citric acid application, also had blood clotting completed within the 120 seconds of replantation (grade 5). In all observations of clotting on the root surfaces, the surface was obscured by either the clot or a film assumed to be plasma protein.5,6 Hemocompatibility of a surface is believed to be deter-
STEINBERG, WILLEY, DRUMMOND
993
mined by the amount and composition of the complex array of plasma proteins deposited by blood on a surface.15 Fibrin clot adhesion to the dentin surface appears to be necessary for complete periodontal regeneration.5,6,13,16 It is possible that lack of, or limited, clot adhesion to the implant surface may have been responsible for the differences in rate of blood clot formation and the amount of surface clot attachment between the artificial implants and the root surfaces. Bone formation and its maturation occur faster on HAcoated Ti implants than on non-coated Ti implants.17,18 Since enhanced bone growth may be preceded by rapid clotting,7,8 this suggests that clotting should have occurred faster on the HA-coated Ti implants than on non-coated Ti implants. Our results do not support the observations of van Steenberghe7 and d'Hoedt8 that there is rapid blood clot production on Ti or HA implant surfaces. This discrepancy may be due to the fact that the current study was performed in vivo in fresh extraction sites, while d'Hoedt's study8 was in vitro. The 120-second time period selected for these experiments and previous studies is an arbitrary time period chosen for convenience of observation and comparisons. At this time, it is not possible to conclude that clot formation must be completed within 120 seconds or other time periods for successful healing to occur. While clot formation appears to be complete within this time frame on natural root surfaces, this does not appear to be so on either of the implant surfaces. In summary, our study suggests that there is a markedly slower rate of blood clot formation on the artificial surfaces when compared to the root surfaces. Interpretation of these observations in terms of healing rate on Ti and HA surfaces when compared to the natural root surface is currently not possible. Our future studies will establish the time required to complete clot formation along with evaluation of the tenacity of blood clots to Ti and HA-coated surfaces. Such studies are essential for comparisons of the initial wound healing events at artificial and natural surfaces interacting with adjacent connective tissue.
Acknowledgments
gratefully acknowledge Dr. L. Mercuri, Head of the Department of Oral Surgery, University of Illinois at Chicago, for his help in obtaining patients, and Mr. R. Wibel for technical assistance with the SEM. This investigation was funded by Calcitek, Inc. Carlsbad, CA., and the George Matula Fund, University of Illinois at Chicago. We
REFERENCES
Implant therapy. In: Proceedings of the World Workshop Periodontics, Chicago: The American Academy of Periodontology; 1989; (VIII) 1-10. Nakamura T, Yamamuro T, Higashi S, Kokubo T, Itoo S. A new glass-ceramics for bone replacement: Evaluation of tissue bonding to
1. Meffert R. in Clinical
2.
bone. J Biomed Mater Res 1985; 19:685-698. 3. Dennissen HW, de Groot K, Makkes P, van den Hooff A,
Klopper
994
J Periodontol December 1992
IN VTVO CLOT FORMATION ON IMPLANTS
PJ. Tissue réponse to dense apatite implants in rats. J Biomed Mater Res 1980; 14:713-721. 4. Hykuna K, Yamamuro T, Kotoura Y, et al. Surface reactions of calcium phosphate ceramics to various solutions. J Biomed Mater Res 1990; 24:471^188. 5. Steinberg AD, Le Breton G, Willey R, Mukherjee S, Lipowski J. Extravascular clot formation and platelet activation on variously treated root surfaces. J Periodontol 1986; 57:516-522. 6. Steinberg AD, Willey R. Scanning electron microscopy observations of initial clot formation on treated root surfaces. J Periodontol 1988;
12.
In: Clark RAF, Henson PM, eds. The Molecular and Cellular Biology of Wound Repair. New York: Plenum Press; 1988: 3-33. Marks R. The healing and non-healing of wound and ulcers of the skin. In: Glynn LE, ed. Tissue Repair and Regeneration New York:
13.
Wikesjö UME, Crigger M,
Elsevier/North Holland, Biomedicai Press; 1981: 309-342.
Steenberghe D. Periodontal aspects of osseointegrated oral implants modum Branemark. Dent Clin North Am 1988; 32:355-370. 8. d'Hoedt R. The influence of implant materials and material surfaces on blood clotting: A scanning electron microscopic study. In: van Steenberghe D, Albrektsson T, Branemark PI, et al., eds. TissueIntegration in Oral and Maxillo-Facial Reconstruction. Amsterdam: Excerpta Medica; 1985; 46-50. 9. Hoffman AS. Blood-biomaterial interactions: An overview. In: Cooper SL, Peppas NA, Hoffman AS, Ratner BD, eds. Biomaterials: Interfacial Phenomena and Applications, Advances in Chemistry Series, vol. 199. Washington: American Chemical Society; 1982; 3-8. 10. Trowbridge HO, Emling RC. The blood leukocytes. Inflammation: A Review of the Process. Chicago: Quintessence Publishing Company; van
1989: 39-62. 11. Clark RAF. Overview and
general considerations of wound repair.
KA.
Early healing
Light
and transmis-
sion electron
14.
59:403-411. 7.
R, Selvig
events at the dentin-connective tissue interface.
15.
Nilveus
microscopy observations. / Periodontol 1991; 62:5-14. Nasjleti CE, Caffesse RG, CasteiiI WA, Lopatin DE, Kowalski CJ. Effect of lyophilized autologous plasma on periodontal healing of replanted teeth. /Periodontol 1986; 57:568-575. Ito Y, Imanishi Y. Blood compatibility of polyurethanes. In: Williams D, ed. Critical Reviews in Biocompatibility. Boca Raton, FL: CRC
Press; 1989; 45-104. 16. Poison AM, Proye MP. Fibrin linkage: A precursor for new attachment. / Periodontol 1983; 54:141-147. 17. Block MS, Kent JN, Kay JF. Evaluation of hydroxyapatite-coated titanium dental implants in dogs. J Oral Maxillofac Surg 1987; 45:601608. 18. Cook
SD, Kay JF, Thomas KA, Jarcho M. Interface mechanics and histology of titanium and hydroxyapatite-coated titanium for dental implant applications. Int J Oral Maxillofac Implants 1987; 2:1-15.
Send reprint requests to: Dr. A.D. Steinberg, University of Illinois at Chicago, College of Dentistry, 801 South Paulina St., Chicago, IL 60612. Accepted for publication, June 12, 1992.
In-Vivo Comparisons of Clot Formation on Titanium and HydroxyapatiteCoated Titanium Arnold D.
Steinberg, * Ruth Willey,T and James L. Drummond*
We investigated in vivo clot formation on the surface of hydroxyapatite (HA)coated titanium (Ti) implants and on non-coated Ti implants. Immediately after tooth extraction implant samples were inserted into the blood clot, in the same extraction site, for 1, 30, 60, or 120 seconds. Samples were processed for scanning electron microscopy (SEM). Qualitative observations of clotting topography were made by direct SEM viewing. Neither of the implant surfaces appeared to differ markedly in the degree of clotting during the 120 seconds of implantation; they revealed very early clot formation and limited clot attachment. These results were compared to the findings obtained in a previous study using identical methods with an intact periodontal ligament (PDL), root planed roots, and roots planed and treated with pH 1 citric acid. The PDL surface had the most rapid clot formation at all time periods. By 120 seconds, all root surfaces had completed clot formation. J Periodontol 1992; 63:990-994.
Key Words: Dental implants; titanium; hydroxyapatite; blood coagulation.
Titanium (Ti) dental implants are designed to enhance rapid bone growth around the implant and provide mechanical retention by osseointegration.1 Hydroxyapatite (HA) coated Ti is also currently used as a dental implant material because its direct and rapid surface interaction with adjacent bone2*4 results in bioactive retention or biointegration.1 The first interaction of an implant surface placed in intimate contact with freshly-prepared bone is with clotting blood. This initial interaction results in a coagulation cascade which, if undisturbed, will undergo organization, incorporation of a variety of cellular elements, and finally, bone formation resulting in osseointegration of the implant. Deviation from this biological order of events may result in fibrous tissue formation around the implant. Thus, we believe it is essential that we understand the initial blood interaction with the implant surface to determine if differences in blood clot formation exist between those surfaces leading to fibrous tissue formation and those surfaces leading to osseointegration. Additionally, such findings must be compared to our human tooth replantations (under similar conditions) having a variety of root surface treatments.5,6 van Steenberghe,7 in a review, suggests that rapid formation of a blood clot favors rapid bone apposition and osseointegration of the dental implant rather than fibrous scar formation. d'Hoedt,8 using scanning electron micros-
*Departments of Periodontics and Biochemistry, University of Illinois Chicago, Chicago, IL. Department of Biological Science. •Department of Operative Dentistry.
at
and freshly drawn human blood, observed in clot formation at 30, 90, and 240 seconds after that vitro, aluminum and Ti oxide surfaces was more on application stainless steel. than on He, too, suggested the imrapid formation for enhanced bone formation. of clot portance rapid This study was designed to evaluate and compare, in vivo, the rapidity of clot formation and maturation on HAcoated Ti implants and on non-coated Ti implants in humans. It was also designed to compare these results with a recent investigation, performed under identical conditions, where intact periodontal ligament (PDL) and periodontally diseased root surfaces with variety of root treatments were studied.6 Evaluation of the clot forming potential of these 2 types of implant surfaces is intended to serve as a measure of potential biocompatibility and provide information about the initial events leading to osseointegration and copy
(SEM)
biointegration. This protocol for human investigation was approved by the University of Illinois Institutional Review Board. The procedure and purpose of the project was described to each patient prior to signing the consent form. MATERIALS AND METHODS Dental Implant Materials The dental implant samples were prepared by the manufacturer5 of material identical to that used in their commer-
§Calcitek, Inc., Carlsbad, CA.
Volume 63 Number 12
STEINBERG, WILLEY,
daily available HA-coated implants. The HA was plasma sprayed onto 2 mm thick Ti (TÌ-6A1-4V) plates and sectioned into pieces 5 mm x 10 mm. The samples had HA (coated) on one surface and only Ti (uncoated) on the other surface. They were then sterilized by gamma irradiation and packaged in individually sealed containers for storage. Patient Selection and Extraction Site Selection
Healthy volunteers undergoing uncomplicated tooth extraction at the Oral Surgery Clinic at the University of Illinois at Chicago Dental School were used in this study. Informed consent for the short-term implantation of the dental implant materials into the sockets of freshly extracted teeth was obtained from all patients. Only patients who had not taken any medication for 10 days were selected. The extraction sites chosen were from an area of the mandible where complete anesthesia could be achieved with block anesthetic. This prevented infusion of local anesthetic and vasoconstrictor drugs into the clotting extraction site. Placement of Implant Into Extraction Site
Immediately after extraction, one sample at a time was inserted into the blood clot in the extraction site for 1, 30, 60, and 120 second time periods. These time periods were selected for comparison with human root replantations in a previous study.6 The "1" second time was the time required to insert the sample into the clot in the extraction site and remove it immediately. The samples were held in the extraction site by locking forceps. One extraction site was used for 4 implant samples for each of the 4 time periods. A blood clot was always present in the extraction socket into which the samples were inserted. Five different patients were used, thus 5 samples were obtained for each time period. Preparation
of
Samples
for SEM
Immediately upon removal of the implant material from the extraction site, the samples were rinsed in sterile saline and fixed initially in 0.1% glutaraldehyde (GA) in 0.1 M phosphate buffer, pH 7.2 for 30 minutes at room temperature. This was followed by immersion into 2% GA in 0.1 M phosphate buffer and refrigerated at 4° C. for 24 hours.6 Stereoscopic microscope observations (20 x ) of the amount of blood clot attachment to the implant surface were made immediately after fixation. Samples were washed in distilled water, mounted on stubs, frozen in liquid nitrogen, and dried for 12 hours in a Pearse freeze drier. After coating with 120 A gold, the samples were examined with an ISI DS-130 SEM at 10 kV. SEM Observations
Qualitative observations were made by direct SEM viewing and
appropriate photographs of clotting topography. These included observations of film deposits, initial platelet attachment and aggregation, the presence of erythrocytes or any other blood cells and fibrin formation, and lysis. It was
DRUMMOND
991
Table 1. Blood Clot Formation on Titanium (Ti) and Hydroxyapatite-Coated Titanium (HA) Implant Surfaces
Time
Material/
Sample
Number
(seconds)
30
60
120
1.5 1.0 1.0 2.5 1.0
1.0 1.5 1.0 1.5 1.0
1.0 1.0 1.0 1.0 1.0
1.0
1.0 1.0 1.5 3.0 1.0
1.0 1.5 2.5 2.0 2.0
1.5 1.0 1.0 1.0 2.5
Ti 1 2 3 4 5 HA 1 2 3
The numbers
2.0 1.5 2.0 1.0
4.0 2.5
(1 through 5) represent an arbitrary rank classification.
assumed that the film deposits observed on some of the surfaces after blood contact were plasma proteins. The observations were made by dividing each implant sample into quadrants and evaluating one arbitrarily chosen site from each quadrant. A mean was then obtained from the numerical evaluation of the 4 observations for each of the implant samples (Table 1). The following rank classification of the degree of clot formation and prepared for assessing the degree of clotting at the observed sites:6 1. Film may or may not cover surface. No blood elements or fibrin observed. 2. Initial clot formation: plasma protein layer may be visible, fibrin strands with or without the presence of scattered and aggregating platelets. 3. Organized clot: surface layer not visible, organized mesh-work consisting of aggregating platelets, fibrin, and
erythrocytes. 4. Retracting clot: dense fibrin mesh, platelets not visible, few erythrocytes. 5. Lysis of clot: dense fibrin mesh disappearing, eryth-
to be present outside the remnants of the fibrin mesh. No statistical evaluations were made of these observations due to the limited number of samples.
rocytes appear
RESULTS The photomicrographs in Figures 1 through 5 show the control surfaces and examples of clotting at the different time periods that Ti and HA were exposed to clotting blood in the extraction sites. On most of the implant surfaces, initial blood clotting was observed and included fibrin formation, along with aggregating platelets or individual platelets. On all Ti surfaces the implant surface was visible, while on the HA surfaces, the implant surface usually was completely or partially obscured by fibrin and/or a film assumed to be plasma protein. The data in Table 1 show that the Ti surfaces had clot grading from 1.0 to 2.5 while the HA surface grading ranged from 1.0 to 4.0. On the HA surface only 2 of the surfaces
992
IN VTVO CLOT FORMATION ON IMPLANTS
Figure
1. Control specimen
showing
Ti
surface (bar
=
10
J Periodontol December 1992
µ/ ).
Figure
4. Control
specimen showing HA-coated
surface (bar
Ti
=
10
-m.
Figure 2. One-second implantation of Ti sample into an extraction site showing limited fibrin formation with scattered and aggregating platelets. 10 µ/ ). The underlying Ti surface is visible (bar =
•¿ Ai extraction site Figure Thirty-second implantation sample showing fibrin formation along with some aggregating platelets. The underlying HA surface appears obscured (bar 10 µ/ ). ·.
5.
of HA
'
into
an
=
had a grade of 3.0 or higher. Thus, no marked differences in the degree of clot formation appeared to be present between the Ti and the HA surfaces at the various time periods. Stereoscopic microscope observations were made to estimate the degree of blood clot covering the implant surfaces. These observations suggested limited but similar degrees of blood clot covering both the Ti or HA coated implant surfaces.
Figure 3. One hundred-twenty second implantation of Ti sample into an extraction site showing fibrin formation and some aggregating platelets. 10 µ/ ). The underlying Ti surface is visible (bar =
DISCUSSION Blood clot formation is the first event in all wound healing. Intravascular blood studies show that initial clot formation begins with plasma protein adsorption at the injured site, rapidly followed by platelet attachment, aggregation, and activation. These platelet events are usually accompanied by the specific extrusion of granule-bound substances.9'10 Platelets are an important initial component of early clot formation at the wound site.9-11 Concomitantly, fibrin strands
Volume 63 Number 12
appear along with an influx of erythrocytes. This rapidly leads to an organized meshwork consisting of platelets, fibrin, and erythrocytes. Clot retraction and lysis rapidly follows.6 Within several hours polymorphonuclear leukocytes accumulate at the wound site and their numbers peak within 24 to 48 hours. They are followed by macrophages which appear to reach their peak at about 3 days.12 The very early events in extravascular blood clotting at the interface of the root surface and supporting connective tissues during healing are poorly understood.5-6,13 However, events similar to those described for other wound sites occur."12 Most periodontal wound healing studies focus on interpretation of observations one day or longer after the initiation of wound healing.14 Wikesjö et al.,13 using dogs having small dentin blocks surgically implanted in bone cavities in edentulous alveolar ridges, observed healing at the dentin-connective tissue interface as early as 10 minutes after wound initiation. This study is focused upon in vivo time periods in humans at the initiation of wound healing and at time periods up to 2 minutes. We suggest that the initiating factors in wound healing, namely clot formation, are responsible for inducing the cascade of events which will finally result in the union of the periodontium with the root surface. We have shown that during the first several minutes of the healing process on root surfaces, platelets are the initial cellular elements to become involved at the site of injury and appear to disappear within 2 minutes as fibrinolysis appears. Very limited numbers of polymorphonuclear leukocytes or other blood cells were observed during this time period.6 Neither the Ti or HA-coated implant surfaces in this study appeared to differ markedly from each other in the degree of clotting during the 120 seconds of implantation into the extraction sites (Table 1). At nearly all time periods only very early clot formation was observed (Figs. 2, 3, and 5) and a limited number of surface sites showed frank, macroscopic clot attachment. On all Ti observations, there were no visible plasma protein deposits to obscure the implant surface (Figs. 2 and 3). On HA-coated surfaces (Fig. 5) variability was found in our ability to observe the underlying implant surface. This suggests that in the Ti samples there either were no such deposits or the deposits were present in such limited amounts that the surface of the implant was still visible. If the implant clotting on Ti and HA coated surfaces in Table 1 is compared to identical experiments with root segments, it can be observed that, on root surfaces with intact PDL, a more advanced clot formation occurred for all time periods and clotting was completed before 120 seconds. Periodontally-diseased root surfaces, treated by root planing or root planing followed by 3 minute pH 1 citric acid application, also had blood clotting completed within the 120 seconds of replantation (grade 5). In all observations of clotting on the root surfaces, the surface was obscured by either the clot or a film assumed to be plasma protein.5,6 Hemocompatibility of a surface is believed to be deter-
STEINBERG, WILLEY, DRUMMOND
993
mined by the amount and composition of the complex array of plasma proteins deposited by blood on a surface.15 Fibrin clot adhesion to the dentin surface appears to be necessary for complete periodontal regeneration.5,6,13,16 It is possible that lack of, or limited, clot adhesion to the implant surface may have been responsible for the differences in rate of blood clot formation and the amount of surface clot attachment between the artificial implants and the root surfaces. Bone formation and its maturation occur faster on HAcoated Ti implants than on non-coated Ti implants.17,18 Since enhanced bone growth may be preceded by rapid clotting,7,8 this suggests that clotting should have occurred faster on the HA-coated Ti implants than on non-coated Ti implants. Our results do not support the observations of van Steenberghe7 and d'Hoedt8 that there is rapid blood clot production on Ti or HA implant surfaces. This discrepancy may be due to the fact that the current study was performed in vivo in fresh extraction sites, while d'Hoedt's study8 was in vitro. The 120-second time period selected for these experiments and previous studies is an arbitrary time period chosen for convenience of observation and comparisons. At this time, it is not possible to conclude that clot formation must be completed within 120 seconds or other time periods for successful healing to occur. While clot formation appears to be complete within this time frame on natural root surfaces, this does not appear to be so on either of the implant surfaces. In summary, our study suggests that there is a markedly slower rate of blood clot formation on the artificial surfaces when compared to the root surfaces. Interpretation of these observations in terms of healing rate on Ti and HA surfaces when compared to the natural root surface is currently not possible. Our future studies will establish the time required to complete clot formation along with evaluation of the tenacity of blood clots to Ti and HA-coated surfaces. Such studies are essential for comparisons of the initial wound healing events at artificial and natural surfaces interacting with adjacent connective tissue.
Acknowledgments
gratefully acknowledge Dr. L. Mercuri, Head of the Department of Oral Surgery, University of Illinois at Chicago, for his help in obtaining patients, and Mr. R. Wibel for technical assistance with the SEM. This investigation was funded by Calcitek, Inc. Carlsbad, CA., and the George Matula Fund, University of Illinois at Chicago. We
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Send reprint requests to: Dr. A.D. Steinberg, University of Illinois at Chicago, College of Dentistry, 801 South Paulina St., Chicago, IL 60612. Accepted for publication, June 12, 1992.
