Success of osteogenesis in bone graft procedures can be enhanced by inhibiting oral bacterial infections through the use of prophylactic bacteriostatic fluoride within the grafting environment. Ideally, the fluoride ion should be chemically sequestered and thus unavailable unless needed at times during the process of early infection. As fluoride within fluorapatite is tightly bound at neutral pH and becomes available only during acidic conditions, fluorapatite is an ideal store for the fluoride ion which becomes released for bacteriostasis only during an acidic environment found with incipient bacterial infection. The purpose of this investigation was to compare the histologic properties of new bone formed surrounding fluorapatite (FA)-coated microcrystalline hydroxyapatite (HA) grafting material with comparable bone formed following the use of control HA material (OsteoGen, Impladent, Ltd, Holliswood, NY). The results of histologic analysis within dog studies here showed no detectable difference in new bone following therapeutic grafting procedures using each of the above 2 mineral coatings.
Infection, either immediately subsequent to placement of dental implants or following initial osseointegration, constitutes a significant problem for both the dentist and patient. The obvious consequence of bone loss in the immediate vicinity of the implant is a major threat to the success of the procedure. The author (W.N.) has tried a variety of therapeutic schemes over the years to mitigate such infections using decontamination procedures and grafting, but from an overall perspective, results have been less than satisfactory.
The literature is replete with investigations that support the fact that fluoride is efficacious in the prevention of dental caries.1–5 Further, fluoride is known to inhibit many other types of oral bacteria, including the Treponemas6 which are implicated, along with numerous bacterial species, in the pathogenesis of periodontal disease. It is clear that in order to promote an ideal microenvironment for the normal healing of bone during implant procedures and thus reduce the chance of superimposed infection, bacterial contamination must be held to a minimum. Therefore, if bacteriostatic fluoride ion could be made available directly to the sensitive osteogenesis microenvironment at the time when it is most vulnerable to incipient bacterial contamination, it is logical to expect that such fluoride presence would be of help in reducing bacterial infection and enhancing clinical success of surgical grafts.
It is well acknowledged that oral bacteria lower the pH of the microenvironment as they infect a surgical bed, thus inhibiting the formation of new bone in the area. Therefore, it is theorized that if the fluoride ion could be introduced directly at the site of osteogenesis, this fluoride would inhibit acid-forming bacteria precisely where it is likely to be of most help,7 thus allowing the grafting procedure to progress uneventfully with the uninhibited production of viable new bone.
Electron microprobe microanalysis8 supports the fact that hydroxyapatite at the tooth surface, when exposed to the fluoride ion, converts to fluorapatite through dissolution of the surface molecules of hydroxyapatite and subsequent fluorapatite recrystallization. The anticaries and bacteriostatic beneficial agent of the fluoride treatment is the fluoride within fluorapatite.
Fluorapatite (FA) is a very insoluble mineral in a neutral medium. However, in the presence of acid; fluoride and phosphate are dissolved slowly and liberated into solution. Normal tissue is neutral to slightly basic. Since dissolution of fluoride from FA requires a lowering of the pH, FA would seem to be an ideal delivery molecule for inhibitory fluoride ions when bacteria colonize and subsequently attempt to acidify a grafting environment and produce biofilms.9 When used as a bone grafting agent, FA would act as a source of fluoride ion released from the acidic microenvironment of early infection, thus inhibiting further infection and allowing grafted bone in the defect to be converted to new viable bone.
Physical properties of fluoride-treated synthetic, commercially available grafting material, fluoridated hydroxyapatite, (OsteoGen, Impladent, Ltd, Holliswood, NY) have been investigated.10 The purpose of this investigation (Part II of a III-part series) was to compare the histologic properties of bone graft osteogenesis in the dog model using a surface layer of nanocrystalline FA with a similar control graft using microcrystalline hydroxyapatite (HA) that lacks any surface fluoride component.
ethods and M aterials
Having completed all the necessary research protocol and anesthesia, the bicuspids and molars were extracted from all 4 quadrants of a beagle dog. Three months later, a conventional CT scan was performed, and a CT-generated model was constructed of the lower mandible (Figure 1). The laboratory prepared osteotomies bilaterally in the simulated plaster mandibular edentulous areas that approximated 12 mm in length, 10 mm in depth, and 7 mm in width (Figure 2). Custom cast titanium cages were constructed using the lost wax method that inlayed precisely into and duplicated the exact position of the cortical plate (Figure 3). Each cage was designed with 8 retention screw holes. The castings were then coated with a 30-micron thick layer of plasma HA. Clear acrylic transfer templates were constructed to transfer the precise anatomy of the osteotomies from the CT model to the actual dog mandible.
The dog was again prepared for surgery in the customary manner. The tissue was flapped and the transfer templates were placed. Using bone saws and high-speed carbide dental burs, the osteotomies were prepared to depth. Cages were soaked in platelet-rich plasma (PRP). The left cage was filled with PRP-soaked crystalline resorbable control HA (OsteoGen). The cage was screwed to place, and the tissue was approximated and sutured. The right cage soaked in PRP was filled with PRP-soaked crystalline fluoridated HA (FA) material.
Synthesizing fluorapatite-coated HA
The process of synthesis involved the steps outlined below.
0.43 g (or approximately 0.5 mL by volume) of sodium fluoride (NaF) powder was placed into 10 mL of water (distilled, saline, or sterile irrigation water). This represents a 4.3% neutral solution of NaF that was used both for the treatment of the titanium implant surface and for reacting with the HA to produce FA-coated HA.
The required amount (1–3 g) of hydroxyapatite was mixed in the 4.3% fluoride solution and reacted for 2 minutes.
The excess fluoride solution was decanted from the FA-coated HA powder.
The powder was washed 3 times with water (distilled, saline, or sterile irrigation water), and the liquid was decanted off each time. The powder was then dried routinely.
The fluoridated hydroxyapatite was now ready for the grafting procedure. This FA powder can be mixed with other grafting material (ie, dense HA, autogenous bone, or allograft material).
Washing fluoridated HA
“Washing” is a chemistry term used for removing any excess reactants from the solution after a chemical reaction is completed. There is often excess fluoride ion in solution after 2 minutes reaction of F ion with the HA. The reaction consists of surface substitution of OH ion with the F ion forming a nanocrystalline tube of FA around the microcrystals of HA. The wash is done to remove any excess fluoride from the solution before using the FA-coated HA for grafting. This is thought to be important since early trials with nonwashed material showed local interference with clotting of blood. When washed, no interference with clotting was observed ever again.
The FA-filled cage was then placed and secured with titanium screws. The tissue was approximated and meticulously sutured to achieve a water-tight seal without any stretching.
The dogs were euthanized 4 months later, and the sectioned jaws (Figure 4) were placed in formalin solution and sent to Dr Jack Lemons (University of Alabama at Birmingham, Center for Metabolic Bone, Birmingham, Ala) for processing and histology. The specimens were fixed in buffered formalin, transferred, sectioned by Exakt (R&M Biomaterials, Nashville, Tenn) processing and imaged by Bioquant optical microscopy.
Clinical observations of the dog jaws at the time the cages were harvested showed a distal dehiscence on the left HA control side at the distal buccal location of the cage (Figure 4). Infection, as evidenced by pus and inflamed tissue, was observed inside the wound and at the edge of the left titanium cage. The right FA side showed no evidence of dehiscence or infection.
The histology showed that both right and left sides exhibited viable new bone within the cage structures. Both sides showed substantial integration of new bone on the HA or FA-coated cast titanium cage screen several millimeters distant from the original bone osteotomy. Both sides show substantial bone formation that intimately engulfed the augmentation microcrystals of both HA and FA. No differences in bone reaction to either HA or FA crystals were observed (Figures 5 through 10).
The notion that fluoride prevents caries has been researched more than any other concept in dentistry. More recently, however, it has been found that fluoride also inhibits the Treponemas,6 which are implicated along with the numerous other species of bacteria in the pathogenesis of periodontal disease. Ten years ago, one of the authors (W.N.) began fluoridating ceramic nonresorbable HA used during augmentation procedures, and experienced mixed results. However, 5 years ago, this author changed the treatment regimen and began to use fluoridated resorbable crystalline HA (OsteoGen). The author noted that the fluoridated material produced superior clinical results when grafting defects created by infection around ailing and failing dental implants as compared with the nonresorbable ceramic material. In one case, an adjacent crowned tooth proved necrotic and caused a draining fistula on an adjacent implant. The tooth was treated endodontically, and a periapical procedure was done to seal the apex concurrently with the revisional surgery. The bone loss was grafted by first performing a decontaminating procedure. The implant was etched with a concentrated solution of citric acid until the surface was pristine clean. The area was then washed briefly with 4.3% NaF solution to achieve as much fluoride exposure (fluorapatite conversion) in the native bone as possible. The area was then washed thoroughly to remove excess fluoride. FA-coated HA was then used to graft the defect. The tissue healed normally, and radiographs taken 3 years postoperatively showed bone growth into the area with no persistent or residual infection.11 Many other similarly infected implants were saved over the years using this method.
The theory of explaining the efficacy of fluorapatite in this application is that the fluoride ion inhibits acid-producing bacteria, thus helping to maintain a normal acid-base balance necessary for proper osteogenesis. Treponemas thrive in an acid environment. When FA is present in the augmentation graft material, acid-producing bacteria cannot produce acid without leaching the fluoride ion from the fluorapatite crystal. As soon as bacterial metabolism begins, pH diminishes, thus allowing fluoride to be released as it is dissolved from fluorapatite in the acid environment. The fluoride ion is then available to exert its inhibitory effects on local bacteria, and as a consequence, the pH is somewhat stabilized and not reduced sufficiently to sustain infection. The bacteria, unable to thrive in this less-than-acidic microenvironment, therefore perish or are vulnerable to be overcome by mechanisms of natural resistance inherent in the inflammatory process.
The above-described decontamination procedure has been utilized successfully for ailing and failing implants for the past 5 years. This, in addition to the subsequent grafting with fluoridated FA, has been so profoundly successful on a clinical basis that the author felt that the science of fluoridated HA should be reviewed in a more disciplined manner.10 Further, in light of the clinical success as reported in the above anecdotal reports, it would seem both logical and prudent to follow these case reports with controlled histologic and clinical studies in an attempt to document and support clinical observations and establish accepted clinical protocols. This paper serves to demonstrate that there is no difference in the bone histology between implants employing HA and implants employing FA-coated HA after an augmentation procedure. Crystals of each were engulfed with new viable bone, and there was no detectable difference in bone histology taken from specimens utilizing either of the two materials.
Additionally, it was observed that the fluoridated right jaw in the experiment was free of clinical infection, but this was not the case in the contralateral control side using nonfluoridated HA. More collaborative studies with adequate controls are needed to support indications here that fluoridation is beneficial as an infection-preventive component in bone grafting procedures.
Another point worthy of note is the serendipitous observation that bone was integrated to the cast HA plasma-coated titanium mesh several millimeters distant from the edge of the original bone osteotomy. This growth of new bone, which coated and integrated to the HA-coated titanium mesh distal to any viable bone, indicates the possibility of an osteogenic potential of HA-coated mesh cages grafted with fluoridated HA augmentation material. This could allow for possible recreation of a missing cortical plate in a catastrophic fracture. Cast HA plasma-coated titanium cages constructed to bridge cages in the bone resulting from fractures, gun shot wounds, or infections might be possible using this methodology. Also, nonunion fractures could possibly be approximated and bridged in a similar manner.12
The purpose of this investigation (Part II of III articles) was to compare the histology of bone-graft procedures using FA-coated synthetic implant material with that of procedures using crystalline HA. Results in this dog study showed there was no difference in bone histology between the new bone integrated in intimate contact with the FA-coated HA and the bone surrounding control HA. Further, it is possible that implant failures due to acid-producing bacterial infection could well be inhibited or prevented if the fluoride ion could be introduced directly and specifically to the sensitive peri-implant microenvironment.
Fluorapatite is proposed as an excellent storage reserve vehicle for delivery of reactive fluoride when needed at the site of bacterial infection. The fluoride ion in the fluorapatite crystal lattice becomes available to inhibit bacteria in an acidic environment; otherwise the ion is sequestered, essentially insoluble and unavailable. Fluorapatite would thus seem to be an excellent fluoride carrier. The purpose of this investigation was to compare the histology of new bone growth around bone graft materials in dogs using FA-coated implants and control HA. The results showed no difference in the bone histology between the two minerals.