J Oral Maxillofac 50359-667.
Surg
1992
Chemical, Physical, and Histologic Studies on Four Commercial Apatites Used for Alveolar Ridge Augmentation E.M. PINHOLT, DDS, MS, DRODONT,* I.E. RUYTER, DRENGSc,t H.R. HAANAES, DDS, MD, DRODONT,$ AND G. BANG, DDS, MD, DRMEDS The purpose of this study was to evaluate four commercial apatite products. Subperiosteal alveolar ridge augmentation was performed on the maxilla of rats by implantation of granules of two dense products and of two porous products, and the tissue response was compared with the material characteristics obtained by chemical analysis and infrared spectrometry. None of the apatites caused osteoinduction or osteoconduction; fibrous encapsulation with multinuclear giant cells was observed around all four types. One of the apatites was fluorapatite and not hydroxylapatite, as claimed by the manufacturer. The tissue response to this implant material was dominated by multinuclear giant cells. several bone substitutes in the same anima1.5-7Material characteristics are often not reported in conjunction with evaluation of tissue responses to commercially available apatite implant materials. The purpose of this investigation was to evaluate the biological reactions to granular Alveograf, Calcitite, Interpore-200, and Algipore when implanted subperiosteally for alveolar ridge augmentation and to correlate
Some calcium phosphates, such as hydroxylapatite (HA) [Ca,o(P04)6(OH)~], are claimed to be biocompatible and osteoconductive.’ Osteoconduction is a healing process in which the implanted material acts as a scaffold that is gradually resorbed during creeping substitution as the healing proceeds from the adjacent living bone.2 With osteoinduction, on the other hand, mesenchymal cells are transformed into cartilage and/ or bone-forming cells by a local stimulating factor.334 Hydroxylapatites may be developed synthetically or by treatment of algae and corals by hydrothermal processes5 Because of chemical and geometric similarities to HA of vertebrate bone and teeth, it has been used widely for implant purposes.’ Most investigations have been performed using one product in one species. Few reports are available using * Associate Professor; previously. Assistant Professor. Department of Oral Surgery and Oral Medicine, Dental Faculty, University of Oslo; Research Fellow, Norwegian Research Council for Science and the Humanities, Institute for Surgical Research, Rikshospitalet, Oslo. Norway. t Senior Scientist, Physical/Chemical Division, NIOM, Scandinavian Institute of Dental Materials, Haslum, Norway. $ Professor and Chairman, Department of Oral Surgery and Oral Medicine, Dental Faculty, University of Oslo, Norway. 4 Professor and Chairman, Department of Oral Pathology and Forensic Odontology, University of Bergen, Haukeland University Hospital, Bergen, Norway. Address correspondence and reprint requests to Dr Pinholt: Department of Oral and Maxillofacial Surgery, Royal Dental College. 20, Notre All&, DK-2200 Copenhagen N, Denmark. 0 1992 American
Association
of Oral and Maxillofacial
FIGURE I. Transverse view of rat maxilla showing location of apatite implants. A, Alveograf; B, Calcitite; c, Interpore-200: D, Algipore.
Surgeons
0278-2391/92/5008-0012$3.00/O
859
880
RIDGE AUGMENTATION BY APATITES
Table 1.
Investigated Apatite Materials
Product Alveograf mesh: 18/40 Calcitite mesh: 20140 Interporemesh: 28135 Algipore HA-Keramik size: 0.5-l mm
Producer
Batch No.
Cook-Waite, New York, NY Calcitek, San Diego, CA Interpore Int, Irvine, CA Friedrichsfeld, Germany
S 008 AC
Origin
Norway, the Inter-pore-200 from Immuno, Copenhagen, Denmark, and the Algipore from Ronvig A/S, Daugaard, Denmark).
Synthetic SURGICAL PROCEDURE
870523
Synthetic
9142
Coral derived
l-10/89
Algae derived
the tissue reaction to the material characteristics obtained by chemical analysis and infrared spectrometry. Material and Methods
The four apatite products were purchased from the manufacturer or from governmental authorized sales representatives in the same way as products are purchased for clinical use (the Alveograf from the manufacturer, the Calcitite from Peter Msller A/S, Oslo,
FIGURE 2. Rat alveolar bone (AB) augmented with granules of Alveograf(AG) covered by oral mucosa, including the epithelium (E). The area formerly occupied by the implant material becomes empty during the decalcification process (hematoxylin-eosin stain, original magnification X 17.5).
Ten male Wistar rats weighing 267 f 11 g were used. They were fed a standard diet (standard diet for rats, Rikshospitalet, The National Hospital, Oslo, Norway) and given water ad libitum. Surgery was done under general anesthesia, Hypnorm (Janssen Pharmaceutics, Beerse, Belgium)/Dormicum (Roche, F. Hoffman-La Roche, AG, Basel, Switzerland) 0.15 mL/ 100 g intramuscularly (1:Hypnorm + 1:sterile water mixed with 1:Dormicum + 1:sterile water, Rikshospitalets Apotek, Oslo, Norway). Incisions were made to bone bilaterally on the premaxilla where the horizontal plate of the hard palate continues into the vertical lateral plate between the incisor and the first molar (Fig 1). Subperiosteal dissection was carried out to create small pockets on each
FIGURE 3. Higher magnification of Figure 2. Ridge augmentation of rat alveolar bone (An)with granulesOfAlVeOgraf(AG).The granules are encapsulated by fibrous connective tissue (FT)with multinuclear giant cells. No osteoinduction or osteoconduction is observed (hematoxylin-eosin stain, original magnification X70).
861
PINHOLT ET AL
side. Posteriorly and laterally on the maxilla in the first molar region an additional incision was made bilaterally and subperiosteal dissection was carried out to create two more pockets. Alveolar ridge augmentation was performed by implanting four granules of an apatite product into each pocket. On the right side, Alveograf was placed anteriorly and Calcitite posteriorly (Table 1). On the left side, Interporewas placed in the anterior pocket and Algipore in the posterior. The surgical sites were closed with Prolene monofilament suture. The control group was comprised of four male Wistar rats.* The sham operations were performed by creating one pocket on the premaxilla, but no implantation was performed.* CLINICAL AND HISTOLOGIC EVALUATIONS
To observe wound healing, clinical evaluation was performed daily the first 3 postoperative days and thereafter on a weekly basis. The animals were weighed at the beginning and at the end of the experimental period. After 8 weeks, the animals were killed. The maxilla was immediately removed from the animal and fixed in 4% neutral formalin. Radiographs were used to locate the implants and possible hard-tissue formation. The specimens were then demineralized in 17% formic acid, dehydrated, and embedded in paraffin. Serial sections were cut at 5 grn and stained with Harris hematoxylin and eosin for light-microscopic examination. INFRARED SPECTROSCOPICALCHARACTERIZATION
The granules of the four proprietary apatite materials were ground to a fine powder and mixed during grind-
FIGURE 5. Rat alveolar bone (AB) augmentation with Calcitite (cr). Three granules of the Calcitite have been implanted into an accidentally created bony defect of the alveolar bone (large arrows). One granule has remained on top of the alveolar ridge (small arrow). Oral mucosa, including the epithehum (E), covers the alveolar bone (hematoxyhn-eosin stain, original magnification x20).
ing with potassium bromide (KBr, Uvasol-spectroscopic grade, Merck, Germany). Pellets with a diameter of 13 mm were made. Infrared (IR) spectra were recorded on a dispersive ratio recording IR-spectrophotometer (model 683 with Model 3500 Data Station, Perkin-Elmer Corp, Norwalk, CT). The IR-spectrophotometer was purged with dry air from a self-generative heatless air drier (model X3HA, Pall Pneumatics Ltd, Portsmouth, England), and operated with slit 6 and noise filter 2 for the 4000 to 200 cm-’ range. CHEMICAL ANALYSIS
FIGURE4. Higher magnificationof Figure3. Rat alveolarbone separatedfromgranulesof Alveograf(AC) by fibrousconnective tissue (FT)containingmultinucleargiant cells (cc). The edgesof the Alveografareresorbed by the multinuclear giant ceils (CC).The areas (AB)
formerly occupied by the microparticles (MC) of the Alveograf are observed inside the multinuclear giant cells. No osteoinduction or osteoconduction is noted (hematoxylineosin stain, original magnification X280).
The apatite samples were dissolved in nitric acid under pressure. The solutions were analyzed semiquantitatively by inductively coupled plasma atomic emission spectrometry (ICP-AES) (ICP Series 2, PerkinElmer Corp). The fluoride content of Algipore was determined quantitatively by ion selective F-electrode analysis (Sintalyzer-System, SINTEF, Trondheim, Norway).
862 Results CLINICALAND HISTOLOGIC EVALUATIONS All animals gained weight during the observation period (weight at the beginning, 267 f 11 g; at end, 443 k 21 g). No infection was observed at any time. All the implants were recovered from the sites of implantation (Fig 2). The region formerly occupied by the implant material became empty during the decalcification process. AlveograJ: Fibrous encapsulation with multinuclear giant cells was observed around the Alveograf (Figs 3,4). Newly formed bone was not observed, but multinuclear giant cells were seen at the edges of the implant site (Fig 4). Calcitite. Granules of Calcitite were in direct contact with, and surrounded by, osseous tissue when implanted into defects created accidentially by pressure
FIGURE 7. Higher magnification of Figure 6. Granule of Calcitite (CT) on top of rat alveolar bone (AB). Fibrous connective tissue (FT) with multinuclear giant cells (GC) envelopes the Calcitite granule. The edges show irregularities in the areas of the giant cells (arrows). The Calcitite does not cause osteoinduction or osteoconduction (hematoxylin-eosin stain, original magnification X 175).
on the alveolar bone (Figs 5,6). The edges of the bone that encased the granules of Calcitite were irregular (Fig 6). Granules on top of the alveolar ridge, and the part of the bony encased granules that were exposed to soft tissue, were covered by fibrous connective tissue containing multinuclear giant cells (Fig 7). New bone formation was not observed. Interpore-200. The Interporegranules were covered by fibrous connective tissue containing multinuclear giant cells (Fig 8). Empty spaces could be seen within the giant cells. These spaces, formerly occupied by the microparticles were now empty due to the decalcification process. No tissue infiltration into
LlGURE6. Higher magnification of Figure 5. Healing of an accidentally created defect in rat alveolar bone (AB) containing granules of Calcitite (CT). Bony tissue has encased the granules, which have irregular edges. The parts of the granules in contact with soft tissue are covered by fibrous connective tissue (FT). No osteoinduction or osteoconduction is observed (hematoxylin-eosin stain, original magnification X90).
FIGURE 8. Rat alveolar bone (AB) separated from granule of Interpore-200 (IP) by fibrous connective tissue (FT) containing multinuclear giant cells (GC).Areas formerly occupied by the microparticIes (MP) are noted inside the multinuclear giant cells (GC). The structure of Interporeis retained in the remnants of the material. There is no evidence that the granule resulted in either osteoinduction or osteoconduction (hematoxylin-eosin stain, original magnification X175).
863
PINHOLT ET AL
remnants of the apatite was noted and no new bone formation was observed. Mgipore. A fibrous capsule dominated by multinuclear giant cells surrounded the granules of Algipore (Figs 9, 10). The structure of the porous implant material was retained. Bony infiltration was not observed. Remnants of a homogeneous, acellular structure were noted (Fig 10). Where the Algipore had been implanted into an accidentally created defect in the alveolar bone, bone ingrowth was observed. Fibrous tissue with multinuclear giant cells had formed between the bone and the implant (Fig 9). Evaluation of the four control animals showed no osteoinduction or osteoconduction.8 IR SPECTROSCOPICALCHARACTERIZAT~ON Figure 11 shows the infrared spectrum Alveogruj of Alveograf with absorption peaks at 3,569 cm-’ and 630 cm-‘, representing monomeric OH- groups in the apatite structure. The absorption bands at I,09 1 cm-‘, 1,045 cm-‘, and 1,04 1 cm-’ are ascribed to PO:- group of HA. The absorption peaks at 568 cm-’ and 605 cm-’ can be assigned to antisymmetric bending motion of the phosphate groups. ” The presence of absorption bands at 436 cm-’ and 473 cm-’ accompanying reduction of OH- absorption at 630 cm-’ during heat treatment of HA is typical for slightly oxygenated HA. ’ ’ The band observed at 342 cm-’ can also be ascribed to a phosphate group of apatite.14 Culcitite. Figure 12 shows the infrared spectrum of Calcitite with a spectrum similar to that of Alveograf. Interpore-200. Figure 11 shows the IR spectrum of Interporewith absorption peaks representing the monomeric OH- groups of HA at 3,573 cm-’ and
(AB) with Algipore (AP) that has kept its structure. Fibrous connective tissue (FT) with multinuclear giant cells (cc) surrounds the implant. Osteoinduction or osteoconduction is not observed (hematoxyhn-eosin stain, original magnitication X25).
FIGURE 10. Rat alveolar bone
630 cm-‘. Absorptions due to carbonate ions of carbonated apatites are well represented. Absorption peaks at 879 cm-‘, 1,460 cm-‘, and 1,548 cm-’ represent CO; absorptions of type A carbonated apatite.‘2S’3 Carbonate absorptions from type B carbonated apatite are present at 872 cm-‘, 1,414 cm-‘, and 1,460 cm-‘. Algipore. Figure 13 shows the infrared spectrum of A&pore with two sharp absorption peaks at 3,645 cm-’ and 3,698 cm-’ representing monomeric OHgroups of a silicate material containing OM-.‘4 The broad band from 3,700 to 2,500 cm-’ is attributed to hydrogen-bonded stretching modes of water. The band at 1,650 cm-’ corresponds to hydrogen bending modes of water. The IR spectrum of Algipore had no absorption peaks representing monomeric OH- groups of HA at 3,570 cm-’ and 630 cm-‘. The spectrum had, however, absorption peaks at 1,425 cm-’ and 1,460 cm-‘, as well as 875 cm-‘, representing carbonate ions in type B sites of carbonated apatite. I5 The phosphate absorption band at 1,050 cm-‘, 1,096 cm-‘, 574 cm-‘, 567 cm-‘, 603 cm-‘, 468 cm-‘, and 350 cm-’ were present, as well as the apatite phosphate absorption bands at 965 cm-‘. COMPOSITION
FIGURE 9. A&ore (AP) implanted as alveolar ridge augmentation on rat alveolar bone (A@. Bone formation is seen around the granule of Algipore placed in an accidentally created defect. Note fibrous tissue (IT) with multinuclear giant cells (cc) on top of alveolar bone and between newly formed bone and implant (hematoxylin-eosin stain, original magnification X 175).
The results of the quantitative determinations of the elements present in the four apatite materials are shown in Table 2. The chemical composition of the synthetic materials Alveografand Calcitite, and the coral-derived Interpore-200, was consistent with a HA structure, whereas the Algipore was different. A fluoride analysis of the Algipore was carried out due to the absence of the absorption peaks at 3,570 cm-’ and 630 em-’ representing the monomeric OH- groups in the apatite structure. This analysis revealed the presence of 3.52
864
FIGURE 1I. 200 (B).
RIDGE AUGMENTATION
Infrared spectra of the Alveograf
(A)
and Interpore-
+ 0.04 wt-% fluoride. The semiquantitative analysis showed the presence of 2.5 wt-% magnesium in addition to calcium and phosphorous (Table 2). Discussion The IR spectra indicated that Alveograf and Calcitite were slightly oxygenated HA. The Interporecontained both type A and type B carbonated apatite accompanied by a reduction of the monomeric hydroxide absorption at 3,573 cm-’ and 630 cm-‘. Carbonate ions occupying the monovalent anionic hydroxide sites are designated as type A and carbonate ions in trivalent anionic phosphate sites as type B. The IR spectrum revealed three well-defined peaks (1,4 14, 1,460, and 1,548 cm-‘) and not the complex spectrum of the type AB carbonated apatite.13 The presence of both type A and type B carbonated apatite in HA formed from coral has been established.16 The presence of type A and type B carbonated HA in human bone has also been reported. I3 It has been claimed that Algipore, in spite of the absence of monomeric hydroxide peaks, is a HA.’ The absence of hydroxide absorption peaks was explained as owing to the large surface area of the material. In the present investigation KBr tablets were made from Algipore ground at room temperature and at - 196°C (in liquid nitrogen). The material was well dispersed in KBr. This should indicate that the IR spectrum is representative of the material. The presence of more than 3.5 wt-% fluoride in the Algipore indicated the possibility that it contained fluorapatite [Ca’O(PO&F~]. It has been shown that the liberational motion of the monomeric OH- ion at 630 cm-’ is not present in the IR spectrum of fluorapatite.“*” The absence of both absorption peaks at 3,670 cm-’ and 630 cm-‘, representing the monomeric OH- ion, was observed in the IR spectrum of Algipore. The absorption, representing the symmetric stretching motion of the phosphate ion, appeared at 965 cm-’ for Algipore and at 960 cm-’
BY APATITES
for Interporeand Alveograf, and at 961 cm-’ for Calcitite. Such a slight increase in this frequency for fluorapatite, as compared with HA, has been observed.” Therefore, it can be concluded that the main component of Algipore is fluorapatite. The existence of fluoride in original marine organisms such as krill has been established. ‘* IQ-ill is a small, shrimplike crustacean that uses plankton, eg, algae, as nourishment. The IR spectrum also indicated the presence of type B carbonated apatite and the ICP-AES (inductively coupled plasma-atomic emission spectrometry) analyses indicated the presence of a magnesium compound. None of the four different granular apatite products in this study were osteoinductive or osteoconductive when implanted subperiosteally on intact bone. When the granules were implanted on the surface of the recipient bone, the host response was fibrous encapsulation with multinuclear giant cells. When granules of dense apatite were located in accidentally created bony cavities, osseous tissue was in direct contact with and surrounded the implant. These healing modes are in accordance with previous findings.’ Most investigations on apatites have been performed by placing the implant into a natural or a created bony cavity, often in the femur or in the mandible of animals or humans. The walls of a cylinder,‘9-21 or a three- or four-walled cavity, 22-25represent an optimal environment for bony healing. Both dense and porous HA products have, under such experimental conditions, been observed in direct contact with bone at the ultrastructural level. 1,2,‘9-21,23,26,27 Furthermore, porous apatite implants have been totally invaded and with time gradually replaced by bone.2,‘9,25 Granules of dense and porous apatites used for alveolar ridge augmentation seem to be incorporated mainly in fibrous tissue and infiltrated by a variable amount of newly formed bone at the juxtacortical part In contrast to implantation of the implant. 8,13*20.21,28-34 into intrabony defects, subperiosteal onlay alveolar
I
4oonYIo~~
.
*
*
6
wm WAYWYBL
uomo~49100
om-’
FIGURE 12. Infrared spectrum of the Cakitite.
865
PINHOLT ET AL
FIGURE 13. Infrared spectrum
implant and surrounding cells.39Protein adhesion onto the substrates probably is involved in the cellular response at the implant surface.39 Surface energies may indirectly determine the interfacial reactions.* Purified bovine bone morphogenetic protein bound to HA (BMP-HA) in in vitro and in vivo studies was unable to induce bone formation in the interior of porous HA.4,40 Klein4’ reported absorption of IgG and complement factor C3 to calcium phosphates as part of the initial healing and degradation of the implant materia1.42The role of these proteins in this context are not fully understood but may imply immunologic reactions. 0sbom25 believes that there exists an epitaxy between bone crystals and the crystal phase of the ceramic, and between protein molecules and the ceramic apatite. Porous apatite blocks have been reported to be infiltrated by bone by a mechanism that could be epitaxial bone growth.43.44The blocks may provide better conditions for this healing mode by keeping the periosteum away from the recipient bone, thereby allowing osseous tissue to regenerate such as in guided tissue regeneration. Algipore blocks and granules have been claimed to be pure HA developed by hydrothermal processing of the carbonate skeleton of algae.5,7 It was reported to be invaded by, and to be in direct contact with, newly formed bone due to a small crystal size and a large surface area containing interconnecting pores of size 30 pm in width and 10 pm in length.’ However, Algipore is not representative of porous HA granules as claimed by Kasperk et a1,5*7as chemical analysis and infrared spectrometry revealed that the material is mainly fluorapatite and, in addition, contains a magnesium compound. Impurities such as magnesium and fluoride ions added to P-tricalcium phosphate were reported to contribute to the stability of the calcium phosphate and to reduce the biodegradation rate.42 Enhanced recruitment and activity of both bone and cartilage resorbing cells was reported after oral supplementation of magnesium in mice.46 This may explain the stability of the Algipore implants and the predominance of multinuclear giant cells. The results of these chemical and the infrared spectrometry analyses of commercially available materials emphasize the importance of standardized procedures
of the Algipore.
ridge augmentation represents an environment comprising one bony wall and the covering mucoperiosteum. The recipient bone is often cortical and the periosteum may not be as osteogenic as is the case with a bony cavity.35 The bony incorporation in the osseous contact site of some of the implants may represent bone formation from the insult of surgery, as reported previGranules of Interporewere reously. 8.20,2’,28-32 sorbed, as indicated by the engulfed microparticles that were seen. This is in contrast to other animal studies where bony ingrowth into part of the implanted granules was reported.30s34 The rat has a high metabolic rate, which makes new bone formation and resorption possible during the observation period of the present study. A fibrous capsule may develop because the granules are lying unstable on top of the bone. Micromovements could create low oxygen tension in the tissue, which favors formation of connective tissue and cartilage instead of bone.36 This has also been reported by Donath et al,20*2’.26 who observed multinuclear giant cells in the fibrous tissue around implant material placed on top or outside of bone. Evaluation of interactions between different calcium phosphates and human fibroblastic cells,37,38as well as between animal osteogenic cells,39 indicate that calcium phosphate stimulates proliferation of human gingival fibroblasts. Physicochemical and electrochemical characteristics of the implant surface influence the type of tissue formed at the interface between Table 2. Product
Semiquantitative Analysis of the Apatite Materials (wt.%) Ca
P
Mg
Alveograf Algipore Calcitite
43 38 41
19 17 19
2.5 0.0011
Interpore-
43
18
0.1
Na
K
Al
0.0096 0.18 0.009
0.008 1 0.67 0.0084
0.048
0.018
0.75
0.27
0.07 1
Sr
Si 0.017 0.056 0.036 0.069
Zn
0.0037
Fe
0.01
Mn
I
0.013
s 0.078 0.25 0.073 0.2
F
3.52
866 for production and control of materials created for implant purposes in humans. Acknowledgment The authors wish to thank Mr K. Nagy at SINTEF, Norwegian Institute of Technology of Trondheim, for the fluoride analysis, MS S. H. Dundas, Nordic Analytical Center, Oslo, for the semiquantitative determination of the other elements, and MS Randi Aarsand, NIOM, for the technical assistance with the IR-spectroscopical analysis.
References 1. Jarcho M: Biomaterial aspects of calcium phosphates. Dent Chn North Am 30:25, 1986 2. Bucholz RW, Carlton A, Holmes RE: Hydroxyapatite and tricalcium phosphate bone graft substitutes. Orthop Clin North Am 18:323, 1987 3. Urist MR: Bone. Formation by autoinduction. Science 150:893, 1965 4. Urist MR, Nilsson 0, Rasmussen J, et al: Bone regeneration under the influence of a bone morphogenetic protein (BMP) beta tricalcium phosphate (TCP) composite in skull trephine defects in dogs. Clin Orthop 214:295, 1987 5. Kasperk C, Ewers R: Tierexperimentelle untersuchungen zur Einheilungstendenz synthetischer, korahiner und ans Algen gewonnener (phykogener) Hydroxlapatitmaterialien. Z Zahnaerztl Implantol 2:242, 1986 6. Janicke S, Wagner W, Wahlmann UW: Histologische Reaktionen nacb Implantation unterschiedlicher dichter Hydroxylapatitgranulate in Unterkiefer and Femur im Tierversuch. Z Zahnarztl Implant01 IV: 101, 1988 7. Kasperk C, Ewers R, Simons B, et al: Algae-derived (phycogene) hydroxylapatite. A comparative histological study. Int J Oral Maxillofac Surg 17:319, 1988 8. Pinholt EM, Bang G, Haanaes HR: Alveolar ridge augmentation bv osteoinduction in rats. Stand J Dent Res 98:434, 1990 9. Fowier BO, Moreno EC, Brown WE: Infra-red spectra of hydroxyapatite, octacalcium phosphate and pyro1yse.d octacalcium phosphate. Arch Oral Biol 11:477, 1966 10. Stutman JM, Termine JD, Posner AS: Vibrational spectra and structure of the phosphate ion in some calcium phosphates. Trans NY Acad Sci 27~669, 1965 11. Ducheyne P, Raemdonck WV, Heughebaert JC, et al: Structural analysis of hydroxyapatite coatings on titanium. Biomaterials 7197, 1986 12. Bone1 G, Monte1 G: Chimie minbrale-Sur une nouvelle apatite carbona& synthetique. CR Acad Sci Paris 258:923, 1964 13. Rey C, Collins B, Goehl T, et al: The carbonate environment in bone mineral: A resolution-enhanced Fourier transform infrared spectroscopy study. Calcif Tissue 45:157, 1989 14. Scholl FK: Hummel/Scholl, Infrared Analysis of Polymers, Resins and Additives. An Atlas. Vo12. Additives and Processing Aids. Mtinchen, Germany, Carl Hanser Verlag, and Weinheim/ Bergstr, Verlag Chemie, 1973, p 76 15. Vignoles M, Bone1 G, Holcomb DW, et al: 1nIluenc.e of preparation conditions on the composition of type B carbonated hydroxyapatite and on the localization of the carbonate ions. C&if Tissue Int 43:33, 1988 16. Roy DM, Linnehan SK: Hydroxyapatite formed from coral skeletal carbonate by hydrothermal exchange. Nature 247: 220, 1974 17. Trombe J-C, Monte1 G: Chimie minerale-Sur les spectre d’ap sorption infrarouge des apatites dont les tunnels contiennent des ions bivalents et des lacunes. CR Acad Sci Paris 276: 127 1, 1973 18. Soevik T, Braekkan OR: Fluoride in Antarctic krill (Euphasia superba) and Atlantic krill (Megunyctiphanes nmegica). J Fish Res Board Can 36:1414, 1979 19. Chiroff RT, White EW, Weber JN, et al: Tissue ingrowth of replamine form implants. J Biomed Mater Res Symp 6:29, 1975
RIDGE AUGMENTATION
BY APATITES
20. Donath K, Rohrer MD, Beck-Mannagetta J: A histologic evaluation of a mandibular cross section one year after augmentation with hydroxyapatite particles. Oral Surg Oral Med Oral Path01 63:651, 1987 21. Donath K, Rohrer MD, Hoermann K: Mobile and immobile hydroxylapatite integration and resorption and its influence on bone. J Oral Implants 13:11, 1987 22. Cranin AN, Ronen E, Shpuntoff R, et al: Hydroxylapatite (H/ A) particulate versus cones as post-extraction implants in humans. J Biomed Mater Res 22: 1165, 1988 23. Kato K, Aoki H, Tabata T, et al: Biocompatibility of apatite ceramics in mandibles. Biomater Med Dev Art Org 7:291, 1979 24. Kenney EB, Lekovic V, Cruranza FA, et al: A comparative clinical study of solid and granular porous hydroxylapatite implants in human periodontal osseous defects. J Biomed Mater Res 22: 1233, 1988 25. Osborn JF: Hydroxylapatitkeramik-Ein osteotroper Werkstoff fur den Knochenersatz. Experimentelle Mund-Kiefer-Gesichts-ChirurgieMikrochirurgische Eingriffe. Stuttgart, Germany, Georg Thieme Verlag, 1983, pp 37-40 26. Donath K, Hoermann K, Kirsch A: Welchen Einfluss hat die Hvdroxvlanatitkeramik auf die Knochenneubildund Dtsch Z Kiefer Gesichts Chir 9:438, 1985 27. Jarcho M, Kay JF, Gumaer KI, et al: Tissue, cellular and subcellular events at a bone-ceramic hydroxylapatite interface. J Bioeng 1:79, 1977 28. Block MS, Kent JN: Healing of mandibular ridge augmentations using hydroxylapatite with and without the addition of autogenous bone in dogs. J Oral Maxillofac Surg 43:3, 1985 29. Chang CS, Matukas VJ, Lemons JE: Histologic study of hydroxylapatite as an implant material for mandibular augmentation. J Oral Maxillofac Surg 41:729, 1983 30. Frame JW, Rout PGJ, Browne RM: Ridge augmentation using solid and porous hydroxylapatite particles with and without autogenous bone or plaster. J Oral Maxillofac Sung 45:77 1, 1987 31. Pinhoh EM, Kwon PHJ: The effect of therapeutic radiation on canine alveolar ridges augmented with hydroxylapatite. J Oral Maxillofac Surg 50:250, 1992 32. Rothstein SS, Paris D, Sage B: Use of Durapatite for the rehabilitation of resorbed alveolar ridges. J Am Dent Assoc 109: 571, 1984 porous 33. White E, Shots EC: Biomaterial aspects of Interporehydroxyapatite. Dent Clin North Am 30:49, 1986 34. Worsaae N, Hjoerting-Hansen E: Genopbygning af Processus Alveolaris med Hydroxylapatite, in Hjoerting-Hansen E (ed): Odontologi 88. Copenhagen, Munksgaard, 1988, pp 159- 169 35. Hjoerting-Hansen E: Studies on Implantation of Anorganic Bone in Cystic Jaw Lesions. Copenhagen, Munksgaard, 1970, pp l-198 36. Burchardt H: The biology of bone graft repair. Clin Orthop 174: 28, 1983 37. Gregoire M, Orly I, Menanteau J, et al: In vitro interactions between calcium phosphate biomaterials and human fibroblastic cells. II. Incidences on the behaviour of cultured gingival cells. Adv Biomat 8:2 15, 1988 38. Orly I, Gregoire M, Menanteau J, et al: In vitro interactions between calcium phosphate biomaterials and human fibroblastic cells. I. Comparative extra and intracellular evolution of 4 synthetic calcium phosphates. Adv Biomater 8:2 11,1988 39. Bagambisa FB, Joos U, Schilh W: The interaction of osteogenic cells with hydroxylapatite implant materials in vitro and in vivo. Int J Oral Maxillofac Implants 5:2 17, 1990 40. Kawamura M, Iwata H, Sato K, et al: Chondro-osteogenetic response to crude bone matrix proteins bound to hydroxylapatite. Clin Orthop 217:281, 1987 41. Klein CP, de Groot K, Vermeiden JP, et al: Interaction of some serum proteins with hydroxylapatite and other materials. J Biomed Mater Res 14705, 1980 42. Klein CP, de Groot K, Driessen AA, et al: A comparative study of different beta-whitlockite ceramics in rabbit cortical bone with regard to their biodegradation behaviour. Biomater 7: 144, 1986
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43. Holmes RE, Hagler HK: Porous hydroxylapatite as a bone graft substitute in mandibular contour augmentation: A histometric study. J Oral Maxillofac Surg 45:421, 1987 44. Holmes RE, Roser SM: Porous hydroxyapatite as a hone graft
substitute in alveolar ridge augmentation: a histometric study. Int J Oral Maxillofac Sung 16:718, 1987
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45. Dahlin C, Linde A, Gottlow J, et al: Healing of bone defects by guided tissue regeneration. Plast Reconstr Surg 81:672, 1988
46. Marie PJ, Hott M: Effect of cakitonin on the magnesium-induced bone resorption in the mouse. Magnesium 6: 100, 1987
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1992
Discussion Chemical, Physical, and Histologic Studies on Four Commercial Apatites Used for Alveolar Ridge Augmentation Mohamed El Deeb, BDS, DOS, MS University of Minnesota, Minneapolis School of Dentistry Synthetic hydroxylapatite (HA) has been used widely in various maxillofacial applications during the last 10 years.ie9 Currently, many products with different shapes are available. Some are porous; some are nonporous. The purpose of the article by Pinholt et al was 1) to evaluate the biological reaction to four different granular HA materials (Alveograf, Calcitite, Interpore-200, and Algipore placed subperiosteally for augmentation of the alveolar ridge and 2) to correlate the tissue reaction to the materials with their characteristics, as obtained by chemical analysis and infrared spectrometry. Pinholt et al state that none of the four types of HA tested in this study caused osteoinduction or osteoconduction, and that fibrous encapsulation, with multinuclear giant cells, was observed around all of them. We have used granular Alveograf, Calcitite, and Interporein preclinical and clinical investigations,2-9 and experience has indicated that nonporous HA, when used in subperiosteal augmentation, generally is associated with little bone formation, if any, at the interface between the implant and the host bone. When HA is placed subperiosteally, the granules only contact bone on one surface and are surrounded by soft tissue in all other directions. Therefore, in these situations, bone forms only at the implantbone interface. On the other hand, when HA is placed within a bony defect, the granules are surrounded by bony walls on all sides except superiorly and bone will form within the defect, surrounding the HA granules. As for the osteoinductive capabilities of the materials used in this study, I agree with Pinholt et al that none have osteoinductive capabilities. We have placed granules of Interpore-200 intramuscularly in the chests of Rhesus monkeys6 and Calcitite material in the chests of rats’ and also found no evidence of bone formation in either study. The authors state that “osteoconduction is a healing process in which the implanted material acts as a scaffold that is gradually resorbed during creeping substitution as the healing proceeds from the adjacent living bone.” I do not believe that a material has to be resorbed completely to be considered an osteoconductive material. Parts or all of the material may remain and act as a scaffold for the formation of new bone, and it may still be considered osteoconductive. Although it may be true that the materials used in this study have no osteoconductive capabilities, I believe that the porous HA has osteoconductive properties, as evidenced by our study in Rhesus monkeys.6 This study showed that when granules of
porous HA are mixed with demineralized bone powder and placed intramuscularly in the chests of Rhesus monkeys, for-
mation of bone is noted within the pores of the material, which acted as a scaffo1d.6 Boyne et al also found that when porous HA was mixed with an osteoinductive material, the bony growth was superior to that obtained by mixing nonporous HA with the same material.’ If a porous HA is used for subperiosteal augmentation of an alveolar ridge, we would expect more bone formation than when nonporous HA is used. Porous HA results in more bone formation than nonporous HA because of the interconnecting pores and also because it reacts differently histochemically. In a previous comparative study, we documented that the type of collagen formed with porous HA differs from the type of collagen formed with a nonporous HA material.’ Our study concluded that porous HA serves as a matrix for ingrowth of host bone, as evidenced by the proliferation of types I and V collagen, whereas nonporous HA elicits a fibrous tissue encapsulation of the implanted material, with collagen types I and III prevailing at-the implant/tissue interface.’ Factors that might affect formation of bone in subperiosteal ridge augmentation using HA are: 1) the amount of material placed; 2) stabilization and lack of mobility of the material; 3) the type of HA used; and 4) the site of the augmentation. The reasons that no bone formed within the material tested in this study may be the following: 1) The authors used only four granules per implant, and these were placed subperiosteally. This amount is extremely limited, bone has difficulty forming around such a small amount of HA. 2) The authors used HA to augment bucahy and superiosteally, without removing the teeth or stabilizing the material. This might not keep the material in close contact with bone and subsequently, it would be surrounded by fibrous tissue. 3) Use of the facial musculature during eating and drinking might create a micromotion that would displace the material further from the augmented site.
References 1. Boyne, Scheer P, Stringer D: Comparison of porous and nonporous hydroxylapatite and xenographs in restoration of ridges. Presented at the AAOMS 67th Annual Meeting, Washington, DC, October 1985 2. El Deeb M, Roszkowski M, El Deeb, ME: Nonporous hydroxylapatite gramdes as an extracranial and extranasal augmenting material in dogs: Technique and initial findings. Cleft Palate J 24:4, 1987 3. El Deeb M, Roszkowski M: Hydroxylapatite granules and blocks as an extracranial augmenting material in rhesus monkeys. J Oral Maxillofac Surg 46:33, 1988
Surg
1992
Chemical, Physical, and Histologic Studies on Four Commercial Apatites Used for Alveolar Ridge Augmentation E.M. PINHOLT, DDS, MS, DRODONT,* I.E. RUYTER, DRENGSc,t H.R. HAANAES, DDS, MD, DRODONT,$ AND G. BANG, DDS, MD, DRMEDS The purpose of this study was to evaluate four commercial apatite products. Subperiosteal alveolar ridge augmentation was performed on the maxilla of rats by implantation of granules of two dense products and of two porous products, and the tissue response was compared with the material characteristics obtained by chemical analysis and infrared spectrometry. None of the apatites caused osteoinduction or osteoconduction; fibrous encapsulation with multinuclear giant cells was observed around all four types. One of the apatites was fluorapatite and not hydroxylapatite, as claimed by the manufacturer. The tissue response to this implant material was dominated by multinuclear giant cells. several bone substitutes in the same anima1.5-7Material characteristics are often not reported in conjunction with evaluation of tissue responses to commercially available apatite implant materials. The purpose of this investigation was to evaluate the biological reactions to granular Alveograf, Calcitite, Interpore-200, and Algipore when implanted subperiosteally for alveolar ridge augmentation and to correlate
Some calcium phosphates, such as hydroxylapatite (HA) [Ca,o(P04)6(OH)~], are claimed to be biocompatible and osteoconductive.’ Osteoconduction is a healing process in which the implanted material acts as a scaffold that is gradually resorbed during creeping substitution as the healing proceeds from the adjacent living bone.2 With osteoinduction, on the other hand, mesenchymal cells are transformed into cartilage and/ or bone-forming cells by a local stimulating factor.334 Hydroxylapatites may be developed synthetically or by treatment of algae and corals by hydrothermal processes5 Because of chemical and geometric similarities to HA of vertebrate bone and teeth, it has been used widely for implant purposes.’ Most investigations have been performed using one product in one species. Few reports are available using * Associate Professor; previously. Assistant Professor. Department of Oral Surgery and Oral Medicine, Dental Faculty, University of Oslo; Research Fellow, Norwegian Research Council for Science and the Humanities, Institute for Surgical Research, Rikshospitalet, Oslo. Norway. t Senior Scientist, Physical/Chemical Division, NIOM, Scandinavian Institute of Dental Materials, Haslum, Norway. $ Professor and Chairman, Department of Oral Surgery and Oral Medicine, Dental Faculty, University of Oslo, Norway. 4 Professor and Chairman, Department of Oral Pathology and Forensic Odontology, University of Bergen, Haukeland University Hospital, Bergen, Norway. Address correspondence and reprint requests to Dr Pinholt: Department of Oral and Maxillofacial Surgery, Royal Dental College. 20, Notre All&, DK-2200 Copenhagen N, Denmark. 0 1992 American
Association
of Oral and Maxillofacial
FIGURE I. Transverse view of rat maxilla showing location of apatite implants. A, Alveograf; B, Calcitite; c, Interpore-200: D, Algipore.
Surgeons
0278-2391/92/5008-0012$3.00/O
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RIDGE AUGMENTATION BY APATITES
Table 1.
Investigated Apatite Materials
Product Alveograf mesh: 18/40 Calcitite mesh: 20140 Interporemesh: 28135 Algipore HA-Keramik size: 0.5-l mm
Producer
Batch No.
Cook-Waite, New York, NY Calcitek, San Diego, CA Interpore Int, Irvine, CA Friedrichsfeld, Germany
S 008 AC
Origin
Norway, the Inter-pore-200 from Immuno, Copenhagen, Denmark, and the Algipore from Ronvig A/S, Daugaard, Denmark).
Synthetic SURGICAL PROCEDURE
870523
Synthetic
9142
Coral derived
l-10/89
Algae derived
the tissue reaction to the material characteristics obtained by chemical analysis and infrared spectrometry. Material and Methods
The four apatite products were purchased from the manufacturer or from governmental authorized sales representatives in the same way as products are purchased for clinical use (the Alveograf from the manufacturer, the Calcitite from Peter Msller A/S, Oslo,
FIGURE 2. Rat alveolar bone (AB) augmented with granules of Alveograf(AG) covered by oral mucosa, including the epithelium (E). The area formerly occupied by the implant material becomes empty during the decalcification process (hematoxylin-eosin stain, original magnification X 17.5).
Ten male Wistar rats weighing 267 f 11 g were used. They were fed a standard diet (standard diet for rats, Rikshospitalet, The National Hospital, Oslo, Norway) and given water ad libitum. Surgery was done under general anesthesia, Hypnorm (Janssen Pharmaceutics, Beerse, Belgium)/Dormicum (Roche, F. Hoffman-La Roche, AG, Basel, Switzerland) 0.15 mL/ 100 g intramuscularly (1:Hypnorm + 1:sterile water mixed with 1:Dormicum + 1:sterile water, Rikshospitalets Apotek, Oslo, Norway). Incisions were made to bone bilaterally on the premaxilla where the horizontal plate of the hard palate continues into the vertical lateral plate between the incisor and the first molar (Fig 1). Subperiosteal dissection was carried out to create small pockets on each
FIGURE 3. Higher magnification of Figure 2. Ridge augmentation of rat alveolar bone (An)with granulesOfAlVeOgraf(AG).The granules are encapsulated by fibrous connective tissue (FT)with multinuclear giant cells. No osteoinduction or osteoconduction is observed (hematoxylin-eosin stain, original magnification X70).
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PINHOLT ET AL
side. Posteriorly and laterally on the maxilla in the first molar region an additional incision was made bilaterally and subperiosteal dissection was carried out to create two more pockets. Alveolar ridge augmentation was performed by implanting four granules of an apatite product into each pocket. On the right side, Alveograf was placed anteriorly and Calcitite posteriorly (Table 1). On the left side, Interporewas placed in the anterior pocket and Algipore in the posterior. The surgical sites were closed with Prolene monofilament suture. The control group was comprised of four male Wistar rats.* The sham operations were performed by creating one pocket on the premaxilla, but no implantation was performed.* CLINICAL AND HISTOLOGIC EVALUATIONS
To observe wound healing, clinical evaluation was performed daily the first 3 postoperative days and thereafter on a weekly basis. The animals were weighed at the beginning and at the end of the experimental period. After 8 weeks, the animals were killed. The maxilla was immediately removed from the animal and fixed in 4% neutral formalin. Radiographs were used to locate the implants and possible hard-tissue formation. The specimens were then demineralized in 17% formic acid, dehydrated, and embedded in paraffin. Serial sections were cut at 5 grn and stained with Harris hematoxylin and eosin for light-microscopic examination. INFRARED SPECTROSCOPICALCHARACTERIZATION
The granules of the four proprietary apatite materials were ground to a fine powder and mixed during grind-
FIGURE 5. Rat alveolar bone (AB) augmentation with Calcitite (cr). Three granules of the Calcitite have been implanted into an accidentally created bony defect of the alveolar bone (large arrows). One granule has remained on top of the alveolar ridge (small arrow). Oral mucosa, including the epithehum (E), covers the alveolar bone (hematoxyhn-eosin stain, original magnification x20).
ing with potassium bromide (KBr, Uvasol-spectroscopic grade, Merck, Germany). Pellets with a diameter of 13 mm were made. Infrared (IR) spectra were recorded on a dispersive ratio recording IR-spectrophotometer (model 683 with Model 3500 Data Station, Perkin-Elmer Corp, Norwalk, CT). The IR-spectrophotometer was purged with dry air from a self-generative heatless air drier (model X3HA, Pall Pneumatics Ltd, Portsmouth, England), and operated with slit 6 and noise filter 2 for the 4000 to 200 cm-’ range. CHEMICAL ANALYSIS
FIGURE4. Higher magnificationof Figure3. Rat alveolarbone separatedfromgranulesof Alveograf(AC) by fibrousconnective tissue (FT)containingmultinucleargiant cells (cc). The edgesof the Alveografareresorbed by the multinuclear giant ceils (CC).The areas (AB)
formerly occupied by the microparticles (MC) of the Alveograf are observed inside the multinuclear giant cells. No osteoinduction or osteoconduction is noted (hematoxylineosin stain, original magnification X280).
The apatite samples were dissolved in nitric acid under pressure. The solutions were analyzed semiquantitatively by inductively coupled plasma atomic emission spectrometry (ICP-AES) (ICP Series 2, PerkinElmer Corp). The fluoride content of Algipore was determined quantitatively by ion selective F-electrode analysis (Sintalyzer-System, SINTEF, Trondheim, Norway).
862 Results CLINICALAND HISTOLOGIC EVALUATIONS All animals gained weight during the observation period (weight at the beginning, 267 f 11 g; at end, 443 k 21 g). No infection was observed at any time. All the implants were recovered from the sites of implantation (Fig 2). The region formerly occupied by the implant material became empty during the decalcification process. AlveograJ: Fibrous encapsulation with multinuclear giant cells was observed around the Alveograf (Figs 3,4). Newly formed bone was not observed, but multinuclear giant cells were seen at the edges of the implant site (Fig 4). Calcitite. Granules of Calcitite were in direct contact with, and surrounded by, osseous tissue when implanted into defects created accidentially by pressure
FIGURE 7. Higher magnification of Figure 6. Granule of Calcitite (CT) on top of rat alveolar bone (AB). Fibrous connective tissue (FT) with multinuclear giant cells (GC) envelopes the Calcitite granule. The edges show irregularities in the areas of the giant cells (arrows). The Calcitite does not cause osteoinduction or osteoconduction (hematoxylin-eosin stain, original magnification X 175).
on the alveolar bone (Figs 5,6). The edges of the bone that encased the granules of Calcitite were irregular (Fig 6). Granules on top of the alveolar ridge, and the part of the bony encased granules that were exposed to soft tissue, were covered by fibrous connective tissue containing multinuclear giant cells (Fig 7). New bone formation was not observed. Interpore-200. The Interporegranules were covered by fibrous connective tissue containing multinuclear giant cells (Fig 8). Empty spaces could be seen within the giant cells. These spaces, formerly occupied by the microparticles were now empty due to the decalcification process. No tissue infiltration into
LlGURE6. Higher magnification of Figure 5. Healing of an accidentally created defect in rat alveolar bone (AB) containing granules of Calcitite (CT). Bony tissue has encased the granules, which have irregular edges. The parts of the granules in contact with soft tissue are covered by fibrous connective tissue (FT). No osteoinduction or osteoconduction is observed (hematoxylin-eosin stain, original magnification X90).
FIGURE 8. Rat alveolar bone (AB) separated from granule of Interpore-200 (IP) by fibrous connective tissue (FT) containing multinuclear giant cells (GC).Areas formerly occupied by the microparticIes (MP) are noted inside the multinuclear giant cells (GC). The structure of Interporeis retained in the remnants of the material. There is no evidence that the granule resulted in either osteoinduction or osteoconduction (hematoxylin-eosin stain, original magnification X175).
863
PINHOLT ET AL
remnants of the apatite was noted and no new bone formation was observed. Mgipore. A fibrous capsule dominated by multinuclear giant cells surrounded the granules of Algipore (Figs 9, 10). The structure of the porous implant material was retained. Bony infiltration was not observed. Remnants of a homogeneous, acellular structure were noted (Fig 10). Where the Algipore had been implanted into an accidentally created defect in the alveolar bone, bone ingrowth was observed. Fibrous tissue with multinuclear giant cells had formed between the bone and the implant (Fig 9). Evaluation of the four control animals showed no osteoinduction or osteoconduction.8 IR SPECTROSCOPICALCHARACTERIZAT~ON Figure 11 shows the infrared spectrum Alveogruj of Alveograf with absorption peaks at 3,569 cm-’ and 630 cm-‘, representing monomeric OH- groups in the apatite structure. The absorption bands at I,09 1 cm-‘, 1,045 cm-‘, and 1,04 1 cm-’ are ascribed to PO:- group of HA. The absorption peaks at 568 cm-’ and 605 cm-’ can be assigned to antisymmetric bending motion of the phosphate groups. ” The presence of absorption bands at 436 cm-’ and 473 cm-’ accompanying reduction of OH- absorption at 630 cm-’ during heat treatment of HA is typical for slightly oxygenated HA. ’ ’ The band observed at 342 cm-’ can also be ascribed to a phosphate group of apatite.14 Culcitite. Figure 12 shows the infrared spectrum of Calcitite with a spectrum similar to that of Alveograf. Interpore-200. Figure 11 shows the IR spectrum of Interporewith absorption peaks representing the monomeric OH- groups of HA at 3,573 cm-’ and
(AB) with Algipore (AP) that has kept its structure. Fibrous connective tissue (FT) with multinuclear giant cells (cc) surrounds the implant. Osteoinduction or osteoconduction is not observed (hematoxyhn-eosin stain, original magnitication X25).
FIGURE 10. Rat alveolar bone
630 cm-‘. Absorptions due to carbonate ions of carbonated apatites are well represented. Absorption peaks at 879 cm-‘, 1,460 cm-‘, and 1,548 cm-’ represent CO; absorptions of type A carbonated apatite.‘2S’3 Carbonate absorptions from type B carbonated apatite are present at 872 cm-‘, 1,414 cm-‘, and 1,460 cm-‘. Algipore. Figure 13 shows the infrared spectrum of A&pore with two sharp absorption peaks at 3,645 cm-’ and 3,698 cm-’ representing monomeric OHgroups of a silicate material containing OM-.‘4 The broad band from 3,700 to 2,500 cm-’ is attributed to hydrogen-bonded stretching modes of water. The band at 1,650 cm-’ corresponds to hydrogen bending modes of water. The IR spectrum of Algipore had no absorption peaks representing monomeric OH- groups of HA at 3,570 cm-’ and 630 cm-‘. The spectrum had, however, absorption peaks at 1,425 cm-’ and 1,460 cm-‘, as well as 875 cm-‘, representing carbonate ions in type B sites of carbonated apatite. I5 The phosphate absorption band at 1,050 cm-‘, 1,096 cm-‘, 574 cm-‘, 567 cm-‘, 603 cm-‘, 468 cm-‘, and 350 cm-’ were present, as well as the apatite phosphate absorption bands at 965 cm-‘. COMPOSITION
FIGURE 9. A&ore (AP) implanted as alveolar ridge augmentation on rat alveolar bone (A@. Bone formation is seen around the granule of Algipore placed in an accidentally created defect. Note fibrous tissue (IT) with multinuclear giant cells (cc) on top of alveolar bone and between newly formed bone and implant (hematoxylin-eosin stain, original magnification X 175).
The results of the quantitative determinations of the elements present in the four apatite materials are shown in Table 2. The chemical composition of the synthetic materials Alveografand Calcitite, and the coral-derived Interpore-200, was consistent with a HA structure, whereas the Algipore was different. A fluoride analysis of the Algipore was carried out due to the absence of the absorption peaks at 3,570 cm-’ and 630 em-’ representing the monomeric OH- groups in the apatite structure. This analysis revealed the presence of 3.52
864
FIGURE 1I. 200 (B).
RIDGE AUGMENTATION
Infrared spectra of the Alveograf
(A)
and Interpore-
+ 0.04 wt-% fluoride. The semiquantitative analysis showed the presence of 2.5 wt-% magnesium in addition to calcium and phosphorous (Table 2). Discussion The IR spectra indicated that Alveograf and Calcitite were slightly oxygenated HA. The Interporecontained both type A and type B carbonated apatite accompanied by a reduction of the monomeric hydroxide absorption at 3,573 cm-’ and 630 cm-‘. Carbonate ions occupying the monovalent anionic hydroxide sites are designated as type A and carbonate ions in trivalent anionic phosphate sites as type B. The IR spectrum revealed three well-defined peaks (1,4 14, 1,460, and 1,548 cm-‘) and not the complex spectrum of the type AB carbonated apatite.13 The presence of both type A and type B carbonated apatite in HA formed from coral has been established.16 The presence of type A and type B carbonated HA in human bone has also been reported. I3 It has been claimed that Algipore, in spite of the absence of monomeric hydroxide peaks, is a HA.’ The absence of hydroxide absorption peaks was explained as owing to the large surface area of the material. In the present investigation KBr tablets were made from Algipore ground at room temperature and at - 196°C (in liquid nitrogen). The material was well dispersed in KBr. This should indicate that the IR spectrum is representative of the material. The presence of more than 3.5 wt-% fluoride in the Algipore indicated the possibility that it contained fluorapatite [Ca’O(PO&F~]. It has been shown that the liberational motion of the monomeric OH- ion at 630 cm-’ is not present in the IR spectrum of fluorapatite.“*” The absence of both absorption peaks at 3,670 cm-’ and 630 cm-‘, representing the monomeric OH- ion, was observed in the IR spectrum of Algipore. The absorption, representing the symmetric stretching motion of the phosphate ion, appeared at 965 cm-’ for Algipore and at 960 cm-’
BY APATITES
for Interporeand Alveograf, and at 961 cm-’ for Calcitite. Such a slight increase in this frequency for fluorapatite, as compared with HA, has been observed.” Therefore, it can be concluded that the main component of Algipore is fluorapatite. The existence of fluoride in original marine organisms such as krill has been established. ‘* IQ-ill is a small, shrimplike crustacean that uses plankton, eg, algae, as nourishment. The IR spectrum also indicated the presence of type B carbonated apatite and the ICP-AES (inductively coupled plasma-atomic emission spectrometry) analyses indicated the presence of a magnesium compound. None of the four different granular apatite products in this study were osteoinductive or osteoconductive when implanted subperiosteally on intact bone. When the granules were implanted on the surface of the recipient bone, the host response was fibrous encapsulation with multinuclear giant cells. When granules of dense apatite were located in accidentally created bony cavities, osseous tissue was in direct contact with and surrounded the implant. These healing modes are in accordance with previous findings.’ Most investigations on apatites have been performed by placing the implant into a natural or a created bony cavity, often in the femur or in the mandible of animals or humans. The walls of a cylinder,‘9-21 or a three- or four-walled cavity, 22-25represent an optimal environment for bony healing. Both dense and porous HA products have, under such experimental conditions, been observed in direct contact with bone at the ultrastructural level. 1,2,‘9-21,23,26,27 Furthermore, porous apatite implants have been totally invaded and with time gradually replaced by bone.2,‘9,25 Granules of dense and porous apatites used for alveolar ridge augmentation seem to be incorporated mainly in fibrous tissue and infiltrated by a variable amount of newly formed bone at the juxtacortical part In contrast to implantation of the implant. 8,13*20.21,28-34 into intrabony defects, subperiosteal onlay alveolar
I
4oonYIo~~
.
*
*
6
wm WAYWYBL
uomo~49100
om-’
FIGURE 12. Infrared spectrum of the Cakitite.
865
PINHOLT ET AL
FIGURE 13. Infrared spectrum
implant and surrounding cells.39Protein adhesion onto the substrates probably is involved in the cellular response at the implant surface.39 Surface energies may indirectly determine the interfacial reactions.* Purified bovine bone morphogenetic protein bound to HA (BMP-HA) in in vitro and in vivo studies was unable to induce bone formation in the interior of porous HA.4,40 Klein4’ reported absorption of IgG and complement factor C3 to calcium phosphates as part of the initial healing and degradation of the implant materia1.42The role of these proteins in this context are not fully understood but may imply immunologic reactions. 0sbom25 believes that there exists an epitaxy between bone crystals and the crystal phase of the ceramic, and between protein molecules and the ceramic apatite. Porous apatite blocks have been reported to be infiltrated by bone by a mechanism that could be epitaxial bone growth.43.44The blocks may provide better conditions for this healing mode by keeping the periosteum away from the recipient bone, thereby allowing osseous tissue to regenerate such as in guided tissue regeneration. Algipore blocks and granules have been claimed to be pure HA developed by hydrothermal processing of the carbonate skeleton of algae.5,7 It was reported to be invaded by, and to be in direct contact with, newly formed bone due to a small crystal size and a large surface area containing interconnecting pores of size 30 pm in width and 10 pm in length.’ However, Algipore is not representative of porous HA granules as claimed by Kasperk et a1,5*7as chemical analysis and infrared spectrometry revealed that the material is mainly fluorapatite and, in addition, contains a magnesium compound. Impurities such as magnesium and fluoride ions added to P-tricalcium phosphate were reported to contribute to the stability of the calcium phosphate and to reduce the biodegradation rate.42 Enhanced recruitment and activity of both bone and cartilage resorbing cells was reported after oral supplementation of magnesium in mice.46 This may explain the stability of the Algipore implants and the predominance of multinuclear giant cells. The results of these chemical and the infrared spectrometry analyses of commercially available materials emphasize the importance of standardized procedures
of the Algipore.
ridge augmentation represents an environment comprising one bony wall and the covering mucoperiosteum. The recipient bone is often cortical and the periosteum may not be as osteogenic as is the case with a bony cavity.35 The bony incorporation in the osseous contact site of some of the implants may represent bone formation from the insult of surgery, as reported previGranules of Interporewere reously. 8.20,2’,28-32 sorbed, as indicated by the engulfed microparticles that were seen. This is in contrast to other animal studies where bony ingrowth into part of the implanted granules was reported.30s34 The rat has a high metabolic rate, which makes new bone formation and resorption possible during the observation period of the present study. A fibrous capsule may develop because the granules are lying unstable on top of the bone. Micromovements could create low oxygen tension in the tissue, which favors formation of connective tissue and cartilage instead of bone.36 This has also been reported by Donath et al,20*2’.26 who observed multinuclear giant cells in the fibrous tissue around implant material placed on top or outside of bone. Evaluation of interactions between different calcium phosphates and human fibroblastic cells,37,38as well as between animal osteogenic cells,39 indicate that calcium phosphate stimulates proliferation of human gingival fibroblasts. Physicochemical and electrochemical characteristics of the implant surface influence the type of tissue formed at the interface between Table 2. Product
Semiquantitative Analysis of the Apatite Materials (wt.%) Ca
P
Mg
Alveograf Algipore Calcitite
43 38 41
19 17 19
2.5 0.0011
Interpore-
43
18
0.1
Na
K
Al
0.0096 0.18 0.009
0.008 1 0.67 0.0084
0.048
0.018
0.75
0.27
0.07 1
Sr
Si 0.017 0.056 0.036 0.069
Zn
0.0037
Fe
0.01
Mn
I
0.013
s 0.078 0.25 0.073 0.2
F
3.52
866 for production and control of materials created for implant purposes in humans. Acknowledgment The authors wish to thank Mr K. Nagy at SINTEF, Norwegian Institute of Technology of Trondheim, for the fluoride analysis, MS S. H. Dundas, Nordic Analytical Center, Oslo, for the semiquantitative determination of the other elements, and MS Randi Aarsand, NIOM, for the technical assistance with the IR-spectroscopical analysis.
References 1. Jarcho M: Biomaterial aspects of calcium phosphates. Dent Chn North Am 30:25, 1986 2. Bucholz RW, Carlton A, Holmes RE: Hydroxyapatite and tricalcium phosphate bone graft substitutes. Orthop Clin North Am 18:323, 1987 3. Urist MR: Bone. Formation by autoinduction. Science 150:893, 1965 4. Urist MR, Nilsson 0, Rasmussen J, et al: Bone regeneration under the influence of a bone morphogenetic protein (BMP) beta tricalcium phosphate (TCP) composite in skull trephine defects in dogs. Clin Orthop 214:295, 1987 5. Kasperk C, Ewers R: Tierexperimentelle untersuchungen zur Einheilungstendenz synthetischer, korahiner und ans Algen gewonnener (phykogener) Hydroxlapatitmaterialien. Z Zahnaerztl Implantol 2:242, 1986 6. Janicke S, Wagner W, Wahlmann UW: Histologische Reaktionen nacb Implantation unterschiedlicher dichter Hydroxylapatitgranulate in Unterkiefer and Femur im Tierversuch. Z Zahnarztl Implant01 IV: 101, 1988 7. Kasperk C, Ewers R, Simons B, et al: Algae-derived (phycogene) hydroxylapatite. A comparative histological study. Int J Oral Maxillofac Surg 17:319, 1988 8. Pinholt EM, Bang G, Haanaes HR: Alveolar ridge augmentation bv osteoinduction in rats. Stand J Dent Res 98:434, 1990 9. Fowier BO, Moreno EC, Brown WE: Infra-red spectra of hydroxyapatite, octacalcium phosphate and pyro1yse.d octacalcium phosphate. Arch Oral Biol 11:477, 1966 10. Stutman JM, Termine JD, Posner AS: Vibrational spectra and structure of the phosphate ion in some calcium phosphates. Trans NY Acad Sci 27~669, 1965 11. Ducheyne P, Raemdonck WV, Heughebaert JC, et al: Structural analysis of hydroxyapatite coatings on titanium. Biomaterials 7197, 1986 12. Bone1 G, Monte1 G: Chimie minbrale-Sur une nouvelle apatite carbona& synthetique. CR Acad Sci Paris 258:923, 1964 13. Rey C, Collins B, Goehl T, et al: The carbonate environment in bone mineral: A resolution-enhanced Fourier transform infrared spectroscopy study. Calcif Tissue 45:157, 1989 14. Scholl FK: Hummel/Scholl, Infrared Analysis of Polymers, Resins and Additives. An Atlas. Vo12. Additives and Processing Aids. Mtinchen, Germany, Carl Hanser Verlag, and Weinheim/ Bergstr, Verlag Chemie, 1973, p 76 15. Vignoles M, Bone1 G, Holcomb DW, et al: 1nIluenc.e of preparation conditions on the composition of type B carbonated hydroxyapatite and on the localization of the carbonate ions. C&if Tissue Int 43:33, 1988 16. Roy DM, Linnehan SK: Hydroxyapatite formed from coral skeletal carbonate by hydrothermal exchange. Nature 247: 220, 1974 17. Trombe J-C, Monte1 G: Chimie minerale-Sur les spectre d’ap sorption infrarouge des apatites dont les tunnels contiennent des ions bivalents et des lacunes. CR Acad Sci Paris 276: 127 1, 1973 18. Soevik T, Braekkan OR: Fluoride in Antarctic krill (Euphasia superba) and Atlantic krill (Megunyctiphanes nmegica). J Fish Res Board Can 36:1414, 1979 19. Chiroff RT, White EW, Weber JN, et al: Tissue ingrowth of replamine form implants. J Biomed Mater Res Symp 6:29, 1975
RIDGE AUGMENTATION
BY APATITES
20. Donath K, Rohrer MD, Beck-Mannagetta J: A histologic evaluation of a mandibular cross section one year after augmentation with hydroxyapatite particles. Oral Surg Oral Med Oral Path01 63:651, 1987 21. Donath K, Rohrer MD, Hoermann K: Mobile and immobile hydroxylapatite integration and resorption and its influence on bone. J Oral Implants 13:11, 1987 22. Cranin AN, Ronen E, Shpuntoff R, et al: Hydroxylapatite (H/ A) particulate versus cones as post-extraction implants in humans. J Biomed Mater Res 22: 1165, 1988 23. Kato K, Aoki H, Tabata T, et al: Biocompatibility of apatite ceramics in mandibles. Biomater Med Dev Art Org 7:291, 1979 24. Kenney EB, Lekovic V, Cruranza FA, et al: A comparative clinical study of solid and granular porous hydroxylapatite implants in human periodontal osseous defects. J Biomed Mater Res 22: 1233, 1988 25. Osborn JF: Hydroxylapatitkeramik-Ein osteotroper Werkstoff fur den Knochenersatz. Experimentelle Mund-Kiefer-Gesichts-ChirurgieMikrochirurgische Eingriffe. Stuttgart, Germany, Georg Thieme Verlag, 1983, pp 37-40 26. Donath K, Hoermann K, Kirsch A: Welchen Einfluss hat die Hvdroxvlanatitkeramik auf die Knochenneubildund Dtsch Z Kiefer Gesichts Chir 9:438, 1985 27. Jarcho M, Kay JF, Gumaer KI, et al: Tissue, cellular and subcellular events at a bone-ceramic hydroxylapatite interface. J Bioeng 1:79, 1977 28. Block MS, Kent JN: Healing of mandibular ridge augmentations using hydroxylapatite with and without the addition of autogenous bone in dogs. J Oral Maxillofac Surg 43:3, 1985 29. Chang CS, Matukas VJ, Lemons JE: Histologic study of hydroxylapatite as an implant material for mandibular augmentation. J Oral Maxillofac Surg 41:729, 1983 30. Frame JW, Rout PGJ, Browne RM: Ridge augmentation using solid and porous hydroxylapatite particles with and without autogenous bone or plaster. J Oral Maxillofac Sung 45:77 1, 1987 31. Pinhoh EM, Kwon PHJ: The effect of therapeutic radiation on canine alveolar ridges augmented with hydroxylapatite. J Oral Maxillofac Surg 50:250, 1992 32. Rothstein SS, Paris D, Sage B: Use of Durapatite for the rehabilitation of resorbed alveolar ridges. J Am Dent Assoc 109: 571, 1984 porous 33. White E, Shots EC: Biomaterial aspects of Interporehydroxyapatite. Dent Clin North Am 30:49, 1986 34. Worsaae N, Hjoerting-Hansen E: Genopbygning af Processus Alveolaris med Hydroxylapatite, in Hjoerting-Hansen E (ed): Odontologi 88. Copenhagen, Munksgaard, 1988, pp 159- 169 35. Hjoerting-Hansen E: Studies on Implantation of Anorganic Bone in Cystic Jaw Lesions. Copenhagen, Munksgaard, 1970, pp l-198 36. Burchardt H: The biology of bone graft repair. Clin Orthop 174: 28, 1983 37. Gregoire M, Orly I, Menanteau J, et al: In vitro interactions between calcium phosphate biomaterials and human fibroblastic cells. II. Incidences on the behaviour of cultured gingival cells. Adv Biomat 8:2 15, 1988 38. Orly I, Gregoire M, Menanteau J, et al: In vitro interactions between calcium phosphate biomaterials and human fibroblastic cells. I. Comparative extra and intracellular evolution of 4 synthetic calcium phosphates. Adv Biomater 8:2 11,1988 39. Bagambisa FB, Joos U, Schilh W: The interaction of osteogenic cells with hydroxylapatite implant materials in vitro and in vivo. Int J Oral Maxillofac Implants 5:2 17, 1990 40. Kawamura M, Iwata H, Sato K, et al: Chondro-osteogenetic response to crude bone matrix proteins bound to hydroxylapatite. Clin Orthop 217:281, 1987 41. Klein CP, de Groot K, Vermeiden JP, et al: Interaction of some serum proteins with hydroxylapatite and other materials. J Biomed Mater Res 14705, 1980 42. Klein CP, de Groot K, Driessen AA, et al: A comparative study of different beta-whitlockite ceramics in rabbit cortical bone with regard to their biodegradation behaviour. Biomater 7: 144, 1986
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43. Holmes RE, Hagler HK: Porous hydroxylapatite as a bone graft substitute in mandibular contour augmentation: A histometric study. J Oral Maxillofac Surg 45:421, 1987 44. Holmes RE, Roser SM: Porous hydroxyapatite as a hone graft
substitute in alveolar ridge augmentation: a histometric study. Int J Oral Maxillofac Sung 16:718, 1987
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Surg
1992
Discussion Chemical, Physical, and Histologic Studies on Four Commercial Apatites Used for Alveolar Ridge Augmentation Mohamed El Deeb, BDS, DOS, MS University of Minnesota, Minneapolis School of Dentistry Synthetic hydroxylapatite (HA) has been used widely in various maxillofacial applications during the last 10 years.ie9 Currently, many products with different shapes are available. Some are porous; some are nonporous. The purpose of the article by Pinholt et al was 1) to evaluate the biological reaction to four different granular HA materials (Alveograf, Calcitite, Interpore-200, and Algipore placed subperiosteally for augmentation of the alveolar ridge and 2) to correlate the tissue reaction to the materials with their characteristics, as obtained by chemical analysis and infrared spectrometry. Pinholt et al state that none of the four types of HA tested in this study caused osteoinduction or osteoconduction, and that fibrous encapsulation, with multinuclear giant cells, was observed around all of them. We have used granular Alveograf, Calcitite, and Interporein preclinical and clinical investigations,2-9 and experience has indicated that nonporous HA, when used in subperiosteal augmentation, generally is associated with little bone formation, if any, at the interface between the implant and the host bone. When HA is placed subperiosteally, the granules only contact bone on one surface and are surrounded by soft tissue in all other directions. Therefore, in these situations, bone forms only at the implantbone interface. On the other hand, when HA is placed within a bony defect, the granules are surrounded by bony walls on all sides except superiorly and bone will form within the defect, surrounding the HA granules. As for the osteoinductive capabilities of the materials used in this study, I agree with Pinholt et al that none have osteoinductive capabilities. We have placed granules of Interpore-200 intramuscularly in the chests of Rhesus monkeys6 and Calcitite material in the chests of rats’ and also found no evidence of bone formation in either study. The authors state that “osteoconduction is a healing process in which the implanted material acts as a scaffold that is gradually resorbed during creeping substitution as the healing proceeds from the adjacent living bone.” I do not believe that a material has to be resorbed completely to be considered an osteoconductive material. Parts or all of the material may remain and act as a scaffold for the formation of new bone, and it may still be considered osteoconductive. Although it may be true that the materials used in this study have no osteoconductive capabilities, I believe that the porous HA has osteoconductive properties, as evidenced by our study in Rhesus monkeys.6 This study showed that when granules of
porous HA are mixed with demineralized bone powder and placed intramuscularly in the chests of Rhesus monkeys, for-
mation of bone is noted within the pores of the material, which acted as a scaffo1d.6 Boyne et al also found that when porous HA was mixed with an osteoinductive material, the bony growth was superior to that obtained by mixing nonporous HA with the same material.’ If a porous HA is used for subperiosteal augmentation of an alveolar ridge, we would expect more bone formation than when nonporous HA is used. Porous HA results in more bone formation than nonporous HA because of the interconnecting pores and also because it reacts differently histochemically. In a previous comparative study, we documented that the type of collagen formed with porous HA differs from the type of collagen formed with a nonporous HA material.’ Our study concluded that porous HA serves as a matrix for ingrowth of host bone, as evidenced by the proliferation of types I and V collagen, whereas nonporous HA elicits a fibrous tissue encapsulation of the implanted material, with collagen types I and III prevailing at-the implant/tissue interface.’ Factors that might affect formation of bone in subperiosteal ridge augmentation using HA are: 1) the amount of material placed; 2) stabilization and lack of mobility of the material; 3) the type of HA used; and 4) the site of the augmentation. The reasons that no bone formed within the material tested in this study may be the following: 1) The authors used only four granules per implant, and these were placed subperiosteally. This amount is extremely limited, bone has difficulty forming around such a small amount of HA. 2) The authors used HA to augment bucahy and superiosteally, without removing the teeth or stabilizing the material. This might not keep the material in close contact with bone and subsequently, it would be surrounded by fibrous tissue. 3) Use of the facial musculature during eating and drinking might create a micromotion that would displace the material further from the augmented site.
References 1. Boyne, Scheer P, Stringer D: Comparison of porous and nonporous hydroxylapatite and xenographs in restoration of ridges. Presented at the AAOMS 67th Annual Meeting, Washington, DC, October 1985 2. El Deeb M, Roszkowski M, El Deeb, ME: Nonporous hydroxylapatite gramdes as an extracranial and extranasal augmenting material in dogs: Technique and initial findings. Cleft Palate J 24:4, 1987 3. El Deeb M, Roszkowski M: Hydroxylapatite granules and blocks as an extracranial augmenting material in rhesus monkeys. J Oral Maxillofac Surg 46:33, 1988
