Osseous response to implanted natural bone mineral and synthetic hydroxylapatite ceramic in the repair of experimental skull bone defects

J Oral Maxillofac

Surg

50:241-249,1992

Osseous Response to Implanted Natural Bone Mineral and Synthetic Hydroxylapatite Ceramic in the Repair of Experimental Skull Bone Defects BJijRN STEN

KLINGE, DMD, PHD,* PER ALBERIUS, ISAKSSON, DMD, MD,* AND JijRGEN

DMD, MD, Pt-tD,t JijNSSON, DMD§

The purpose of this study was to assess and compare the osseous responses to implanted particles of resorbable anorganic xenograft bone mineral and nonresorbable dense synthetic hydroxylapatite of two different granule sizes. Four trephine calvarial defects were produced in each of 13 adult rabbits. The experimental materials were subsequently implanted in three defects, leaving the fourth defect for control purposes. Six animals were killed 4 weeks after surgery and seven at 14 weeks. The tissue responses were assessed by contact radiography, light microscopy, and histometry. The biocompatibility of the implants was confirmed. All defects healed uneventfully, although the resorbable hydroxylapatite seemed to promote initial bone regeneration. The importance to orthognathic surgery of early and effective healing of bone gaps, as well as of the advantage of implant resorbability to bone remodeling, are discussed.

To avoid these problems, various allogeneic and alloplastic materials have been developed; interest especially has focused on the latter materials in search of suitable and biocompatible substitutes for bone grafts. Hydroxylapatite (HA), a compound that contributes the major portion of rigid mineral structure to bone and teeth, seems to be a bone graft substitute with wide applications. Calcium phosphate ceramics have been used in a variety of forms for more than a decade. The dense and porous HA forms have lately been recommended for bone grafting in orthognathic surgery.5-8 A biological affinity with mammalian bone minerals has been reported? although the ceramic itself is suggested to have no osteogenic ability,“-” but is believed to permit osteoconduction in areas that are adjacent to host bone.‘*-I4 However, to what extent is bony healing affected by resorption of the graft? Is bony repair enhanced? Unlike synthetic HA, natural HA products may undergo physiological remodeling” and resorption. Recently, an anorganic resorbable natural bone mineral (Bio-Oss, Gestlich Pharma, Switzerland) has been introduced. The objective of this study was to assess and compare the osseous responses to particles of resorbable natural bone mineral and nonresorbable synthetic HA. This was accomplished in an animal model.

Treatment of craniofacial and maxillofacial deformities, as well as congenital deficiencies, has a long history of experimentation with various grafts and implants. In the adult skull, large defects resulting from trauma, neoplasms, or infection will not heal spontaneously. Similarly, osteotomy gaps in the facial skeleton often require grafting to stabilize the osseous segments and enhance healing. In attempts to achieve rapid and proper bony healing, several forms of autogeneic bone transplants have been used.’ However, procurement of such bone has some disadvantages, eg, the necessity of a second surgical site, morbidity and potential deformity of the donor site, an increased operative time, and donor availability limitations. Furthermore, major resorption of autogeneic bone grafts has been reported in patients subjected to craniofacial reconstruction.2-4

* Head, Department of Laboratory Animal Research, Faculty of Odontology, University of Lund, Malmo, Sweden, t Associate Professor, Department of Plastic Surgery, MAS, Maim& Sweden. $ Head, Department of Oral Surgery, Lasarettet, Halmstad, Sweden. 0 Research Assistant, Department of Laboratory Animal Research, Faculty of Odontology, University of Lund, Malmo, Sweden. Address correspondence and reprint requests to Dr Alberius: Department of Plastic Surgery, MAS, S-2 I4 0 I Malmo, Sweden. 0 1992 American

Association

of Oral and Maxillofacial

Surgeons

0278-2391/92/5003-0007$3.00/O

241

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OSSEOUS RESPONSETO BONE SUBSTITUTE

Materials and Methods ANIMALS

Thirteen adult male and female New Zealand white rabbits (3.2 * 0.6 kg) were kept in standard laboratory conditions of light-dark schedule and relative humidity. Stock diet and tap water were provided ad libitum. Six animals were killed by an overdose of pentobarbital at 4 weeks and seven at 14 weeks after surgery. Each subgroup had similar sex and body weight distribution. EXPERIMENTAL MATERIALS

Two different calcium phosphate materials were used in the study; synthetic dense HA of two different granule sizes (0.3 to 0.6 mm and 0.6 to 1.Omm, respectively; Apaceram, Asahi Optical Co, Ltd, Japan) and resorbable porous hydroxylapatite (particle size, 0.25 to 1.0 mm; Bio-Oss) Bio-Oss is an anorganic xenograft material of bovine origin with a structure similar to cancellous bone. SURGERY The surgical procedure has been presented in detail elsewhere.‘(j In brief, four 5-mm-diameter, full-thickness (approximately 2 mm) skull defects were made in the frontal and parietal bones with a trephine (Fig 1) under neuroleptanalgesia supplemented with intramuscular diazepam. The circular bone plugs were gently removed and extreme care was exercised to avoid injury to the dura mater and its fibrous attachments to the inner table of the calvarial bone. Postoperative antibiotics (Streptocillin, vet 0.1 mL; dihydrostreptomycin, 0.25 g/mL; and benzylpenicilline 0.2 g/mL; Novo Industri a/s, Denmark) were administered once daily for 1 week. The size of the defects was selected to resemble the typical magnitude of an osteotomy gap in maxillofacial surgery. The selected diameter made possible simultaneous investigation of all experimental implants in each animal, ie, each test site was compared with the remaining experimental sites and the control defect. In this project, no attempt was made to produce any critical size defects, defects of a size that will not heal during the lifetime of the animal,17 as the osteoinductive potential of the implant material had already been tested and found to be nonexistent.‘0~“,‘8~19The strategy ~sed~~~~’ gives the possibility to evaluate the interaction between spontaneous bone regeneration and the influence of the implant on the healing response. EXPERIMENTALOUTLINE The defects were rinsed with sterile saline and then either filled with an implant or left empty. The peri-

FIGURE 1. Dorsal view of the rabbitcalvarium showing the positioning of the experimentaldefects.N, Nasal bone;F, frontalbone; P, parietalbone; SC.coronal suture.

osteum was repositioned and the incision closed. The defects were evaluated at 4 or 14 weeks following implantation. Four experimental groups were constructed: group 1, defects filled with resorbable natural bone mineral (rHA); group 2, defects filled with dense HA with a granule size of 0.3 to 0.6 mm (sHA); group 3, defects filled with dense hydroxylapatite with a granule size of 0.6 to 1.0 mm (IHA); and group 4, untreated control defects. The filling material was placed in a different pattern in each animal to avoid any bias that may have arisen because of site. ANALYSIS OF DATA

Osseous repair was evaluated by contact radiography,22 histology, and histometry. Contact radiographs were obtained by placing the calvarial specimens on a 5 X 7-cm Kodak Ektaspeed Intraoral film. The radiographic apparatus, Siemens Heliodent (Siemens, Germany), was set at 70 kV, with an exposure time of 0.2 seconds. The contact radiographs of each defect were independently scored by two observers to fit into a predetermined healing category (0, no healing; 1, observ-

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ET AL

able new bone mineralization; 2, bone mineralization covering the major part of the defect; 3, defect completely filled with mineralized bone); for the first two categories, amalgamation between implant and surrounding bone was considered not to have occurred, whereas grades 3 and 4 constituted amalgamation. Ordinarily, immediate mutual agreement on the classification of the healing result was reached, but when the evaluation was not unanimous, the defect was reevaluated until mutual agreement was reached. Following radiography, the calvarial specimens were fixed in buffered formalin, decalcified in formic acid, trimmed, and embedded in paraffin. Serial sections 7 pm thick, were cut transversally in the midpart of the lesion and stained with hematoxylin and eosin. All slides were evaluated twice by each of two independent observers without prior knowledge of which type of implant to be examined. Hereafter, all slides exhibiting divergent grading between observers (near 15%) were studied simultaneously by these observers to determine the definitive grading score. The healing pattern was analyzed with particular reference to fibrous tissue involvement, marginal bone cell necrosis, new bone production, graft incorporation, bone marrow redevelopment, and bone maturation. A grading systemz2 was applied to assess bone regeneration (0, none; 1, small amounts; 2, moderate amounts; 3, profuse amounts; 4, bridging) and bone marrow redevelopment (0, none; 1, beginning to appear; 2, present in moderate amounts; 3, colonization by red marrow or fat). The histologic sections from the rabbits killed 4 weeks after surgery were evaluated by a video image digital analysis system (Oculus TM-200, Coreco, Inc, Toronto, Canada). Measurements of the bone density of representative tissue areas demonstrating the regenerating bone in the defect were compared with identically sized areas of surgically noninvolved bone distant from the defect but in the same microscopic slide. The image analyzer converted the image of the selected area into digital grey values and, subsequently, these were correlated to the number of pixels. The result was calculated as a test-to-control ratio for each animal and, thereafter, the ratios were statistically evaluated. The analysis was randomly repeated in 16 slides to assure precision. The advanced bony healing found after 14 weeks did not allow for exact definition of the margin of the defects and, consequently, histometric analysis was not accurate and hence is not reported. The control skull defects were assessed for reference purposes concerning the amount of bone in the defects. SCANNING ELECTRON MICROSCOPY

The implant materials were fixed for 2 hours in 1% osmium tetroxide in 0.15 M cacodylate buffer at room temperature. After repeated washes in buffer, the de-

hydration was performed in an ethanol series. The specimens were subsequently dried in a Balzer critical point drier, mounted with epoxy glue on holders, and sputtered in a Polaron E 5400 high-resolution coater with gold/palladium (40:60). The preparations were studied in a JEOL T 330 scanning electron microscope. Results The materials tested showed slightly dissimilar surface structure (Fig 2). All incisions heated uneventfully. Gentle manual palpation disclosed no instability of implanted materials. RADIOGRAPHIC EXAMINATION

The control defects showed limited healing 4 weeks after surgery, but after 14 weeks most defects were covered by a rather homogenous mineralized tissue. The implants varied in their potential to incorporate. At 4 weeks, the sHA granules exhibited a better amalgamation than the larger ones, although the implantation of rHA implants was most successful and resulted in the best amalgamation to contiguous bone (Fig 3). After 14 weeks, all implants seemed well amalgamated to the surrounding bony margins; the size of the implanted granules appeared now to be only of minor importance to the regenerative response. However, both types of dense granules (sHA and 1HA) remained as distinct radiopacities. The rHA implants amalgamated best to the border of the defects and were much less radiopaque, with a density similar to the surrounding bone (Fig 4). The extent of amalgamation of the implanted materials to surrounding bone as evaluated radiographically is presented in Table 1. The amount of amalgamation (RA) was expressed as the ratio of the number of amalgamated implants to the total number of implants of each material. HISTOLOGIC EXAMINATION

At 4 weeks, the interstitial space between the implants was infiltrated by loose, immature fibrous connective tissue, which was slightly denser around the large HA granules (1HA). The rich cellularity and vascularity of the fibrous tissue seemed similar between groups. After 14 weeks, the fibrous tissue was less abundant and appeared in slender bands between the bone trabeculae, the composition and extent not being markedly different between experimental groups. These observations are based on many sections, but no attempt was made to measure the different cell types present. Moderate marginal bone necrosis and accompanying local bone resorption were observed at 4 weeks in most specimens, especially in the 1HA and rHA implant groups; this bone was completely revitalized after

244

OSSEOUS RESPONSE TO BONE SUBSTITUTE

FIGURE 2. Scanning electron micrographs showing the materials implanted. A, rHA; B, sHA; C, IHA. Note the different surface structure of the rHA, sHa, and 1Ha implants (horizontal bar corresponds to 10 pm, original magnification X2,000).

14 weeks. Generally, the initial inflammatory cellular response was mild in the adjacent tissue stroma and a few scattered giant cells were sometimes identified within the implant proximity. At 14 weeks, the inflammatory response had become minimal. No cerebral herniation was detected. After 4 weeks bone formation was obvious at the defect margin, along the periosteal surface adjacent to the margin of the defect, and in solitary bony islands scattered on the dura. Bony repair was highly variable, although osteoblasts were seen on the surface of all implants tested. In all groups, occasional specimens showed bone to traverse the defect, but this was restricted to a thin layer along the dural surface. In all animals, the majority of the bone produced emanated from the margins of the defects. Osseous production was most extensive along the surfaces of the small HA

(sHA; Fig 5) and rHA (Fig 6) implants. Bone was usually in direct apposition to the implants. The bone was mostly of the woven type, but lamellar apposition covering the HA implants and the dural islands was also noted. The histomorphometric analysis showed the rHA implant to exhibit a significantly higher ratio of bone density than controls, but differences between remaining groups were not significant. The measurements of bone density in the sHA and 1HA groups were markedly dispersed. The vascular content in bone was comparable between groups. Already at 4 weeks a beginning redevelopment of the bone marrow was observed, particularly in the 1HA group (Fig 7). After 14 weeks, all implants were completely integrated into the surrounding bone, which generally appeared more mature. The sHA group showed the highest vascularity. The marrow reappearance was most

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FIGURE 3. Radiograph showing the calvarial tissue block 4 weeks after surgery. A beginning integration of the implant materials to the surrounding bone is evident. The resorbable HA implant (I) seems to be the most successful. 1, rHA; 2, sHA: 3, IHA; 4, control.

extensive in the IHA and the rHA implant groups and preceded controls slightly. All specimens in each group had complete bony coverage of the defect, although the total amount of bone formation around the nonresorbable implants seemed to lag behind that seen in rHA implants. The architecture of the bone tissue differed slightly between groups. The sHA group showed wide bone trabeculae, similar in size to the surrounding unoperated bone and controls. The 1HA implant group seemed more disorganized, with slender bone trabeculae and poorly developed cancellous bone between the compact outer and inner bone layers. Also, the amount of fibrous tissue relative to bone appeared highest in this group. The rHA implants appeared to have degraded progressively and were only detectable irregularly. Osteoclasts in moderate amounts were found close to the implant-tissue interface (Fig 8). This group displayed thin but dense mature trabeculae. Grading of the extent of bone regeneration and bone marrow redevelopment is presented in Table 1.

FIGURE 4. Radiograph at 14 weeks. The nonresorbable granules (2, sHA: 3,1HA) are clearly visible throughout their respective defects, whereas the rHA (I) appears to have degraded considerably. The degree of mineralization in the latter defect compares favorably with the control defect (4).

prove bony regeneration. Primarily, these implants are placed for maxillary inferior repositioning, large magnitudes of maxillary advancement or transverse expansion, and for segmental osteotomies in the maxilla. Theoretically, a rapid and mature bony healing of the osteotomy line will give positional stability of the segments and minimize the tendency for relapse while, on the other hand, uncontrolled resorption of the imTable 1. Bony Regeneration, Bone Marrow Redevelopment, Bone Density, and Radiographic Assessment of implant Amalgamation to Contiguous Bone in Experimental Skull Defects rHA wk4 (n = 6)

wk 14 (n = 7)

sHA

IHA

Control

BR BMR RA

3.5 (0.6) 1.0 (-) 0.83

2.5 (1.3) 1.0 (0.8) 0.50

2.8 (1.0) 1.2 (0.8) 0

2.5 (1.3) 0.5 (0.6) -

BD BR BMR RA

0.64 (0.07)’ 4.0 (-) 2.0 (0.6) 1.0

0.32 (0.32) 4.0 (-_) 1.3 (0.5) 1.0

0.28 (0.36) 4.0 (-_) 2.2 (0.8) 1.0

0.02 (0.02) 4.0 (--_) 1.5 (0.8) -

Discussion Various bone graft substitutes are routinely used in midface osteotomy gaps and continuity defects to im-

Measurements are mean (SD). Abbreviations: BD, bone density: BMR, bone marrow redevelopment: BR, bony regeneration: RA. radiographic assessment. * P < .05 relativeto controls (Wilcoxon signed rank test).

246

FIGURE 5. Microphotograph showing regeneration in the dense HA implantation group with small granule size (sHA) at 4 weeks. The area of the implant is covered by cellular and irregular bone (hematoxylin-eosin stain, original magnification X63).

planted material may completely destroy the operative result by jeopardizing the stability of the surgical segments. The purpose of the present investigation was to compare the effect of particulate resorbable and nonresorbable HA in an experimental model of active osteogenesis under comparable healing conditions (in terms of the amount of compact and cancellous bone tissue) to the frontal maxillary region. Previous investigations studying porous HA have mostly employed coralline or synthetic phosphate ceramics. We decided to study resorbable anorganic natural bone mineral, newly introduced into the oral surgical field,23,24relative to nonresorbable dense HA-implant granules. The HA particulate form was selected to enable complete osseous regeneration. In onlay experimental’3*25 and clinical* studies, a superficial bony ingrowth of only 20% to 30% has been registered in mandibular and maxillary dense HA blocks by histometry. Further-

FIGURE 6. Resorbable natural bone mineral (rHA) 4 weeks after implantation into calvarial defect. Notice lingering thin bone trabeculae covered by osteoblasts. The implant material has been dissolved during section preparation (hematoxylin-eosin stain, original magnification X63).

OSSEOUS RESPONSE TO BONE SUBSTITUTE

FIGURE 7. Illustration of IHA implant 4 weeks after implantation. Notice the beginning reappearance of the bone marrow and the thin bone trabeculae covered by osteoblasts partly encompassing the implanted material (i) (hematoxylin-eosin stain, original magnification X63).

more, Salyer and Hall8 concluded that a full replacement of dense HA blocks with regenerating bone is not a realistic expectation. We chose to use the adult rabbit calvarium, which, like the major parts of the frontal midface, contains only modest amounts of bone marrow. Generally, bone marrow may facilitate bone formation,26-28 although it is not necessary for its occurrence.29’30 For example, Uchida et a131implanted calcium HA into small bur holes in the skulls of rats and rabbits but new bone was rarely seen within the ceramic pores. They suggested this lack of healing was the result of the skulls being almost devoid of bone marrow. Similarly, Ohgushi et al28implanted porous HA blocks in rat femoral defects, but found these to exhibit no healing potential unless the implants were combined with marrow. Interestingly, we found the major part of the healing process

FIGURE 8. Resorbable hydroxylapatite (rHA) 14 weeks after implantation. Notice the small remnant of the implant centrally; a few osteoclasts can be seen at the periphery. The bone marrow is redeveloping (hematoxylin-eosin stain, original magnification X63).

KLINGE

ET AL

to emanate from the diploe. At first, the periosteal bone production resulted in the formation of an umbrellalike structure of endocranial and ectocranial compact bone seemingly with modest importance to the basic primary interconnection of the bone edges. Later, though, complete union of the external and internal plates was observed. The ideal bone graft substitute material from a biological and biochemical point of view is one that is completely replaced by new bone from the host. Calcium phosphate compounds are attractive because of their biocompatibility and chemical and physical resemblance to bone mineral. Specific characteristics such as the surface structure, shown by the scanning electron microscopic study, may vary considerably between implants. Yet the impact of such differences is impossible to define strictly. However, the result of this investigation corroborates previous investigations32s33 as to the biocompatibility of these compounds. We found that only a mild initial host inflammatory response was provoked at 4 weeks, and subsided almost completely at 14 weeks. At the intervals tested, no evidence of resorption of host bone adjacent to the implants was detected, nor was any deviation from the normal morphologic appearance evident. Furthermore, the fibrous tissue response was similar to that reported in earlier studies. Consequently, in this study, the general reactions of the tissues surrounding the implants were in accord with previous reports, which implies that a sound and reliable basis for the evaluation of the bone tissue response to the experimental materials was achieved. The use of decalcified sections for histologic observation permitted detailed analysis of the regenerative process. We found that osteoid and mineralization fronts were localized peripherally to the implants, the apposition of which subsequently occurred to produce complete bony union over the defects. No tendency of bone induction was detectable, although the resorbable implant seemed to promote initial bony healing. We noted definite regenerative activity already after four weeks despite the maturity of the animals used. Similarly, Finn et a134and Butts et a13’observed beginning bone production after 2 to 3 weeks in adult mongrel dogs subjected to alveolar ridge augmentation or interpositional mandibular implantation with porous HA. Furthermore, Bhaskar et a136found bone tissue to be deposited within 2 weeks on the lattice of tibia1 implants in adult rats. In contrast, Alper et al,37 investigating unilateral radial defects in rats, found encapsulation of porous HA powder without even signs of osteoconduction. These divergent results point to the importance, especially in the mature animal, of direct contact between the surface of the implanted material and the adjoining, healthy, vital bone tissue. The pores of porous HA bone substitute provide a

247 passive scaffold for vascular ingrowth, which then leads to osteogenic cell ingrowth and bone apposition.38 Bone ingrowth into the pores of calcium phosphate ceramics has now been reported by numerous investigators using a variety of materials and animal models. The optimum pore size of porous bone substitutes has been discussed by many investigators. Flatley et a139considered this to be 500 pm and Uchida et a127found a better penetration of bone with larger pore sizes (2 10 to 300 pm) than with smaller sizes (150 to 2 10 pm). Hulbert et a140reported the best bone tissue ingrowth to occur with pore size of greater than 100 pm. Also, Klawitter et a14’defined the optimum pore size to be 100 to 135 pm. Daculsi and Passuti:* comparing porosities from 100 to 600 pm, found that the 100~pm macropores allowed for bone ingrowth, although the larger sizes were invaded by bone more rapidly. The structural similarity of Bio-Oss to cancellous bone implies that the pore size of this implant is well within this range and explains to a substantial extent the early and effective bone apposition observed in these implants. Moreover, Kita et a144found the angular structure of the rHA granules to prevent dense packing, which would also allow for a greater infiltration of new bone. Although the porosity leads to improved bone ingrowth, it is also associated with implant degradation; the macroporosity seen in porous HA materials signifies increased degradation. 44,45The variability in resorbability is considered to be a function of composition, porosity, and crystallinity,46 although the definitive mechanism is debated. 38 From a physiological point of view, moderate resorbability would be less detrimental to the adaptive remodeling of the surrounding bone because no interference from the implanted material would occur while, on the contrary, the permanent nature of dense HA might negatively affect the mechanical stresses in adjacent bone, eventually leading to nonphysiological remodeling. The markedly improved healing observed in the rHA group raises the question of whether factors from the wound area stimulatory to bone regeneration are activated in conjunction with HA degradation. Some differences in the regenerative pattern between the experimental groups were obvious. At 4 weeks, the rHA implant was fairly well (five of six defects) amalgamated to the surrounding bone radiologically, and microscopically the bone regeneration was impressive and the redevelopment of the bone marrow comparable with the other groups. These observations were substantiated by the histomorphometric analysis. The healing of the two groups testing dense HA implants was equivalent to that of controls, except that the bone marrow reappearance seemed to have progressed further in the former groups. Radiographically, 1HA implants showed nonamalgamation. After 14 weeks all defects had healed. rHA and 1HA implants

248 showed a more normalized bone marrow appearance than remaining groups, although a complete normalization was not yet accomplished. Kita et a1,43when investigating the reactions of Bio-Oss implanted into drill holes in the rabbit knee, noted a greater incidence of marrow regeneration in defects filled with rHA than with dense HA materials. Nevertheless, the implantation of rHA seems to promote and enhance healing in membranous defects composed of mostly compact bone surfaces. Moreover, the presence of osteoclastlike cells on the surface of rHA implants as observed by us and Boyne et al47 suggests that natural bone mineral implants remodel in a more physiological manner.15 Furthermore, the great variability registered for bone density measurements in the sHA and 1HA groups is perplexing. Probably, these data reflect the difference in physical (surface topography) and biochemical properties of synthetic materials relative to natural bone (rHA and control groups). Some comparisons of porous and dense HA have been reported earlier. For example, El Deeb and Holmes4* performed zygomatic and mandibular augmentations in rhesus monkeys and reported that dense HA developed a fibrous capsule around the implant while bony ingrowth occurred solely in the porous surface. Frame et al49 performed ridge augmentation in edentulous areas of dog jaws and observed no significant difference in the amount of bone ingrowth into HA particles. The new bone was formed in those parts of the implants that were adjacent to the underlying alveolar bone. Krejci et alSo clinically evaluated granular dense and block porous HA implants in the treatment of human periodontal bony defects and found improvement in only those sites treated with dense HA. We, on the other hand, observed definite long-term bone regeneration around all implants regardless of granule size or resorbability, although initial bone regeneration in and around rHA implants was more extensive. Consequently, interest is now focused on the clinical application of anorganic natural bone mineral, used alone or as an autograft extender, for interpositional implantation in maxillofacial osteotomy defects. Acknowledgment We thank Dr Eric Hallberg, Department of Zoology, University of Lund, for generous help with the scanning electron microscopic investigation. The Apaceram implant material was kindly provided by Bioimplant Scandinavia AB, Malmo, Sweden.

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OSSEOUS RESPONSE TO BONE SUBSTITUTE

4. Schultz RC: Reconstruction of facial deformities with alloplastic material. Ann Plast Surg 7:434, 1981 5. Kent JN, Quinn JH, Zide MF, et al: Alveolar ridge augmentation using nonresorbable hydroxylapatite with or without autogenous cancellous bone. J Oral Maxillofac Sum 41:629, 1983 6. Wolford LM, Wardrop RW, Hartog JM: Con&e porous hydroxylapatite as a bone grafl substitute in orthognathic surgery. J Oral Maxillofac Sure 45: 1034. 1987 7. Rosen HM: Porous, bl&k hydroxyapatite as an interpositional bone graft substitute in orthognathic surgery. Plast Reconstr Surg 83:985, 1989 8. Salyer KE, Hall CD: Porous hydroxyapatite as an onlay bonegraft substitute for maxillofacial surgery. Plast Reconstr Surg 84:236, 1989 9. Jarcho M, Kay JF, Gumaer KI, et al: Tissue, cellular and subcellular events at a bone-ceramic hydroxylapatite interface. J Bioeng 1:79, 1977 10. McDavid PT, Boone ME, Kafmwy AH, et al: Effect of autogenous marrow and calcitonin on reactions to a ceramic. J Dent Res 58: 1478, 1979 11. Nade S, Armstrong L, McCartney E, et ah Osteogenesis after bone and bone marrow transplantation. The ability of ceramic materials to sustain osteogenesis from transplanted bone marrow cells: Preliminary studies. Clin Orthop 18 1:255, 1983 12. Cameron HU, Macnab I, Pilliar RM: Evaluation ofbiodegradable ceramic. J Biomed Mater Res 11:179, 1977 13. Holmes R, Hagler H: Porous hydroxyapatite as a bone graft sub stitute in maxillary augmentation: A histometric study. J Craniomaxillofac Surg 16:199, 1988 14. Holmes RE, Hagler HK: Porous hydroxyapatite as a bone graft substitute in cranial reconstruction: A histometric study. Plast Reconstr Surg 8 1:662,1988 15. Spector M: Characterization of calcium phosphate bioceramic implants. Luzem, Switzerland, Symposium on “Modem Trends in Bone Graft Substitute Materials,” May 11, 1990 16. Alberius P, Klinge B, Isaksson S: Management of craniotomy in young rabbits. Lab Anim 23:70, 1989 17. Schmitz JP, Hollinger JO: The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop 205:299, 1986 18. Jarcho M: Calcium phosphate ceramics as hard tissue prosthetics. Clin Orthop 157:259, 198 1 19. El Deeb M, Roszkowski MT, El Hakim I: Subcutaneous implantation of hydroxylapatite/collagen in induced diabetic and non-diabetic rats. Int J Oral Maxillofac Sum 19:113, 1990 20. Glowacki J, Altobelli D, Mulliken JB: Fate of-mineralized and demineralized osseous implants in cranial defects. Calcif Tissue Int 33:71, 1981 21. 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 22. Alberius P, Isaksson S, Klinge B, et al: Regeneration of cranial suture and bone plate lesions in rabbits. Implications for positioning of osteotomies. J Craniomaxihofac Surg 18:271, 1990 23. Nentwig G-H, Gassner A: 2.5 years of clinical experience in the therapy of cystic alveolar defects with a spongious bovine hydroxyapatite implant material. Heidelberg, Germany, 2nd international symposium on ceramics in medicine, September 10-l 1, 1989 24. Boyne PJ: Comparison of Bio-Oss and other implant materials in the maintenance of the alveolar ridge of the mandible in man. Luzem, Switzerland, International symposium on “Modem Trends in Bone Graft Substitute Materials,” May 11, 1990 25. Holmes RE, Hagler HK: Porous hydroxyapatite as a bone graft substitute in mandibular contour augmentation: A histometric study. J Oral Maxillofac Surg 45:421, 1987 26. Burwell RG: Studies in the transplantation of bone: VIII. Treated composite homograft-autografts of cancellous bone; An analysis of inductive mechanisms in bone transplantation. J Bone Joint Surg 48B:532, 1966 27. Uchida A, Nade S, McCartney E, et al: Bone ingrowth into three different ceramics implanted into the tibia of rats and rabbits. J Orthop Res 3:65, 1985

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