purified fibrillar collagen composite implants

oral surgery oral medicine oral pathology with

sections

OPI

oral and maxillofacial and endodontics

radiology

oral surgery Editor: ROBERT B. SHIRA, DDS School of Dental Medicine,

Tufts University

I Kneeland Street Boston, Massachusetts 02111

Histologic evaluation of the bone/graft interface after mandibular augmentation with hydroxylapatite/purified fibrillar collagen composite implants Donald R. Mehlisch, MD, DDS,a Alan S. Leider, DDS, MA,b and W. Eugene Roberts, DDS, PhD,C Austin, Texas, and San Francisco, Cali’ BIOMEDICAL

RESEARCH

GROUP

AND

UNIVERSITY

OF THE

PACIFIC

Samples of the bone/graft interface were evaluated histologically in five patients 1 year afler mandibular ridge augmentation with a composite of hydroxylapatite particles in a matrix of purified fibrillar collagen (HAIPFC). The resulting defects were refilled with HA/PFC after the biopsy specimens were obtained. Histologic examination of the specimens yielded no evidence of purified fibrillar collagen. Hydroxylapatite particles were surrounded by dense fibrous host connective tissue, trabeculae of woven and lamellar bone, or both. HA/PFC was found to be biocompatible with human tissue and receptive to direct bone apposition on the hydroxylapatite particles. (ORAL

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lveolar ridge atrophy resulting from alveolar bone resorption is prevalent among an estimated 20 million edentulous denture wearers in the United States and affects both denture retention and stability.’ Severe maxillary or mandibular ridge atrophy necessitates surgical reconstruction with the use

Supported by The Collagen Corporation, Palo Alto, Calif. BBiomedical Research Group, Inc., Austin. bDirector, Pacific Oral Pathology Laboratory, University Pacific, San Francisco. CDirector, Pacific Bone Research Laboratory, University Pacific, San Francisco. 7/12/14157

of the of the

of one or more of the materials available for bone repair. One such material is hydroxylapatite (HA), which in particulate implants forms a stable graft when placed subperiosteally against host bone. Numerous animal studies have provided histologic evidence of the long-term biocompatibility of particulate HA and of its .favorable interaction with both soft tissue and bone.2-9 In exhaustive animal testing of the soft tissue interaction, Drobeck and coworkers2 implanted HA (in both multifaceted particle and disk forms) subcutaneously along the spines of rats, and in dogs between the shoulders, on the hips, and subperiosteally in the mandible. The lack of a clinical inflammatory response was corroborated by the ab685

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Fig. 1. Diagram of augmented alveolar ridge with HA/ PFC composite implant that demonstrates excision of biopsy specimen from bone/implant interface.

Fig. 2. Immediate reaugmentation of site of biopsy at bone/implant interface with segment of HA/PFC composite implant.

sence of histologic evidence for such a response. Both particles and disks were eventually surrounded by dense fibrous collagen capsules. Kurashina and coworkers3 confirmed the formation of a “biologic seal” around canine perimucosal HA implants. This response was characterized by epithelial attachment and connection of supra-alveolar collagen fibers to the implant surface. Although Misiek and associates4 noted transient mild inflammation with implants of round particle HA in buccal soft tissue pouches in beagles, the reaction had disappeared by 6 months and the particles were enclosed in a collagenous matrix. Nine months after placing HA subperiosteally in dogs, Chang and colleagues5 noted considerable bony growth into the graft with no apparent foreign body

Fig. 3. Patient 117. A, Photomicrograph showing dense fibrous connective surrounding HA particles of HA/PFC composite implant. There is no evidence of PFC component of graft. Host connective tissue is closely associated with ceramic particles, but occasional foreign body cells (arrowheadsj intervene at particulate surface. Mononuclear infiltrate was absent in this specimen. (Decalcified section, H & E stain; original magnification, X40.) B, High-magnification photomicrograph showing fibrovascular connective tissue of varying density surrounding portion of single HA particle. Vascular channels are indicated by arrowheads. (Decalcified section, H & E stain; original magnification, x100.)

reaction or resorption. Investigating the bone/HA interface, Gumaer and other& implanted both granular and solid HA in holes drilled into canine femur bone that just entered the marrow cavity. The implants were completely surrounded by mature lamellar bone 6 to 8 years later; it appeared that host bone incorporated the HA and remodeled in a manner similar to normal bone. As in the soft tissue implants, no inflammatory reaction was observed, demonstrating excellent compatibility of HA with bone tissue. West and Brustein7 implanted coralline-form HA into intrabony pockets in two dogs; at 6 to 8 months the implants were completely surrounded and invaded by fibrovascular tissue, and often enclosed in woven

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4. Patient 119. A, Photomicrograph of section from biopsy specimen showing dense fibrous connective tissue (CTJ associated with surface of HA particle. Again, there is no evidenceof inflammatory processrelated to ceramic or surrounding tissue. (Decalcified [50 pm] section, H & E stain; original magnification, X100.) B, Section from adjacent region of same biopsy specimen shown in Fig. 4, A. Observedenseviable lamellar bone (LB) in direct opposition with several HA particles. (Decalcified section, H & E stain; original magnification, X 100.)

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Fig.

5. Patient 103. Photomicrograph showing new bone (NB) surrounding HA particles in left field and dense connective tissue (CT) surrounding particles in right field. Arrowheads indicate line of demarcation between preexisting alveolar bone (PL?)and new bone (NB). (Undecalcified section, H & E stain; original magnification, x20.) Fig.

composite, a combination of dense HA and PFC, in human beings. bone. Channels within these implants usually had vascular cores enveloped in connective tissue, which was further enclosed in a “collar” of woven bone. Again, no inflammatory reaction was noted. Canine mandibular ridge augmentation with coralline HA by Piecuch and associates8 demonstrated greater bony ingrowth than with previously used ceramic materials, and a similar trial by Block and Kentlo showed an osseoinductive response by means of HA with and without autogenous bone. The present study is the first histologic evaluation of the interface of host bone and graft after implantation of an HA/purified fibrillar collagen (PFC)* “Alveoform

biograft

(bone

grafting

matrix).

MATERIAL

AND METHODS

In a clinical trial initiated in 1985,77 patients had alveolar ridge augmentation with an HA/PFC composite without autologous bone. 1L ‘* The grafts used were sterile, preformed implants that contained a protein matrix of highly purified, fibrillar bovine collagen into which was incorporated a mineral component consisting of high-density, particulate HA. The specific collagen preparation that was combined with HA has been marketed in injectable form since 1981; its safety, as well as that of particulate HA, has been extensively documented. 13 Of the 77 participants in the original trial, five were solicited for biopsy and subsequent histologic evalu-

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Fig. 6. Patient 129. Photomicrograph showing preexisting alveolar bone (PB), new bony ingrowth (NB) from alveolar ridge inferiorly, and dense connective tissue in superior aspect of implant. Bone implant interface iarrowheads) suggests normal remodeling of new and preexisting alveolar bone. (Undecalcified section, H & E stain; original magnification, X20.)

Fig. 7. Microradiograph of adjacent section from same biopsy specimen shown in Fig. 6 also demonstrating bony ingrowth (NB) and fibrous connective tissue (CT) beyond zone of direct bone apposition on HA particles. Several particles have scalloped margins consistent with moderate demineralization of HA. (Decalcified section, H & E stain; original magnification, X20.)

ation of the mandibular alveolar bone and HA/PFC composite implant interface. These particular patients underwent implantation at least 1 year before reentry and were representative of the original study group with respect to extent of ridge atrophy before augmentation, age (one 75 years old, one 46 years old, and three in the mid-60s), and sex (four females, one male). All patients had unremarkable medical histories, were told of potential risks and benefits of the procedure, and gave informed consent. Preoperative baseline examination included panoramic and true lateral cephalometric radiographs. Photographs and study casts were taken to document the clinical condition of the alveolar ridge. A study cast was also obtained for use in constructing a surgical splint to help recontour the biopsy site. In all cases, the biopsy site was restored to its presurgical condition by placing additional HA/PFC composite into the defect. Antibiotic therapy was initiated 24 hours before surgery and maintained until 5 days after the proce-

dure. Participants were treated as outpatients and received local anesthesia with or without intravenous sedation. Mucoperiosteal incisions were made horizontally along the buccal vestibule anterior to the mental foramen, approximately at the level of the original crest of the preaugmented alveolar ridge. Exposure of the HA/PFC composite implant and mandibular bone was accomplished by careful dissection. Periosteum and fibrous tissue were dissected from the implant and mandibular bone, and a section of basal bone and implant 4 to 6 mm in height was removed (Fig. 1). The specimen was taken at an angle to the side of the buccal vestibule to preserve the crest of the implant at the lingual plate and sustain structural ridge support. To allow adequate interface evaluation, the specimen generally included a minimum of 2 mm of basal bone and 2 mm of HA/PFC composite. After biopsy, the resected area of bone and implant was reaugmented with HA/PFC composite (Fig. 2) and the mucosal flaps were closed with silk sutures.

Histology

Volume 70 Number 6 Table

I. Summary of histologic findings in patients treated with HA/PFC

Patient No.

117 119 103 126 129 *No

of HAIpFC

interface

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composite

Type of host tissue injiltrating HA particles

Biopsy of bone/graft interface obtained?

Bone

Fibrous connective tissue

No Yes Yes Yes Yes

No Yes Yes No Yes

Yes Yes Yes Yes Yes

host bone was excised in this specimen;

composite bone/graft

1

only

implant

Extent of injlammation

Minimal Minimal Minimal Minimal Minimal

Host bone status

* Normal Normal Normal Normal

was taken.

Patients wore a temporary splint for 24 hours to help contour the new HA/PFC composite implant in the surgical defect, after which they resumed wearing dentures with soft linings. Sutures were removed 5 to 7 days after surgery, and a preliminary evaluation of healing was made. A final evaluation 1 month after surgery verified proper healing, ridge contour, and denture acceptability. After healing was complete, dentures were relined. At the time of surgery, specimens were blotted dry and marked with an indelible ink pen on the upper right quadrant (the buccal aspect) and also on the most posterior region to record anatomic orientation. The biopsy specimens were fixed in 10% neutral buffered formalin to prepare both undecalcified and decalcified sections. Undecalcified specimens were embedded in high-density methyl methacrylate (Polysciences, Inc., Warrington, Pa.); sections were cut at 50 to 70 pm and left unstained or stained with hematoxylin and eosin (H&E). These sections were examined with ordinary light microscopy as well as polarized light. Microradiographs of 70 pm undecalcified sections from two patients were prepared by exposure on a Kodak high-resolution spectroscopic plate (50 minutes, 27 kVp, 3 mA) before dehydrating and mounting for direct microscopy. Other specimens decalcified in formic acid and sodium citrate solution were processed for paraffin embedding, sectioned at 5 to 7 pm and stained with H&E; 3 pm sections from the residual undecalcified blocks were also stained with H&E. These thin decalcified and undecalcified sections were used to increase resolution of local cell and tissue reactions to the implants. RESULTS

All five patients tolerated the surgical excision of a 4 x 6 mm biopsy specimen from the implant/bone interface and had no postoperative sequelae. Reaugmentation of the biopsy site was accomplished by the reimplantation of a segment of HA/PFC composite (Fig. 2). All patients were able to resume denture functioning after postoperative healing occurred and a denture reline was completed.

Histologic evaluation of both undecalcified and decalcified sections from the implant/bone interface suggested a high level of biocompatibility with soft tissues and bone. Figs. 3 to 9 are photomicrographs of specimens from each of the five patients and microradiographs of specimens from two patients. Table I summarizes the histologic findings in the five patients studied. The PFC component of the HA/PFC composite had been replaced by host fibrous connective tissue in regions of every sample. The connective tissue was moderately dense, well vascularized, and generally in immediate contact with the HA particles as illustrated in Fig. 3, A and B. Inflammation was minimal in all specimens, as evidenced by a mild, scattered, mononuclear cell infiltrate or an occasional multinucleated giant cell (Fig. 3, A). In three of the five specimens, host connective tissue and bone had infiltrated different regions of the implant (Figs. 4 to 6). The newly deposited bone associated with the implant was classified as woven and/or lamellar. Direct opposition of bone on HA particles indicates a high degree of biocompatibility of the implant. Rigid osseous fixation of some of the particles indicates the HA was stabilized within the implant. Bone has low tolerance for micromotion.9, l4 Stabilization of HA particles within a collagen matrix appears to be a favorable attribute for osseous ingrowth (Figs. 5 and 6). Microradiographs of a section from one patient (Table I, No. 129) showed both bone and connective tissue ingrowth into the graft (Fig. 7) whereas only connective tissue ingrowth was observed in a second patient (Table I, No. 126; Fig. 8). In both cases, the HA particles were scalloped, indicative of moderate demineralization of the ceramic. The osseous surface of the alveolar ridge was clearly revealed in the microradiograph and an adjacent thin (3 pm) decalcified section from patient 126 (Figs. 8 and 9). Approximately 70% of the ridge surface was smooth and 30% scalloped, suggestive of modeling activity consistent with normal bone turnover in an edentulous alveolar ridge. Hence the architecture of adjacent

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Fig. 8. Patient 126. Microradiograph at a higher magnification from patient showing only connective tissue (CTJ ingrowth at surface of alveolar ridge (AR). Some surface demineralization larrowheads) is apparent in central HA particle. Ridge is generally smooth, but a few crypts suggest normal surface modeling. (Decalcified section, H & E stain; original magnification, X40.)

mineralized tissues was unaffected and consistent with biocompatibility of the graft. Fluorescent bone labels were not administered to patients in this study so that a dynamic interpretation of bone-forming activity was not possible. However, histologic evaluation of sections at all levels clearly indicates that HA/PFC composite provides a suitable environment for bony and fibrous fixation at the ridge/implant interface. DISCUSSION

HA was clinically tested in human beings for the augmentation of the atrophic alveolar ridge after the biocompatibility and safety were demonstrated in animal models. Since the initial successful clinical trials of alveolar ridge augmentation by Kent and colleagues, 15,i6 limited human histologic data have been available to the practitioner. Our study supports the findings of several animal studies, along with a few human trials, that fibrous connective tissue and bony

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Fig. 9. Photomicrograph of section from same case shown in Fig. 8 demonstrating HA particle, dense connective tissue (CT), and normal alveolar ridge bone (AR). (Decalcified section, H & E stain; original magnification, x100.)

growth from the host eventually infiltrate implanted particulate HA. This fixation results in a rigid and firmly anchored alveolar ridge implant. Although it was not possible to demonstrate the precise mechanism by which this bony ingrowth occurred, the processapparently involves initial invasion of the implant by fibroblast-like cells that deposit collagenous fibrous tissue followed in time by woven and then lamellar bone formation, progressing from the host bone outward. Foreign body reaction was minimal to nonexistent, confirming that both HA and PFC were biocompatible with human tissue, as has been abundantly demonstrated in other studies.13, I4 In an earlier study, Beirne and Greenspan” performed a vestibuloplasty and removed from each of five patients a 3 mm specimen of HA implanted for 3 to 15 months. Histologic findings showed fibrous connective scar tissue containing macrophages in all specimens and, in one specimen, multinucleated giant cells surrounded the HA particles. No bone/implant interface was present. This mild inflammatory response observed by Beirne and coworkers”, ‘* was similar to that noted by Froum and associates19 when human

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periodontal defects were filled with HA; no bond was observed between graft and bone. By contrast, several other investigators have observed new bone formation associated with HA particles. In a specimen taken 5 months after augmentation, Chao and Poon20 observed new bone formation between host bone and the implant with mature connective tissue between individual particles; no foreign body reaction was noted. Histologic examination of a single human specimen at 1 year after implantation with HA revealed bone formation around the particles and new bone replacing the interparticulate fibrous tissue, in addition to a mild localized lymphocytic infiltrate reportedly caused by denture pressure. Similarly, Page and Laskin2’ found new compact bone with well-formed lamellae and haversian systems extending from the alveolar ridge directly into the implant and enclosing the HA particles. In an animal study, Gumaer and colleagues noted interdigitating connective tissue stalks at the bone/ HA interface that contained multinucleated cells (possibly osteoclasts). The authors commented that although this phenomenon was rarely seen, it might represent “limited resorptive activity on the surfaces of a few isolated granules.” This tentative evidence of resorption has not been supported by findings from any other animal study. Donath and coworkers22 examined a human specimen that was cross-sectioned from the site of a recent mandibular fracture 1 year after a bone defect at the site was filled with dense, particulate HA. Direct bone apposition onto HA particles was only noted when host bone and implant were in close proximity and not in areas where particles were mobile, suggesting that new bone growth proceeds from the host bone toward the implant. Mobile particles were surrounded with connective tissue alone. Particles encased in new bone showed perpendicular bonding of collagen fibers between the lamellar bone and the surface of the ceramic. All particles of HA had border zones of dissolution into small crystalline granules (rubble zones), and an active process of HA resorption was evidenced by the appearance of HA granules within the cytoplasm of mononuclear phagocytes in the haversian canals. The authors speculated that impurity of the HA used may have contributed to the well-established resorption process. Although scalloped margins of the HA particles consistent with limited demineralization were observed with HA/PFC composite, neither rubble zones nor phagocytized granules were noted. Thus, although our findings do call into question previous assumptions of the nonresorbable property of HA, the observed demineralization of the particles seems consistent with normal bone turnover in the mandible. It appears that HA may be subject to the same gradual processes of resorption and replacement by new bone

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that characterize the life of the host bone itself. Further investigation of this possibility must rest with future studies. As in the original study of alveolar ridge augmentation with HA/PFC composite,” the cohesive yet moldable form of the HA/PFC was advantageous, allowing rapid and uncomplicated replacement of the material subjected to biopsy, so that patients could resume use of soft-lined dentures after only 24 hours of wearing a temporary splint. HA/PFC composite appears to be an excellent biocompatible material capable of osseointegration with host bone and an excellent means by which the atrophic alveolar ridge may be augmented. We thank Susanne Smith, Estelita Deleon, and Mary Gonzalez for their technical assistance during this project.

REFERENCES

1. Prosthodontic care: number and types of denture wearers, 1975. Chicago: Bureau of Economic and Research Statistics, American Dental Association, 1976. 2. Drobeck HP, Rothstein SS, Gumaer KI, Sherer AD, Slighter RG. Histologic observation of soft tissue responses to implanted, multifaceted particles and discs of hydroxylapatite. J Oral Maxillofac Sura 1984:42:143-9. 3. Kurashina K, de Lange GL, de Putter C, de Groot K. Reaction of surrounding gingiva to perimucosal implants of dense hydroxylapatite in dogs. Biomaterials 1984;5:215-20. 4. Misiek DJ. Kent JN. Carr RF. Soft tissue resoonses to hydroxylapatite particles of different shapes. J Oral Maxillofat Surg 1984;42:150-60. 5. Chang C-S, Matukas VJ, Lemons JE. Histologic study of hydroxylapatite as an implant material for mandibular augmentation. J Oral Maxillofac Surg 1983;41:729-37. 6. Gumaer KI, Sherer AD, Slighter RG, Rothstein SS, Drobeck HP. Tissue response in dogs to dense hydroxylapatite implantation in the femur. J Oral Maxillofac Surg 1986;44:618-27. 7. West TL, Brustein DD. Freeze-dried bone and coralline implants compared in the dog. J Periodontol 1985;56:348-5 1. 8. Piecuch JF, Topazian RG, Skoly S, Wolfe S. Experimental ridge augmentation with porous hydroxylapatite implants. J Dent Res 1983;62:148-54. 9. Roberts WE, Turley PK, Brezniate N, Fielder PJ. Bone physiology and metabolism. Calif Dent Assoc J 1987;15:54-61. 10. Block MS, Kent JN. Healing of mandibular ridge augmentations using hydroxylapatite with and without autogenous bone in dogs. J Oral Maxillofac Surg 1985;43:3-7. 11. Mehlisch DR, Taylor TD, Leibold DG, et al. Evaluation of collagen/hydroxylapatite for augmenting deficient alveolar ridges: a preliminary report. J Oral Maxillofac Surg 1987; 45:408-13. 12. Mehlisch DR, Taylor TD, Leibold DG, et al. Collagen/ hydroxylapatite implant for augmenting deficient alveolar ridges: 12-month clinical data. J Oral Maxillofac Surg 1988;46:839-43. 13. DeLustro F, Smith ST, Sundsmo J, Salem G, Kincaid S, Ellingsworth L. Reaction to injectable collagen: results in animal models and clinical use. Plast Reconstr Surg 1987;79:581-92. 14. Brunski JB. Biomaterials and biomechanics. Calif Dent Assoc J 1988;16:66-75. 15. Kent JN, Quinn JH, Zide MF, Finger lM, Jarcho M, Rothstein SS. Correction of alveolar ridge deficiencies with nonresorbable hydroxylapatite. J Am Dent Assoc 1982; 105:993-1001. 16. Kent JN, Quinn JH, MF, Guerra LR, Boyne PJ. Alveolar ridge augmentation using nonresorbable hydroxylapatite with

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or without autogenous cancellous bone. J Oral Maxillofac Surg 1983;41:629-42. Beirne OR, Greenspan JS. Histologic evaluation of tissue response to hydroxylapatite implanted on human mandibles. J Dent Res 1985;64: 1152-4. Beirne OR, Curtis TA, Greenspan JS. Mandibular augmentation with hydroxylapatite. J Prosthet Dent 1986;55:362-7. Froum SJ, Kushner L, Scopp IW, Stahl SS. Human clinical and histological responses to Durapatite implants in intraosseous lesions, case reports. J Periodontol 1982;53:719-25. Chao S-Y, Poon C-K. Histologic study of tissue response to implanted hydroxylapatite in two patients. J Oral Maxillofac Surg 1987;45:359-62. Page DG, Laskin DM. Tissue response at the bone-implant in-

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terface in a hydroxylapatite augmented mandibular ridge. J Oral Maxillofac Surg 1987;45:356-8. 22. Donath K. Rohrer M, Beck-Mannagetta J. A histological evaluation of a mandibular cross section 1 year after augmentation with hydroxyapatite particles. ORAL SURG ORAL MED ORAL

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