Copyright S Munksgaard 1996 Journal of Clinical Periodontology.  ISSN: 0303-6979

J Clin Periodontal 1996; 23:615-620
Printed in Denmark.
All rights reserved.

Stuart J.Froum Department of Surgical Sciences (Periodontics)
and Implants. New York University Dental Center.
345 E. 24th Street. New York. NY 10010-4099. USA

Human histologic evaluation of
HTR polymer and freeze-dried
bone allograft

A case report

Froum SJ: Human histologic evaluation of HTR polymer and freeze-dried bone allograft. A cast report. J Clin Periodontol 1996. 23. 615-620. © Munksgaard. 1996.

Abstract. This case report compares the results of placement of HTR™ (Hard Tissue Replacement Synthetic Bone™) and freeze-dried bone allograft (FDBA) in the same patient. 2 notches were made in each of 6 teeth, 1 at the gingival margin and the other at the most apical level of calculus. Soft tissue responses with both materials included probing depth reduction, gingival shrinkage and gain in clinical attachment. Histological sections of block extraction sites 30 months after placement of either material showed no signs of new attachment. Little or no inflammation was present with both materials. There was also an absence of all FDBA particles which assumes complete resorption of this material prior to 30 months. Gingival shrinkage resulted in the exposure of the gingival and calculus notch in all FDBA treated sites. Gingival epithelium was found adjacent to the gingival notch in all HTR™ treated sites. The calculus notch in HTR™ treated sites was lined by junctional epithelium with connective tissue and bone opposing the adhesion. HTR™ particles were present and surrounded by connective tissue or bone. HTR™ appears to serve as a scaffold for new bone formation when in close contact with alveolar bone.

Key words: human histology: HTR: freeze-dried bone allograft Accepted for publication 3 August 1995

Various bone replacement grafts have been used to facilitate restoration of bone and, in some instances, periodontal support lost to periodontal disease. Reviews by Froum & Gomez (1993) and Garrett & Bogle (1994) support the use of various bone graft and bone substitute materials to treat such defects, the latter noting that "conclusive evidence now exists that some tissue regeneration occurs after regenerative procedures, in which bone grafts are used". Hancock (1989) documented this with autogeneous and allogenic bone grafts in the management of vertical type osseous defects. Freeze dried bone allograft FDBA and HTR™ (Hard Tissue Replacement) Synthetic Bone™ alloplast (Bioplant Inc.. New York. NY) are 2 materials for which positive clinical results have been reported. Sepe et al. (1978), Sanders et al. (1983) have described clinical results with FDBA alone and in combination with autogenous bone. In reviewing the results, Mellonig et al. (1991) reported 50% to 100% bone fill in 220 of 327 test sites treated with FDBA and surgically re-entry 6 months or more later. Rumelhart et al. (1989) found no statistically significant difference in clinical bone fill, levels of probing depth reduction or gain in attachment in a comparison of FDBA with decalcified freeze dried bone allograft (DFDBA).  Although conclusive histologic evidence of regeneration following use of DFDBA has been presented (Bowers et al 1989a, 1989b). no such studies with FDBA have been reported. Treatment of human infrabony and furcation defects with HTR1 Nl have shown favorable clinical results. Yukna (1991) compared HTR™ with open flap debridement in 21 patients using 6-month re-entry data.  HTR™ resulted in greater mean defect fill, (2.2 mm versus 1.00 mm), decreased probing depth (3.2 mm versus 2.3 mm) and increased gain in clinical attachment (1.9 mm versus 1.0 mm) versus the flap debridement control. More recently. Yukna (1994) compared HTR™ to autogenous osseous coagulum (AOC) at 15 paired mandibular Class II furcations in 9 subjects and reported improved results with HTR™ in enhancing horizontal furcation fill (1.9 mm versus 0.8 mm) and in % of defect fill (44.4% versus 17.1%). Improved clinical results with HTR™ are however, not universal. In a 12-month, 15-patient clinical evaluation of bilateral infrabony defects treated with either open flap debridement or HTR™ polymer grafts. Shahmiri et al. (1992) found no statistical significant differences in pocket responses. While human histologic studies have demonstrated bone formation around HTR™ particles (Yukna & Greer 1992. Suzki et al. 1989), Stahl et al. (1990) have reported a "gamut of healing responses“ in human block sections taken after 4 to 26 weeks. HTR™ particles were surrounded for the most part by dense collagen with peripheral bone formation seen only occasionally. Clinical closure consisted a of long epithelial adhesion at 7 sites and limited new attachment in 4 instances. The present case report describes the 30-month post-healing clinical and histological outcomes of the use of FDBA or HTR™ at 6 intraosseous defects in the same individual.

Material and Methods

A 56-year-old man presented with a medical history which included angina (last attack 3 years earlier) and an allergy to penicillin. He had stopped a cardizam regimen I year earlier. His physicians reported no contra-indications to treatment or medication and no restriction on the use of epinephrine in obtaining local anesthesia. The patient's chief complaints were occasional pain in the region of the right maxillary premolar. gingival bleeding and difficulty in chewing because of his missing maxillary molars.

Table 1. Presurgical clinical data of FDBA and HTR™ grafted sites (mm).

Table 2. Clinical measurements (mm) at sites 30 months after placement of FDBA and HTR™, before block sections.

 
He reported "swelling and pus" palatal to the maxillary incisors. Although whole-mouth periodontal treatment was provided, only that at teeth 13, 12, 11, 21, 22, 23 is considered here. A fixed splint was in place from tooth 16 to tooth 22 and a single temporary crown on #23. Teeth 14 and 15 had pockets extending to the apices. Although obviously hopeless, as they were symptom-free and in good function, they were retained, symptoms permitting. They were scheduled for removal when all other remaining maxillary teeth were to be extracted. Teeth 12-22 had probing depths of 5 8 mm and responded to light probing with bleeding and exudate.  Independent evaluation by 2 members of the Department of Diagnosis and the Department of Prosthodontics concurred with the patient's wish that all of the remaining maxillary teeth should be extracted. The patient insisted that "he wants no further problems" and desired a complete maxillary denture fitted. Based on that information, he was referred for possible participation in our evaluation study.

Fig. I. Pre-surgical clinical appearance after removal of tooth no. 16 (pontic). A fixed splint extends from tooth 15 to tooth no. 22. A temporary crown is present on tooth no. 23.

Fig. 2. Flap reflection reveals defects on teeth nos. 21 to 23. Note supracrestal calculus.

Fig 3. Histologic overview of mesial surface of tooth no. 13, 30 months after FDBA placement. Note the sulcular epithelium apical to the remnants of the calculus notch (arrow). Hematoxylin-eosin stain. 35Xmagnification.

Fig 4. Histological appearance of mesial of tooth no. 13 apical to Fig. 3 with no evidence of new attachment, new cementum, or graft particles. Hematoxylin-eosin stain. 35Xmagnifcation.

The nature of the study was explained to the patient, whereby 2 types of bone replacement materials would he used and the teeth subsequently extracted in block section, 6 to 12 months following surgery. This protocol would change only if symptoms demanded early extraction. The patient was informed that he could withdraw from the study at any time and signed a consent form. All clinical measurements were made by the author. Scaling and oral hygiene instructions were provided 1 month prior to surgery. The maxillary right 1st molar pontic was severed from the splint at the time of scaling to reduce the occlusal stress on the right premolar teeth (Fig. 1). No attempt was made to remove subgingival calculus. Oral hygiene instructions were repeated weekly until surgery.

Measurements

1 month after scaling and oral hygiene instruction, pre-surgical measurements were recorded for teeth #13-23 (Table I). Dentinal notches were made with a no. 1/2 round bur at the gingival margin immediately apical to the crown margin on each tooth at the greatest probing depth site of each test tooth. Measurements were made with a Boley gauge, silver point and locking pliers to the nearest 0.1 mm (Froum et al. 1982). The distance from the gingival notch to the base of the pocket was recorded, as was the degree of mobility of the splint and individual tooth. Following flap reflection, a 2nd notch was made through the most apical point of calculus at the invoked root site (Froum et al. 1983). The distances from that notch to the bone crest and from the notch to the deepest point of the osseous defect were recorded. Soft tissue measurements were repeated from the most coronal notch 1 month before block section.

Surgical procedure

Under local anesthesia, buccal and palatal full thickness mucoperiosteal flaps were reflected (Fig. 2). Following root/calculus notching, the root and osseous defect were thoroughly debrided. The root was debrided of all visible hard and soft deposits to the base of the osseous defects. Debridement was completed with ultrasonic and hand instruments. Intra-marrow penetration was performed at 2 sites (mesial surfaces of teeth nos. 12, 22). Spontaneous bleeding from the walls of the bony crest was evident at all other sites. 3 adjacent sites (13, 12, 11) were tilled with FDBA (crushed cortical bone. 200 350 µm. University of Miami, Tissue Bank, Miami, FL) and the 3 other sites (21. 22. 231 were filled with HTR™ (#40). To minimize the risk of one material migrating into the site of another, it was decided against use of different materials at alternate sides. The 2 bordering sites receiving different materials, tooth 11 (lingual) and tooth 21 (lingual) had "contained" circumferential defects from which material migration seemed unlikely. Both materials were prepared with saline solution at least 30 min before implantation. Sues were overfilled with graft materials and covered completely by the coronally positioned and sutured flap. A periodontal dressing (Coe Park. GC America Inc., Chicago. IL) was placed. Tetracycline 250 mg qid was prescribed for 2 weeks, after which the sutures were removed. Professional plaque removal was provided weekly for 6 weeks and then at 2-week intervals for 6 months. The patient was then unable to return for about 2 years, but reported professional "cleanings" at 3- to 6- month intervals and some compliance in home care regimen. Scaling was then provided 3x over a 4-week period and appropriate measurements were recorded and radiographs were taken. The maxillary teeth, numbers (13, 12, 11, 22, 23), were then extracted in block section. All individual specimens were decalcified and prepared for histologic examination. Step-serial mesially cut sections (8 µm) of teeth 13, 12, 22, and 23 and step-serial buccal-lingual sections of teeth 11 and 21 were prepared and stained selectively with hematoxylin- eosin and Mallory-trichrome stains.

Results
Clinical observations

Preoperative probing and bone osseous depths ranged between 6.9 mm and 8.2 mm, and 2.9 mm and 4.6 mm. respectively. No mobility of either the splinted teeth or the single canine was observed at any stage. There was no bleeding on probing recorded before block section removal, and post-surgical healing was uneventful. By 30 months, all sites showed significant pocket reductions ami gain in clinical attachment level (Table 2). Post-surgical probing depths at sites treated with FDBA were between 3.5 to 4.5 mm. and those treated with HTR™. 3.2 to 3.6 mm. Gain in attachment was between 1.9 and 2.5 mm. and between 3.5 and 3.7 mm, with FDBA and HTR™ sites, respectively. Gingival shrinkage was between 1.1 and 1.8 mm at FDBA sites, and between 0.1 and 1.0 at HTR™ sites.

Histological Observations
Freeze dried bone allograft

Post surgical-shrinkage led to exposure of the gingival and calculus notches in all specimens. Sulcular epithelium was located apical to the calculus notch (Fig. 3). Further apical to that notch, attachment consisted of junctional epithelium and inserted collagen fibers with no evidence of cementogenesis either supra-or sub-crestally (Fig. 4).

Fig 5 Histological appearance of mesial of tooth no. 23, 30 months after HTR™ graft placement. Note presence of long junctional epithelium at the apical aspect of the calculus notch. Hematoxylin-eosin stain, 35x magnification.

Fig. 6. Histologic overview of site of mesial of tooth no. 22. Note bone surrounding the HTR™ particle at the osseous crest and the limited inflammation seen in the soft tissue opposite the HTR™ particle. Mallory trichrome stain, 35xmagnification.

Fig. 7.  High magnification of crestal bone as shown in Fig. 5. Note that the HTR™ particle is surrounded at the crrst with bone that apparently abuts the particle at its periphery. Hematoxylin-eosin stain. 125 - magnification.

Minimal inflammation was present in the underlying connective tissue with no evidence of FDBA particles in any section. Although no evidence of active osteogenesis was present, reversal lines were noted along the periodontal aspect of the alveolar bone and along the crest.

Hard tissue replacement Synthetic Bone™ alloplast \

The gingival epithelium faced the coronal notch at all sites and a long junctional epithelium was seen extending to the apical aspect of the calculus notch (Fig. 5). Particles of HTR™ were present, some of which were in close proximity to the crest, and surrounded by a connective tissue capsule with limited crestal remodeling and new bone formation. However, other sections showed particles surrounded by lamellar bone, suggestive of crestal remodeling and new bone formation (Figs. 6. 7). Minimal gingivil inflammation was seen in all sections, with no evidence of cementogenesis opposite the alveolar crests.

Discussion

The results described here are based on only 6 sites in a single patient and cannot be considered universal. Both bone replacement materials, FDBA and HTR™ Synthetic Bone ,alloplast, resulted in improved clinical parameters: decreased probing depth and new clinical attachment. Gingival shrinkage occurred at all sites and little gingival inflammation was evident. The patient's presentation with minimal deposits, inflammation and bleeding on probing after a 2-year absence was an indication that he did have professional supportive therapy (as he reported) and did maintain a high level of homecare. However, in view of the pre-extraction scaling and root planing, the degree to which bone replacement materials affected the healing responses remains unclear. The observation that neither material led to periodontal regeneration or new attachment, is at variance with a previous report which documented limited new attachment with HTR™ material in 4 of 11 block sections (Stahl et al. 1990). The negative response reported here may relate to the position of the calculus notches which were coronal to the existing alveolar crest at the time of implant surgery. Had it been formed, new attachment would have been supracrestal. Such new attachment has been shown to occur to a limited degree only in select cases with iliac marrow (Dragoo & Sullivan 1983) and with barrier membranes and coronal anchorage (Stahl & Froum 1991). There was no evidence of any FDBA particles in any section 30 months after implantation. This is of interest in light of a recent publication (Becker et al. 1994) which reported little or no resorption of decalcified Freeze-dried bone 3 to 13 months after insertion in fresh human extraction sockets. The difference between their histological results and those described here may arise from differences in grafted sites, material used (DFDBA, FDBA), the time of histological examination (13 months versus 30 months), or a combination of these factors. HTR™ particles were present in all sections 30 months after surgery and appeared to be well tolerated, with little or no surrounding inflammation. Such particles, when surrounded by connective tissue, were in close proximity to the crestal bone position. Other particles were partially or completely surrounded by bone. This suggests that bone grew around them as part of osseous remodeling. The lack of cementogenesis indicates that new attachment did not form at any of the experimental sites with either of the materials examined. Osteoclasts were not observed, even 30 months after surgery, and there was no evidence of resorption of HTR™ particles. It is impossible, in the present case, to determine which variables were responsible for the clinical improvements seen following surgery. Several authors (Nyman et al. 1975, Rosling et al. 1975, Polson 1978, Froum et al. 1982) have reported significant gains in clinical attachment, probing depth reduction and osseous till, following open debridement alone. Perhaps the thorough debridement performed here is responsible for the clinical results. Human histological studies, however, have found little or no new bone growth with open debridement alone (Stahl et al. 1982). In the present study, new bone growth was evident at sites where HTR particles had been deposited, and remained close to the alveolar bone.

Zusammenfassung

Histologische Auswertung von HTR Polymer und gefriergetrocknetem allogenen Knochenimplantat am Menschen. Ein Fallbericht. Mit diesem Fallbericht wird beabsichtigt, die Resultate des Einbringens von HTR™ (Hartgewebeersatz mit synthetischem Knochen™), am gleichen Patienten mit gefriergetrocknetem, allogenen Knochenimplantat (FDBA) zu vergleichen. An jedem einzelnen einer Gesamtheit von 6 Zahnen wurden 2 Kerben angebracht, die eine am Gingivalsaum und die andere an dem am weitesten apikal gelegenen Zahnsteinniveau. Reduktion der Sondierungstiefe, Schrumpfung der Gingiva und klinischer Attachmentgewinn wurden als Reaktion des Weichgewebes auf die beiden Knocherersatzmaterialien aufgefaβt und evaluiert. Histologische Schnitte der 30 Monate nach dem Einbringen beider Materialien vorgenommenen Blockextraktion, erbrachten keine Hinweise auf neues.  Attachment. Beide Materialien verursachten keine odor nur geringe Entzundung. Weiterhin wurden keine FDBA Partikel gesehen, was die Annahme nahelegt, daβ die vollstandige Resorption dieses Materials vor dem Ablauf der 30 Monate stattfand. An allen mit FDBA behandelten Stellen. hatte das Schrumpten der Gingiva die freilegung: die am Gingivasaum und am apikalen Zahnsteinniveau gesetzten Kerben zur Folge. An allen mit HTR™ behandelten Stellen wurde direkt neben der Gingivakerbe gelegenes Gingivaepithel vorgefunden.  An den mit HTR™ behandelten Stellen wurde weiterhin die Zahnsteinkerbe mit, von Bindegewebe durchsetztem, Saumepithel und adhasionshinderndem Knochen abgedeckt. Von Bindegewebe oder Knochen umgebene HTR™ Panikel waren vorhanden. In engem Kontakt mit dem Alveolarknochen befindliches HTR™ scheint als gerust fur die Neubildung von Knochen zu dienen.

Resume

Evaluation histologique humaine d'allogreffe d'os seche et congele et d'un polymere HTR. Un cas clinique.  Ce cas compare les resultats du placement de HTR™ (os synthetique de remplacement de tissus durs) et de FDBA (allogreffe d'os ie- CIIL' el congele) chez le meme patient. Deux entailles ont ete effectuees au niveau de six dents, l'unc au niveau de la gencive marginale et l’autre au niveau le plus apical du tartre. Les reponses des tissus mous avec les deux materiaux comprenaient la reduction de profondeur au sondage, la diminution de I'inflammation gingivale et le gain d'attache clinique. Des coupes histologiques des sites obtenus trente mois apres le placement des deux materiaux ne montraient aucun signe de nouvelle attaché. Peu ou pas d'inflammation etait presente avec les deux types de materiaux. II y avait egalement une absence de particules FDBA, ce qui pourrait indiquer une resorption complete de ce materiel avant treinte mois. La diminution de l’oedeme gingival a resulte a I'exposition des entailles gingivales et de sous-tartre de tous les sites traites par FDBA. L'epithelium gingival a ete trouve adjacent a l'entaille gingivale de tous les sites traites HTR™. L'entaille sous-tarte des sites traites HTR™ etait recouverte par de I'epithilium de jonction avec du tissu conjonctif" et de l'os contre cette adhesion. Les particules HTR™ etaient presentes et entourees par du tissu conjonctif ou de l"os. HTR™ semble done servir d'echaffaudage a la neoformation osseuse lorsqu'il est place en contact intime avec l'os alveolaire.

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