Easy Graft : "Biocompatibility of Beta-Tricalcium Phosphate Root Replicas in Porcine Tooth...

8/9/2019 Easy Graft : "Biocompatibility of Beta-Tricalcium Phosphate Root Replicas in Porcine Tooth Extraction Sockets-A Corr

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Biocompatibility ofb-Tricalcium

Phosphate Root Replicas in PorcineTooth Extraction Sockets A Correlative Histological,Ultrastructural, and X-ray

Microanalytical Pilot Study

P. N. RAMACHANDRAN NAIR* AND HANS-ULRICH LUDERInstitute of Oral Biology

Section for Oral Structures and Development

Centre of Dental and Oral Medicine

University of Zurich, Zurich, Switzerland

FABRIZIO A. MASPERO Department of Materials

Swiss Federal Institute of Technology (ETH)

Zurich, Switzerland

JURGEN H. FISCHERInstitute of Experimental Medicine

University of Koln, Koln, Germany

JENS SCHUGClinic for Preventive Dentistry, Periodontics and Cariology

Centre of Dental and Oral Medicine

University of Zurich, Zurich, Switzerland

ABSTRACT: This investigation studies porcine tissue response in toothextraction sockets treated with root replicas made out of-tricalcium phosphate(-TCP; -Ca3(PO4)2) granules, molded and held together by thermal fusion of

*Author to whom correspondence should be addressed. E-mail: [email protected]

JOURNAL OFBIOMATERIALS APPLICATIONS Volume 00 2006 1

0885-3282/06/00 000118 $10.00/0 DOI: 10.1177/0885328206054167 2006 SAGE Publications

J Biomater Appl OnlineFirst, published on January 27, 2006 as doi:10.1177/0885328206054167

Copyright 2006 by SAGE Publications.


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a thin film of polyglycolicpolylactic acid copolymer. Six left mandibular thirdincisors (n6) of experimental pigs are treated with the root replicas and fourcontralateral incisors are used as nontreated controls (n4). Two animals each

were killed at 20, 40, and 60 weeks of observation periods. The mandibular jawsegments were prepared in toto for light microscopy by resin embedding andserial ground sectioning. Additionally, one -TCP-treated socket at 60 weeks wasthoroughly investigated by correlative light, electron microscopic and electronprobe X-ray microanalysis to assess the bioabsorbability and host removal of thereplica material from the implant site. The extraction wounds of the animalshealed satisfactorily with very little histologically observable differences in thehealing pattern of the test and control sites. The -TCP was completely removedfrom extracellular sites, but at 60 weeks, remnants of it were found in thecytoplasm of multinucleated giant cells. The root replicas made out of -TCPwere biocompatible and bioabsorbable. Osseous healing occurred both in the test

and control sockets, but the healing process was delayed due to the presence of-TCP particles.

KEY WORDS: alveolar process atrophy, alveolar ridge preservation,biocompatibility, oral implants, oral wound healing, osseointegrateddental prostheses, tooth extraction sockets.

INTRODUCTION

The long-term prognostic, functional, and aesthetic success of the

conventional noninvasive and osseointegrated dental prosthesesdepends on the presence of sufficient volume of healthy jaw bone [1].The latter relates to proper postextraction healing and rehabilitation ofthe alveolar process. The morphological events of human alveolar sockethealing after tooth extraction have been well documented [2]. It has longbeen recognized that healing of tooth extraction sockets is associatedwith a catabolic remodelling of the residual alveolar process that resultsin loss of large volume of the jaw bone [3]. This atrophy of the alveolarprocess has been described as a . . . chronic progressive, irreversible and

disabling disease. . .

[4] or reduction of residual ridges (RRR, [5]). Theetiology of the condition is unknown. Nevertheless, both systemic andlocal factors have been suggested to be involved [5,6]. The rate of RRR israpid immediately after tooth extraction and the process is particularlypronounced in the anterior maxilla.

Various preoperative and intraoperative measures have beenattempted to preserve the alveolar process. Careful oral hygiene andprecautions that minimize trauma during tooth extraction procedureslimit postoperative inflammation and associated bone loss. Other

measures include: (a) retention of the natural roots so as to preservethe alveolar process, (b) insertion of prefabricated semianalogousroot form implants, and (c) application of various forms of guided bone

2 P. N. R. NAIR ET AL.


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regeneration (GBR) techniques. Alloplastic bone substitutes [717] andbone or osseous derivatives of isogenous [1,1820], allogenous [7,11,21,22], and xenogenous [2325] origin have been used as osseoinductive

and/or osteoconductive materials in various GBR techniques. Numerousattempts have been aimed at maintaining [7,915,18,2329] or regener-ating (or augmenting) [1,8,16,17,1922, 30,31] the alveolar process.Most of the publications were human case reports [7,10,14,16,18,20,22,23,25,28,29,32] or animal studies [9,12,17,19,21, 26,30] with varyingand often conflicting outcomes assessed by clinical and radiographiccriteria. In several cases, there was histological examination of thetissues removed during implant surgery [1,7,10,11,1619,21,2325,30].

Biodegradable osteosynthesis devices have been successfully used

for internal fixation of fractures in maxillofacial and orthopedicsurgeries [33,34]. The idea has been advanced [35] that custom-made biodegradable root replicas applied as immediate implants inextraction sockets could preserve the alveolar process. The animalexperimental application of polyglycolic acid (PGA) and subsequentlimited human therapeutic usage of polylactic acid (PLA) root replicashave been reported to preserve the alveolar ridge [35,36]. Copolymersof polyglycolicpolylactic acids (PLGA) eventually replaced the homo-polymers for such clinical applications [15,37].

Clinical reports on bioabsorbable synthetic polymers, albeit limited,have been positive, but investigations on PLGA showed an in vitro dropof pH of a phosphate buffered (PBS) medium to well below 3 as a resultof degradation [38] of the copolymers into acidic monomers. In the body,the latter are expected to be metabolized yielding energy, CO2, andwater via the citric acid cycle [15]. However, an in vivo lowering of pHaround the implanted root replicas can happen as a result of imbalancein the release and elimination of the acidic degradation products withpossible adverse effects, such as demineralization of the residual bone

[37], inflammation, and even necrosis of tissues surrounding the replica.Research [38] has shown that granules of -tricalcium phosphate

(-TCP, -Ca3(PO4)2) can be held together by thermal fusion ofa very thin film of PLGA coating of the granules. The resultingagglomeration of -TCP granules can be shaped to any form by usingappropriate molds. On melting, the thin PLGA film would gluethe -TCP granules together exposing the surface of the latter,but would not lower the pH of the surroundings on degradation.The near spherical shape of the -TCP granules ensures an

interstitial porosity of the resultant product open to the exterior.Therefore, the -TCP with low amounts of PLGA as a binding agenthas been suggested [38] to provide an almost neutral degradable

Biocompatibility of-Tricalcium Phosphate Root Replicas in Swine 3


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material to treat tooth extraction sockets for the prevention ofalveolar bone loss.

It is mandatory to study the reaction of mammalian tissues to

chemicals and foreign objects that are intended for various implanttherapies before such materials are clinically applied on a large scale inhuman and animal patients. Therefore, the objectives of this investi-gation were: (a) to study the histological response to treating toothextraction sockets with freshly prepared root replicas of -TCP in aminiature swine model and (b) to answer the question as to whether ornot the -TCP is completely removed from the site of implantation.

MATERIALS AND METHODS

Animals and Teeth

A total of 10 teeth from six healthy adult miniature swines (Table 1)were involved in this study. The pigs were kept under appropriate farmconditions with food and water ad libitum. Two animals were randomlyallotted to each of the three observation groups, namely, 20, 40, and60 weeks, respectively. Physical examination of the animals, photo-,

radiographic recordings and subsequent invasive oral surgical proce-dures were done under general anesthesia at the scheduled times,in accordance with German legislation on protection of animals,veterinary professional standards and support. In addition to generalanesthesia, infiltration local anesthesia was used during tooth extrac-tion to minimize bleeding and postoperative pain. In all six animals, theextraction site of the left mandibular third incisor (tooth 33) was treatedwith a root replica that was freshly prepared as described below. In four

Table 1. Summary of the animal data.

Animal

Observation

(weeks)

Age

(years) Sex

Test

socket

Control

socket

SRP-1 20 6 M* 33 43

SRP-2 20 6 F 33

SRP-3 40 3 F 33 43

SRP-4 40 3 F 33 43

SRP-5 60 3 F 33 43

SRP-6 60 6 F 33

M* castrated male; F female.Tooth 33 is mandibular left third incisor; tooth 43 is mandibular right third incisor.



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of the six animals, the contralateral extraction site of tooth 43, was nottreated with the root replica as a split mouth control. The animals werenot used for any other experimental purpose. It is noteworthy that third

mandibular incisors are the smallest of porcine teeth that are amenableto extraction without extensive trauma to the teeth and the surroundingbones. Our own attempt to extract a second mandibular incisor in oneanimal resulted in only a fractured crown of the tooth. Therefore, apartfrom ethical considerations, the number of test and control socketscould not be increased by making use of more extraction sockets in theexperimental animals involved.

Preparation of the Root Replicas

After extraction, the six test teeth were rinsed briefly in running coldwater and the soft tissue attached to the root surface was removedmechanically. The replicas were prepared chairside with -TCPgranules coated with a thin layer of PLGA (PLGA 50 : 50, BoehringerIngelheim, Ingelheim, Germany) as described in detail elsewhere [38].Briefly, the granules (500800mm) were poured into the freshlyprepared root impression mold of the extracted tooth. The mold

containing the granules was then heated to a temperature of 80

C.The PLGA surface film of the granules melt at 70C so that the granulesglue together to form a root-shaped body on cooling. The replicasrevealed an interconnected porosity of about 50% (v/v) with an averagepore diameter of 200 mm. Preparation of the root replica molds tookabout 5 min.

Clinical Procedures

After careful extraction of the teeth to avoid damaging the alveolarprocesses of the animals, root replicas were inserted into the alveoli aftera few minutes. The edges of the gingival wounds were held togetherby sutures that also retained the replicas in the alveoli. The sutureswere removed after 10 days of application. Empty contralateral alveolarsockets, sutured after tooth extraction, served as controls. The animalswere clinically examined, photographed, and radiographed at variousintervals during the observation periods. After appropriate period ofobservations, the animals were killed under general anesthesia at the

determined times (Table 1). Thereafter, the lower jaws were dissectedout, briefly rinsed in cold water and immersed in large volumes of half-strength Karnovskys fixative [39]. The fixative solution was changed



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after 24 h and the specimens were further fixed in cold storage (4C) for46 weeks.

Tissue Processing

The lower jaws of the six animals were divided along the midlineinto left and right halves using a diamond disc (Vari/Cut, LeoCorporation, St. Joseph, USA). Each half was then trimmed toremove the mandibular portion distal to the canines. Using a bandsaw (Exakt, Norderstedt, Germany) the mesial segments of the man-dibles so prepared were further subdivided mesiodistally through themidplane of the extraction sockets of teeth 33 and 43, respectively.

In the case of one animal of 60 weeks of observation period, a centralslice of about 0.8 mm thickness was removed from the test sideand saved for Epon (Fluka AG, Buchs, Switzerland) embedding.The buccal and lingual segments of each mandibular specimen weredehydrated in ascending grades of ethanol, thoroughly infiltrated withand thereafter embedded in the commercial resin Technovit 7200 VLC (Kulzer, Wehrheim, Germany). The 0.8-mm thick central lower jaw segment removed from the test side of the animal with 60 weeksof observation period was substantially trimmed to reduce the speci-

men size to the area of the healing extraction socket of the tooth33 that could be clearly orientated in a stereomicroscope. The non-demineralized specimen was postfixed in 1.33% osmium tetroxide(OsO4) buffered in 0.067 M s-collidine, dehydrated in ascending gradesof ethanol and embedded in Epon.

Scanning Electron Microscopy (SEM)

The cutting faces of the polymerized Technovit blocks were polished

with silicon carbide grinding paper followed by a polishing clothwith diamond paste of 3, 1, and 0.25 grain size. The polished blockfaces were then coated with a carbon layer of about 1015 nm thick-ness using a MED020/EVM030 electron beam evaporator (BAL-TEC,Balzers, Lichtenstein) and examined in the backscatter mode ina Stereoscan 180 scanning electron microscope (SEM, Cambridge,Dortmund, Germany) equipped with a four-quadrant backscatterdetector setup to show atomic number contrast. Digital electronmicrographs were prepared at 1520 kV of accelerating voltage and

a working distance of about 20 mm using a personal computer con-nected to the SEM and the software WinDISS (Point Electronic, Halle,Germany).



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Ground Sections and Light Microscopy

Mesiodistal serial sections of about 50 mm thickness were prepared

using the microgrinding system [40] (Exakt, Norderstedt, Germany).The sections were stained in toluidine blue and photomicrographedusing a photomicroscope M420 (Leica, Glattbrugg, Switzerland)equipped with a Coolpix 4500 (Nikon Corporation, Tokyo, Japan)digital camera.

Transmission Electron Microscopy (TEM) andEnergy Dispersive Analysis of X-rays (EDAX)

From the Epon block, 11.5mm thick survey sections were pre-pared using a histodiamond knife (Diatome AG, Biel, Switzerland)and the Reichert Ultracut E microtome (Leica, Glattbrugg,Switzerland). The sections were stained in periodic acid-Schiff (PAS)and methylene blue-Azur II and photomicrographed in bright andpolarized lights using a Dialux 20 photomicroscope (Leica, Glattbrugg,Switzerland) equipped with the digital camera Progress C14(Jenoptik, Eching, Germany) and an electronic imaging system

(ImageAccess, Imagic, Glattbrugg, Switzerland). After locating anideal site for ultrastructural evaluation and analysis, the selected areain the epon block was target trimmed and thin sectioned with theReichert Ultracut E microtome (Leica, Glattbrugg, Switzerland).Some of the thin sections were double contrasted with lead anduranium salts [41,42] and examined in a Philips EM400T trans-mission electron microscope (Philips, Endoven, The Netherlands).Noncontrasted thin sections were used for EDAX with the aid of aPhilips CM120 scanning transmission electron microscope (STEM,

Philips, Endoven, The Netherlands).

RESULTS

Healing of the extraction wounds of the test and control sites inall the animals was satisfactory by qualitative visual and radiographicassessments at regular intervals. No signs of microbial infection,exudation, or dehiscence of the wound could be observed at thetime of suture removal. Macroscopically, the gingival dimensions and

bony contours maintained the respective preoperative appearances ascould be judged by standardized photographic and radiographicmeans.



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General Histology

Treatment of the porcine tooth extraction sockets with -TCP

(Figure 1(A)(C)) did not result in clear histomorphologically observ-able advantages in the osseous healing pattern (Figure 1(A)(C)) incomparison to untreated contralateral control sites (Figure 1(D)(F)).At 20 weeks postoperatively, a distinct extraction socket was visible inboth the test (Figure 1(A)) and control (Figure 1(D)) specimens.Spicules of bone could be observed to project into the extractionsockets from the bottom and lateral walls of the alveoli. Horizontalextension of the alveolar ridge seemed to narrow the cervical openingof the extraction socket. At 40 weeks (Figure 1(B) and (E)) the sockets

were partially traversed by trabecular bone that formed a looseosseous network. The latter became closer fitting at 60 weeks(Figure 1(C) and (F)) of observation. A complete closure of the cervicalopening of the alveolar ridge could be observed in the test sites at40 and 60 weeks (Figure 1(B) and (C)), compared to interruptionsnoticeable in ridge healing of the contralateral controls (Figure 1(E)and (F)).

SEM Visualization

Backscattered imaging in SEM at 20 weeks revealed distinctextraction sockets at the test (Figure 2(A)) and control sites. Theformer contained a substantial amount of electron-dense fine particlesdistributed throughout the socket. At 60 weeks (Figure 2(B)), a distincttrabecular network of bone could be observed around the socket areathat was much smaller than that in the 20-week specimens. The60-week specimens (Figure 2(B) and (C)) contained similar electron-

dense particles as those observed at 20 weeks, but in much lesserquantity. At a higher magnification (Figure 2(C)), the jaw bonedisplayed in two distinct areas of electron densities. While most of thebone was of high electron density, an osseous layer of varying thickness,but of distinctly lesser electron density, lined the healing and remodelingfront of the socket wall.

Polarization Microscopy and TEM Visualization

The cellular and subcellular details of the test socket at 60 weeks wereanalyzed using various microtechniques in Epon embedded semithin(1.5 mm) and thin (80 nm) sections. The healing socket consisted of



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Figure 1. Overview photomicrographs showing the histological status of healing porcine

extraction sockets (ES) of tooth 33 in animals at 20 (A), 40 (B), and 60 (C) weeks

postextraction that were implanted with biodegradable root replicas. Contralateral

extraction sockets of tooth (43), not treated with root replicas, provided the controls

(DF). There were no histologically observable differences in the osseous healing of the

extraction sockets, except for the complete closure (B, C) of the alveolar ridge in the test

cases as against the interruptions noticeable in the ridge healing of contralateral controls(arrows in E, F). 32, 42 left and right mandibular second incisors, respectively. Original

magnifications: (A)(F), 4.8.



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trabecular bone delimiting large pockets of well-vascularized soft

connective tissue that contained plenty of fat globules (Figure 3(A)).Except for the presence of occasional lymphocytes, the area was free ofinfiltrating inflammatory cells, such as polymorphonuclear leucocytes

Figure 2. Low magnification backscattered scanning electron micrographs (A, B) of

the surfaces of two synthetic resin embedded specimens. Note the extraction sockets (ES)

of teeth 33 at 20 (A) and 60 (B) weeks of observation periods, respectively. Figure 2(A)

originates from that shown histologically in Figure 1(A). The rectangular demarcated area

in (B) is magnified in (C). Note the highly electron dense particles at 20 weeks post-

extraction (arrow in A). Much less material is still visible in the 60-week postextraction

specimen (arrows in B, C). The repairing osseous socket wall has a lower electron density

(arrowheads) than the rest of the bone. Original magnifications: BO bone. (A) 17.5,

(B) 15, (C) 62.



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(PMN) and plasma cells. But a large number of multinucleated giantcells, in isolated clusters, could be observed (Figure 3(B) and (C)). Theirnuclei were distributed randomly throughout the cytoplasm. The cyto-plasm revealed inclusion bodies that appeared empty in bright field(Figure 3(C)) but intensely birefringent in polarized light (Figure 3(D))so that the area of the section appeared to be studded with brightly

illuminating particles against a dark background. Electron microscopi-cally (Figure 4(A)), the inclusion bodies contained a highly electron-dense material that remained intact within the inclusion bodies when

Figure 3. Photomicrographs of a semithin section from the area of the extraction socket

of tooth 33 at 60 weeks postextraction, illustrated in Figure 2(B). The rectangular

demarcated area in (A) is magnified in (B). The trabecular bone (BO), blood vessels (BV),

fat globules (FG), and cellular elements are visible. A higher magnification of the

demarcated area in (B) (shown in C) reveals profiles of giant cells in bright field vision.

In polarized light, the same field shows numerous birefringent bodies (BB) in giant cells.

Original magnifications: (A) 11, (B) 100, (C, D) 520.



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Figure 4. A transmission electron micrograph (A) showing profiles of giant cell cytoplasm

(GC) and nuclei (NU). Note the highly electron-dense material in numerous inclusion

bodies (IB) in the cytoplasm. X-ray microanalysis (B) of the electron-dense material

at the intersection of the two hairlines in the inset (arrow) revealed the presence of

predominantly calcium and phosphorus elements at the site. The same analysis done

at the intranuclear (C, arrow in the nucleus) and intact cytoplasmic control sites did not

reveal the presence of elemental calcium and phosphorus. Original magnification:

(A) 4400.



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these were delimited by plasma membranes. However, when pushedaway from the original sites during sectioning, the electron-densematerial showed signs of disintegration. The birefringent electron-dense

bodies were observed only within the giant cells and not in theinterstitial extracellular areas.

X-ray Microanalysis

The giant cell inclusion bodies (Figure 4(B)), several cytoplasmic andnuclear sites (Figure 4(C)) were subjected to element analysis using aSTEM/EDAX unit. The dye-resistant, birefringent, and electron-dense

inclusion bodies consistently revealed peaks of energy range ofelemental calcium (Ca) and phosphorus (P). Only one significant peakof X-ray emission, in the range of carbon (C), could be registered fromthe cytoplasmic and nuclear sites.

DISCUSSION

This study presents correlative histological, ultrastructural, andX-ray microanalytical data on the biocompatibility and host removal of

-TCP-made root replicas applied as an immediate implant after toothextraction in a miniature swine model.

The idea [35] of applying biodegradable root replicas as immediateimplants in extraction sockets to preserve the alveolar process has notbeen adequately tested by independent animal and human clinicaltrials. Only two publications [35,36] describe the treatment ofextraction sockets with biodegradable root replicas. In an experimentalstudy in rabbits [35], it was found that insertion of custom-made rootreplicas made out of PGA into the extraction sockets of second

maxillary incisors prevented palatal collapse of the implanted area.Contralateral control sockets that did not receive any root replicasshowed clear collapse of the area. Subsequently, in a woman patient[36], one extraction socket was treated with a chairside-made PLA rootreplica. Only radiographic follow-up was possible and it was reportedthat ridge height could be preserved during an observation period of84 weeks. In a larger clinical trial [37] with eight human patients, solidand porous forms of PLGA copolymers were used as root replicas beforeinsertion of definite metallic implants. In some of the cases, an initial

decline in the radiodensity of the alveolar processes surrounding theextraction sockets could be observed, which suggested initial bonedemineralization.



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The first aim of this study was to visualize potential tissue reactionand healing pattern of porcine tooth extraction sockets treated with-TCP root replicas at predetermined observation periods. Serial

ground sections of anterior mandibular regions of the experimentalpigs containing treated and contralateral nontreated sites at 20, 40,and 60 weeks showed gradual bone healing of the extraction sockets. Noclearly observable advantages in the healing pattern of the extractionsockets could be found between the test and control sites by the conven-tional bright field microscopy. The absence of complications, such astissue necrosis or signs of inflammation indicates biocompatibility ofthe root replicas. No quantitative assessment of the tissue componentsof the healing alveolar sockets was done, since the small sample size is

unlikely to yield any more meaningful biological data of appropriatestatistical reliability.

The second objective was to investigate whether or not the -TCPwas completely removed from the site of implantation during a clinicallyrelevant period of observation. As cellular details and the fine particlesof-TCP could not be adequately resolved in 50-mm histological groundsections, other suitable microanalytical methods were used to deter-mine the degree of -TCP removal. Visualization of the treated andnontreated control sockets in backscattered mode of SEM clearly

showed the presence of fine electron-dense particles in test sites, theamount of which declined with increasing observation periods. Lowamounts of electron-dense material were still present in the specimensof the longest observation period. However, backscatter images couldnot clarify whether the particles were intra- or extracellular in nature.

The presence of clusters of multinucleated giant cells in the soft tissueof the healing socket at 60 weeks indicated a foreign body host response.The physical properties of the inclusion bodies, such as dye resistance,birefringence, and high electron density are suggestive of the presence

of inorganic element(s). Electron probe microanalysis consistentlyconfirmed the presence of calcium (Ca) and phosphorus (P). The factthat at 60 weeks, the Ca and P containing birefringent and electron-dense particles were observed only within giant cells, suggests that mostof the -TCP of the root replica were biodegraded and/or bioabsorbedby the host. Taken together, the evidence suggests that the Ca and Pobserved in the inclusion bodies of the giant cells were remnants of-TCP that were eliminated by the host cells. These intracellularremnants of the -TCP may not be of clinical significance, because the

particles were within host cells that should be biologically eliminated indue course, or would be removed during surgical re-entry for placementof an osseointegrated implant.



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CONCLUSIONS

The results of this study allow us to conclude that root replicas made

out of -TCP are biocompatible and bioabsorbable. Osseous healingoccurred in both the test and control sockets, but the healing processappears to be delayed by the presence of-TCP particles.

ACKNOWLEDGMENTS

We are indebted to Dr K. Ruffieux, Department of Materials, FederalInstitute of Technology, Zurich, Switzerland, for the excellent coordina-tion of this project and to Dr S. Jeschkeit and Dr C. Funke, Instituteof Experimental Medicine, University of Koln, Koln, Germany, fortheir veterinary professional support. Our thanks are due toMr H.-P. Gautschi, Central Laboratory for Electron Microscopy,University of Zurich, Zurich, Switzerland, for his help in electronprobe X-ray microanalysis; to Mrs J. Hofmann-Lobsiger and Mrs M.Amstad-Jossi for the excellent technical assistance. We are indebted toDr David Figdor, Monash University, Clayton, Victoria, Australia forthe critical reading and editing of the manuscript.

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