Tierärztliche Hochschule Hannover
Primäre Rekonstruktion mandibulärer Kontinuitätsdefekte durch eine ß-Trikalziumphosphat-Matrix beim Schaf
INAUGURAL-DISSERTATION zur Erlangung des Grades einer Doktorin
der Veterinärmedizin
- Doctor medicinae veterinariae - (Dr. med. vet.)
vorgelegt von Mirja Christine Nolff
Neuss
Hannover 2009
Wissenschaftliche Betreuung: 1. Univ. Prof. Dr. M. Fehr
Tierärztliche Hochschule Hannover
2. Univ. Prof. Dr. Dr. N.-C. Gellrich Medizinische Hochschule Hannover
3. Dr. G. Hauschild
Universitätsklinikum Münster
1. Gutachter: Univ. Prof. Dr. M. Fehr
2. Gutachter: Univ. Prof. Dr. H. Gasse
Tag der mündlichen Prüfung: 28.05.2009
Meinen Eltern
Inhaltsverzeichnis
Diese Dissertation basiert auf zwei Veröffentlichungen in international anerkannten Fachzeitschriften mit Gutachtersystem (peer review).
1 Einleitung
122 Manuskript I
17‘Comparison of two ß-TCP composite grafts used for reconstruction of mandibular critical size bone defects.’
2.1 Summary 18
2.2 Introduction 19
2.3 Material and Methods 21
2.4 Results 27
2.5 Discussion 35
2.6 Footnotes 39
2.7 References 40
3 Manuskript II
45‘Comparison of Computed Tomography and Microradiography for Graft Evaluation after Reconstruction of Critical Size
Bone Defects using ß-Tricalcium-Phosphate.’
Summary 46
Introduction 47
Material and Methods 50
Results 57
Discussion 65
Conclusion 70
Footnotes 71
References 72
4 Diskussion 80
4.1 Methode 80
4.1.1 Tiermodell 80
4.1.2 Defektmodell 83
4.1.3Wahl des chirurgischen Modells 84
4.1.4 Versuchsstruktur 85
4.1.5 Wahl der Auswertungsmethoden 86
4.2 Diskussion der Ergebnisse 89
4.2.1. OP verlauf und Heilungsphase 89
4.2.2. Ergebnisse der feingeweblichen Untersuchungen 91 4.2.3. Ergebnisse der Validierung des diagnostischen Wertes
der Computer Tomographie 95
4.2.4. Praktische Bedeutung der Ergebnisse der durchgeführten
Studie 100
5 Zusammenfassung 101
6 Summary 104
7 Literaturübersicht 107
8 Anhang 114
8.1 Bestätigungen der Verlage 114
8.1.1. Bestätigung Manuskript I 114
8.1.2. Bestätigung Manuskript II 115
8.2 Erklärungen über die erbrachten Eigenleistungen gemäß § 8 Promotions 116 Ordnung der Tierärztlichen Hochschule Hannover
8.3 Tabellarische Darstellungen der Original-Daten 118
8.3.1 Messungen Histomorphometrie 118
8.3.2 Messungen CT und µ-Radiografie 120
9 Danksagung 124
Abkürzungsverzeichnis
BA/TA Bone Area / Tissue Area
BSE Bovine Sponigiforme Encephalopathie CA/TA Cartilage Area / Tissue Area
°C Grad Celsius
cm Zentimeter
CT Computer Tomographie ; Computer Tomography
FA/TA Fibrous Tissue Area / Tissue Area
HA Hydroxylapatit
HIV Humanes Immunudefizienz Virus
HU Hounsfield Units
IM intramuskulär
IV intravenös
Kg Kilogramm
l Liter
mg Milligramm
min Minute
ml Milliliter
MMA Methylmethacrylate, Methylmethacrylat
MSC Mesenchymal Stem Cell
SA/TA Scaffold Area /Tissue Area
SC subcutan
std Standardabweichung
stdgeo geometrische Standardabweichung
q24h alle 24 Stunden q48h alle 48 Stunden ß-TCP ß-Trikalziumphosphat
ß-TCPB ß- Trikalziumphosphat mit Blut
ß-TCPB/BM/CB ß-Trikalziumphosphat mit Blut, Knochenmark und Spongiosa
µg Mikrogramm
µm Mikrometer
x Standardabweichung
xgeo geometrische Standardabweichung
% Prozent
Die vorläufigen Ergebnisse der zugrunde liegenden Studie wurden im Rahmen folgender Kongresse präsentiert:
10th International Symposium / Biomaterials NRW 2008: Fundamentals and Clinical Applications im März 2008 in Essen.
Vortrag unter dem Titel: ‚Comparison of Conventional Computed Tomography and Microradiography for ß-TCP Graft Evaluation.’
14th Swiss Conference on Biomaterials (Swiss Society for Biomaterials 2008) im Mai 2008 in Basel.
Posterpräsentation unter dem Titel:
‚Comparison of two Different ß-TCP Composites for Reconstruction of Ovine Mandibular Continuity Defects.’ , EUR CELL MATER, Vol. 16, Supplement 1, 2008, p.16
Vortragspräsentation unter dem Titel:
‘Comparison of Computed Tomography and Microradiography for ß-TCP Graft Evaluation after Mandibular Reconstruction.’
EUR CELL MATER, VOL 16, SUPPLEMENT 1,2008, p.42
Jahrestagung der Deutschen Gesellschaft für Biomaterialien Im November 2008 in Hamburg.
Posterpräsentationen unter dem Titel:
‚Comparison of two Different ß-TCP Composites for Reconstruction of Ovine Mandibular Continuity Defects.’
‚Comparison of Computed Tomography and Microradiography for ß-TCP Graft Evaluation after Mandibular Reconstruction.’
1 Einleitung
Die adäquate Versorgung ausgedehnter Knochendefekte nach Trauma, ablativer Tumorchirurgie oder ausgedehnten Entzündungen stellt auch heute noch weltweit eine große Herausforderung für die rekonstruktive Chirurgie dar. Der Ersatz von Knochengewebe kann in jeder Lokalisation notwendig werden und betrifft damit verschiedenste chirurgische Disziplinen. Die Rekonstruktion von mandibulären Defekten stellt hierbei aufgrund der funktionellen Beteiligung, die der Mandibel an Vokalisation, Mastikation und Abschluckvorgang zukommt, sowie der komplexen Bewegung und der damit verbundenen Kraftverteilung innerhalb des Knochens eine besondere Schwierigkeit dar (DECHAMPLAIN 1973; STRONG et al. 2003).
Trotz jahrzehntelanger Forschung auf dem Gebiet des Knochenersatzes gelten freie, autogene, corticospongiöse Transplantate oder gestielte, osteomusculocutane Plastiken nach wie vor als Goldstandard (STEVENSON 1999).
Autogener, corticospongiöser Knochen unterstützt die Defektheilung sowohl passiv als auch aktiv. Mit einem autogenen Transplantat werden ein dreidimensionales, osteokonduktives Gerüst, vitale osteogene Zellen und osteoinduktive Wachstumsfaktoren der organischen Matrix übertragen (RODRIGUEZ-MERCHAN u.
FORRIOL 2004; KRAUS u. KIRKER-HEAD 2006). Ein gestieltes Transplantat bietet zusätzlich den Vorteil eines vitalen Weichteilmantels, der mit intakter Vaskularisation transplantiert werden kann und insbesondere bei stark geschädigtem Transplantatlager eine adäquate nutritive Versorgung des übertragenen Knochens gewährleistet (PELEG u. LOPEZ 2006).
Allerdings ist die Entnahme autogener Transplantate mit einer erheblichen Morbidität assoziiert. Der betroffene Patient wird einem zusätzlichen Eingriff ausgesetzt, der mit Schmerzen und Einschränkungen verbunden ist, und muss Risiken wie Frakturgefahr und Nervenschädigung in Kauf nehmen (YOUNGER u. CHAPMAN 1989; BANWART et al. 1995; KLINE u. WOLFE 1995). Zudem ist die Menge des zur Verfügung stehenden Knochens, insbesondere bei Kindern, begrenzt. Bei Transplantatabstoßung kann die Prozedur daher nur in begrenztem Umfang wiederholt werden (DELLOYE et al. 1992).
In der Vergangenheit wurden vielfältige Alternativen zum autogenen Knochen auf ihre Eignung als Ersatzmaterial untersucht, die sich grob wie folgt gruppieren lassen:
allogener Knochen, xenogener Knochen sowie die große Gruppe der synthetischen Knochenersatzmaterialien (RUEGER 1998; CORNELL 1999; BAUER u. MUSCHLER 2000).
Der Einsatz von allo- und xenogenem Knochen wird aufgrund potentieller Übertragungsgefahr bestimmter Virus- oder Prionenerkrankungen (HIV,BSE) in den letzten Jahren kontrovers diskutiert (TOMFORD 1995; BOYCE et al. 1999;
TOMFORD u. MANKIN 1999, HAUSCHILD u. BADER 2004), obwohl in verschiedenen Studien bestätigt wurde, dass nach adäquater Vorbehandlung des Knochenmaterials kein klinisch relevantes Risiko einer Virusübertragung besteht (WENZ et al. 2001). Ein weiterer erheblicher Nachteil von allogenen und xenogenen Transplantaten ist in der Tatsache begründet, dass hierbei im Gegensatz zu autogenem Knochen totes Gewebe transplantiert wird. Dieses tote Gerüst kann
teilweise als Fremdkörper im Knochen. Verglichen mit autogenen Transplantaten besteht insgesamt ein höheres Risiko von Transplantatabstoßung oder Fraktur (WHEELER u. ENNEKING 2005).
Innerhalb der großen Gruppe der synthetischen Ersatzmaterialien erscheinen keramische Werkstoffe besonders geeignet. Insbesondere die beiden Hauptvertreter dieser Gruppe, Hydroxylapatit (HA) und ß-Trikalziumphosphat (ß-TCP), zeichnen sich durch eine hervorragende Biokompatibilität aus. Sie sind osteokonduktiv und ihre chemische Zusammensetzung ähnelt nativem Knochen (JOHNSON et al. 1996;
JENSEN et al. 2006). Optimale Gesamtporosität, Porengröße sowie Interkonnektivität wurden in zahlreichen Studien bestimmt und moderne Produktionsverfahren ermöglichen die Herstellung von keramischen Implantaten mit optimalen strukturellen Eigenschaften in jeder gewünschten drei-dimensionalen Form (EGGLI et al. 1988; LU et al. 1999; HUTMACHER 2001; BLOEMERS 2002; HING 2005). Zusätzlich zu den genannten Vorteilen zeichnet sich ß-TCP außerdem durch seine Biodegradierbarkeit aus. Biodegradierbare Materialien dienen zunächst dem strukturellen Ersatz verlorenen Knochenmaterials und werden im Rahmen der Heilung fortschreitend abgebaut. Der Degradationsprozess ermöglicht ein natürliches Remodelling, an dessen Ende kein Fremdmaterial mehr im Regenerat verbleibt, das potentielle adverse Reaktionen verursachen könnte oder die mechanische Integrität des Regenerates negativ beeinflussen würde (RENOOIJ et al. 1985; HING 2005).
Als Nachteile dieser Materialien sind im Wesentlichen mangelnde mechanische Belastbarkeit und die hohe Röntgendichte zu nennen.
Ein keramisches Knochenersatzmaterial, unabhängig ob HA oder ß-TCP, ist struktur- wie materialbedingt initial nicht in der Lage, die mechanische Belastung im Bereich
der Defektzone zu tragen (CHU et al. 2002; BIGNON et al. 2003). Daher ist in der überwiegenden Zahl der Fälle eine stabile Osteosynthese notwendig, um die Kraftübertragung auf das Transplantat so stark wie möglich zu minimieren. Generell gilt, dass ein keramischer Ersatzstoff stabil genug sein muss, um den Vorgang der Implantation zu überstehen und die eigene Micro- und Makrostruktur zu erhalten.
Mechanische Stabilität des betroffenen Knochenbereiches wird im weiteren Verlauf der Heilung durch Remodelling des neu gebildeten Knochens erreicht (HING 2005).
Aufgrund der strukturellen Ähnlichkeit von ß-TCP, HA und spongiösem Knochen haben alle Materialien eine ähnliche Röntgendichte. Eine zuverlässige Überwachung von Integration, Osteoneogenese und Degradation innerhalb einer implantierten keramischen Matrix auf Basis der bislang klinisch eingesetzten bildbgebenden Verfahren erscheint daher fragwürdig. Obwohl verschiedenste Autoren dieses Problem benennen und fordern, dass die Genauigkeit radiologischer Methoden bei der Beurteilung der genannten Parameter bestimmt wird, fehlen derartige Studien in der Literatur weitgehend (JOHNSON et al. 1996; RUEGER et al. 1998; BLOEMERS 2002; GOLDSTEIN 2002). Es mangelt allerdings nicht an Veröffentlichungen, die den Erfolg verschiedener keramischer Knochenersatzstoffe radiologisch beurteilen, ohne zuvor eine entsprechende Validierung durchgeführt zu haben.
Keramische Knochenersatzstoffe bieten im Gegensatz zu autogenem Knochen lediglich ein osteokonduktives Gerüst; per se liefern sie weder osteogene Zellen noch osteoinduktive Wachstumsfaktoren (JOHNSON et al. 1996; BLOKHUIS et al.
2000; HING 2005). Beide Komponenten können allerdings einem keramischen
(BRUDER u. FOX 1999; TAY et al. 1999). Zudem kann ab einer kritischen Implantatgröße eine ausreichende nutritive Versorgung der Zellen im Kern des Gerüstes nach Implantation nicht mehr gewährleistet werden (SIPE et al. 2002). Es erscheint zum derzeitigen Standpunkt sinnvoll, nach Alternativen zu suchen, die eine möglichst einfache, kostengünstige Kombination von passiver Trägermatrix und aktiven Komponenten wie osteogenen Zellen und osteoinduktiven Faktoren ad tabulam ermöglichen.
Ziel dieser Studie war es, die Eignung von zwei verschiedenen, perioperativ ad tabulam hergestellten ß-TCP-Komposit-Matrices zur Rekonstruktion von mandibulären Kontinuitätsdefekten kritischer Größe zu testen. Hierzu wurden insbesondere Osteointegration, Osteoneogenese sowie Transplantatdegradation bestimmt. Des Weiteren wurde ein objektiver Vergleich von CT - Messungen des Regenerates mit in-vitro bestimmten Werten vorgenommen, um die Genauigkeit des CT als repräsentative Diagnostik-Methode zur Evaluierung des Regenerationsfortschrittes in vivo zu bestimmen.
2 Manuskript I
Eingereicht am 20.04.2008 und akzeptiert zur Veröffentlichung am 24.06.2008 (siehe Seite 114) in ‘Veterinary Comparative Orthopedics and Trauma’, Impact Factor 0,777 (2007). Erschienen in V.C.O.T. 22 (2): 96-102. 2009.
Comparison of two different ß-TCP composite grafts used for reconstruction of mandibular critical size bone defects.
Mirja Christine Nolff 1,2,*, Dr. Dr. Horst Kokemueller 1, Dr. Gregor Hauschild 2,3, Prof. Dr.
Michael Fehr 2, Dr. Dr. Kai- Hendrick Bormann 1, Dr. Karl Rohn 4, Simon Spalthoff 1, Prof. Dr.
Dr. Martin Rücker 1 , Prof. Dr. Dr. Nils -Claudius Gellrich1
1 Department of Oral and Maxillofacial Surgery, Hannover Medical School, 30625 Hannover, Germany
2 Small Animal Clinic, University of Veterinary Medicine Hannover, 30173, Germany
3 Department of Orthopedics, University of Münster, 48149 Münster , Germany
4 Department of Biometry, Epidemiology and Information Processing; University of Veterinary Medicine Hannover, 30559 Hannover, Germany
* Corresponding author
Key words: Mandibular continuity resection, ß-Tricalcium Phosphate Composite,
Summary.
Objective: To compare osseointegration of blood perfused ß-Tricalcium Phosphate cylinders (ß-TCPB) with similar composites that were additionally loaded with cancellous bone and bone marrow (ß-TCPB/BM/CB) for mandibular reconstruction.
Methods: Twelve German Black-Headed Sheep with an average weight of 72.5 +/- 10 kg underwent segmental resection of the right hemi-mandible. Animals assigned to group A (n=6) were reconstructed using ß-TCPB while sheep assigned to group B received ß-TCPB/BM/CB grafts. Tissue quality was histologically assessed and bone-, scaffold-, cartilage- and fibrous-tissue area were estimated using semiautomated histomorphometrical software.
Results: ß-TCPB/BM/CB grafts exhibited significant (p<0.01) higher amounts of bone formation than ß-TCPB. Animals assigned to group B achieved defect union and a high grade of bone maturation. Residual ceramic remnants were rare and disconnected. Bone maturity within group A was inferior and none of the specimens showed defect union. The defect center was still occupied by a ceramic core.
Clinical Significance: TCPB/BM/CB composites may qualify as a promising alternative to autograft bone for mandibular reconstruction in human and veterinary medicine.
Introduction.
Increased understanding of the main reasons for impaired bone healing resulted in continuous improvement of different bone graft substitutesin order to overcome the shortcomings of the current gold standard autogenous cancellous bone (1-4).
Autograft bone is limited regarding its availability and harvesting is associated with considerable donor side morbidity and prolonged surgery time (5, 6). Based on the composition of trabecular bone, alternative methods for bone substitution were divided into cellular-, factor- and matrix-based approaches (7-10). The high impact of osteogenic cells on healing of large or otherwise compromised bone defects has led to the development of techniques that combine osteogenic cells and appropriate scaffolds (9, 11, 12). One of the first approaches to utilize osteoprecursor cells to increase bone formation was the use of unfractioned bone marrow aspirate over a century ago. Since then the usage of bone marrow proved efficient in numerous trials (13, 14), but recent approaches mainly concentrate on in vitro methods for cell purification and expansion for scaffold loading (11, 15-17). Unfortunately, these in vitro tissue engineered constructs have exhibited problems with physical properties, maintenance of cell prototype and host immune response (18-22). Additionally, according to Tay et al. (23), in an environment where cost containment becomes an issue in patient care, the use of this powerful compound in any significant amount may become prohibitive. Bone marrow represents an easily accessible and cost effective source for osteogenic cells that can be combined with appropriate
ceramics have proven to be especially attractive regarding osteoconduction and biocompatibility (1, 15, 24, 25). The current study was designed to compare osseointegration and degradation of a blood loaded ß-TCP composite (ß-TCPB) with a similar composite that was additionally loaded with bone marrow and cancellous bone positioned within a through-bore-hole along the central line (ß-TCPB/BM/CB) after reconstruction of critical size mandibular defects. We assumed that osseointegration of the ß-TCPB/BM/CB composite would quantitatively and qualitatively exceed the result of the ß-TCPB composite.
Materials and Methods.
Experimental Design.
Animal experiments were conducted under an ethic committee approved protocol in accordance with German federal animal welfare legislation. Twelve healthy skeletally mature (age 2-4 years, mean age 3.75 ± 0,59 years) female German Black-headed Sheep with an average weight of 72.5 +/- 7,4 kg were included in the study. Animals were randomly divided into two groups, each animal receiving partial resection and restoration of the right hemi-mandible. Animals assigned to group A (n=6) were grafted with ß-TCPB while defects in group B (n=6) were reconstructed using predrilled ß-TCPB/BM/CB composites (Fig. 1). All animals were sacrificed 12 weeks after surgery and bone-, ceramic-, soft tissue- as well as cartilage area was assessed.
Fig.1. ß-TCPB cylinders after blood aspiration prior to implantation (A) and pre-drilled ß-TCPB/BM/CB composites after blood aspiration and loading with morselized bone and bone marrow (B).
Surgical Procedure.
After intravenous induction (1 ml midazolam, 5 mg/kg propofol) anesthesia was maintained with isoflurane delivered in 100% oxygen (1l/min) and all animals received buprenorphine (10 µg/kg, IM) and carprofen (4 mg/kg, ½ IV, ½ SC) for peri- operative analgesia. During surgery animals received additional fentanyl boluses (0,005 mg/kg, IV). Systolic, diastolic and mean blood pressure, electrocardiogram as well as rectal temperature and hemoglobin oxygen saturation were continuously monitored. After aseptical preparation of the surgical field the lateral and medial aspect of the right mandibular body and angle were exposed by a subangular incision. Before resection two Compact 2.4 UniLOCK a mandibular reconstruction plates were pre-positioned in order to maintain correct position of the mandible. A retromolar segmental resection of the right mandible was performed in order to create a triangular defect, measuring 2.7cm at the lower border and 1.5 cm at the retromolar area. The intervening segment was removed and the titanium plates were reapplied to fixate the proximal and distal segments before reconstruction. Defects were reconstructed using either a ß-TCPB cylinder of 2.5 cm length and 2 cm diameter (group A) or a similar ß-TCPB/BM/CB cylinder (group B) with a central passage of 0.7 cm diameter (Fig. 2).
Fig. 2. Osteotomy and reconstruction of the mandibular critical size defect. (A) Removal of the osteotomized segment and (B) reconstruction of the defect with the bone graft substitute.
Blood was aspirated through the cylindrical ß-TCP blocks using special syringes included in the ß-TCP kit. The cancellous bone material used for group B was harvested from the iliac crest by the use of two bone biopsies (0.5 cm diameter), morselized by an electrical bone mill b, mixed with amorphous marrow aspirated from the depth of the biopsy areas and loaded into the central passage of the predrilled blood soaked cylinders. All cylinders were fitted into the basal aspect of the defect and secured to the plate using resorbable Vicryl 0 cerclages. The soft tissue wounds were closed in layers using resorbable Vicryl 2.0 sutures.
Post-operative Care
An additional bolus of buprenorphine (10 µg/kg IM) was given before recovery to maintain analgesia. All sheep were allowed unlimited activity as well as access to food and water immediately after surgery. Pre-operative prophylactic penicillin (0.04
(buprenorphin) days. The healing process was assessed by daily physical examinations, daily pain scoring based on observation of behavioral changes and changes in ruminating patterns as well as repeated weight controls during the first three weeks. Physical examinations and weight controls were continued until sacrifice. Twelve weeks after surgery all animals were euthanized after deep sedation (Midazolam 1 mg/kg, IM, Propofol 5 mg/kg, IV, pentobarbital 80 mg/kg, IV) and mandibular segments were retrieved for histological evaluation.
Specimen Processing
Mandibular segments were fixated in 3.5% neutral buffered formalin for a week. After rinsing and dehydration using increasing concentrations of ethanol followed by acetone specimens were infiltrated and embedded in methylmethacrylate (MMA) under vacuum. Slow polymerization was completed in a drying-chamber at 37°C.
MMA embedded defects where sectioned along the dorsal plane using a modified inner-hole diamond saw c. Undecalcified slices of 30 µm were surface stained with alizarine-methylene blue for standard light microscopy and histomorphometric analyses.
Histological and histomorphometrical evaluation
Five slides per animal including the central slide, the two slides next to the initial ceramic borders marked by the remaining suture material and two intermediate slides positioned between central slide and surface were used for further evaluation. Digital
images of each slide were obtained using a Zeiss AxioImager MI Microscope fitted with an AxioCam MRc digital camera and AxioVision 4.5 softwared. The AxioVision module MosaiX was used to scan the total specimen (4x3 cm per slide).
Electronically created images of the entire defect area provided the basis for further analysis. A region of interest measuring 2.5 x 1.5 cm was marked orienting at the remaining cortices rostral and caudal of the defect. Total bone area (BA/TA), ceramic area (SA/TA), cartilage area (CA/TA) as well as fibrous tissue area (FA/TA) were quantified using the image-analysis software Analysis 3.0 e. Additionally all slides were blinded and randomly reviewed for qualitative histological evaluation.
Statistical Analysis
Serial slides retrieved from ten animals were included in histomorphometric evaluation and statistical analysis. Goodness of fit for normal distribution of model residuals of BA/TA, CA/TA, FA/TA and SA/TA was assessed using Q-Q-plots and Kolmogorov-Smirnov test. Arithmetic means (x) and standard deviation (std) were calculated for normal distributed parameters. For lognormal distributed parameters such as SA/TA or CA/TA logarithmic transformation was performed prior to analysis and geometric mean (xgeo) as well as geometric standard deviation (stdgeo) were calculated. The impact of the different graft types (TCPB vs. TCPB/BM/CB) and slide position (surface, intermediate, central) was examined using two-way analysis of variance with post hoc t-test for five pair wise comparisons between corresponding
Results.
Soft tissue swelling resolved within a week enabling palpation of the defect area.
None of the animals developed signs of instability or increased pain at the surgical site. All animals started sufficient ingestion and rumination immediately after recovery and continuously gained weight during the maintenance time. At sacrifice inflammation of the grafted area resulting in sequestration of the ceramic became obvious in two cases [group A (n=1) and b (n=1)]. Data from these animals were excluded from further evaluation. Fracture of the reconstruction plates resulting in increased callus formation at the defect area without further impairment of mandibular contour occurred in two animals of each group leaving three sheep per group that underwent bone healing under stable conditions. Statistical analysis revealed no significant difference in the amount of tissue types between specimens retrieved from stable and unstable defects in each group, thus the mechanical unstable animals were not separated and five specimens per group were included in further evaluation.
Fig. 3. Histological overview sections of defects grafted with ß-TCPB (A) and ß-TCPB/BM/CB (B). Bone is developing around the cut ends and along the enveloping fibrous tissue at the lateral graft surface after reconstruction with a TCPB composite (A). The specimen retrieved from an animal that underwent reconstruction with ß-TCPB/BM/CB displays bony union of the defect, residual ceramic is mainly
osseointegrated. Alizarin-red Methylene-blue stain, cc: cis cortex, tc: trans cortex, s:residual ß-TCP scaffold,c:cartilage, OS: osteosynthetic material , red arrows:sections within the scaffold with direct bone ceramic interface.
Descriptive Histology.
Bone in growth into the defect was rare within group A. New bone deposition mainly occurred close to the borders of the osteotomy site and bony organization varied from mature lamellar bone at the junction to the intact cortex to highly immature bone at the periphery of the bony outgrowths. Following the outer surface of the ceramic new bone started to envelope the scaffold. Osteogenic activity was evident due to the presence of osteoid seams and osteoblasts aligned along the mineralized surfaces but direct bone to scaffold contact was rare due to an intervening layer of fibrous tissue (Fig. 3).
Bone apposition within the pores could only be seen in areas of direct bone-scaffold contact and was proceeded by in- growth of fibrous or chondroid tissue (Fig. 4.A).
The core of the ceramic graft was intact and the void pore spaces were completely filled by highly cellular vascularized fibrous tissue. None of the specimens included in group A achieved defect union.
Dense lamellar bone bridged the defect in group B (Fig. 3.B). Similar to group A, osteoid deposition by aligning osteoblasts indicated ongoing bone formation. Main parts of the ß-TCP graft had been replaced by new bone leaving only small,
extracellular remnants of the initially grafted material. In contrast to group A, ceramic surfaces had intimate contact with the newly formed bone (Fig. 4.B).
Porous bone patterns similar to the scaffold architecture and matrix remnants within the woven bone could be seen in close contact to remaining scaffold borders (Fig. 5).
Residual ceramic granules were often accompanied by a moderate amount of histiozytes but no multinucleated giant cells or other histological evidence of a foreign body reaction could be seen.
Fig. 4. Bone-ceramic interface after a healing period of twelve weeks in ß-TCPB composites (A). Bone deposition within the scaffold was rare and the graft was enveloped in fibrous tissue. In contrast, the main parts of the residual ceramic were integrated within newly formed bone when ß-TCPB/BM/CB
composite was used (B).Alizarin-red Methylene-blue stain,s: ß-TCP scaffold, b: mineralizedbone c:cartilage ft: fibrous tissue, red arrows: ongoing enchondral ossification.
Fig. 5. Newly formed woven bone within the defect of an animal grafted with ß-TCP bone marrow composite, red arrows mark incorporated residual ß-TCP within the mineralized bone matrix.
Alizarin-red Methylene-blue stain.
Histomorphometry
Results of the histomorphometric analysis are illustrated in Figure 6 and Table 1.
The distribution of newly formed bone and remaining scaffold area across the defect was variable. Bone deposition within group A was most prominent at the two marginal sections and declined with increasing proximity to the defect center where higher amounts of residual ceramic were present. Average BA/TA calculation for the
two specimens that underwent healing under instable conditions was slightly, but not significantly higher at each position than for the rest of the group.
Total Bone Area a
0 10 20 30 40 50 60 70 80 90 100
1 2 3 4 5
Slide Number
% TA/BA Group A
TA/BA Group B
Ceramic Area b
0 10 20 30 40 50 60 70 80 90 100
1 2 3 4 5
Slide Number
% SA/TA Group A
SA/TA Group B
Connective Tissue Area a
0 10 20 30 40 50 60 70 80 90 100
1 2 3 4 5
Slide Number
% FA/TA Group A
FA/TA Group B
Cartilage Area b
0 10 20 30 40 50 60 70 80 90 100
1 2 3 4 5
Slide Number
% CA/TA Group A
CA/TA Group B
Figure 6. Histomorphometrical evaluation of mean area fractions of bone (a), residual ß-TCP (b), cartilage (c) and fibrous tissue (d) at the five positions within the defect.
a means and standard deviation
b geometric means and geometric standard deviation
Group B presented comparable amounts of BA/TA as well as SA/TA among all slides. The two specimens that underwent healing without rigid fixation due to plate fractures had lower estimates for BA/TA and SA/TA within group B, but still exceeded average estimates within group A. Osteointegration and degradation of the ceramic
graft in group B exceeded group A, showing significant (p<0.01) higher amounts of bone formation and less residual graft material within the three central slides while no significant difference could be estimated for the two marginal slides regarding these two parameters between the groups. Estimates for cartilage area (CA/TA) as well as fibrous tissue area (FA/TA), including blood vessels, did not significantly differ between the two groups (Table 1).
Table Significance of pair-wise comparisons between group A and B.
Probability Values
BA/TA SA/TA FA/TA CA/TA
Slide 1 0.2350 0.06 0.1559 0.1295
Slide 2 0.0005* 0.0013* 0.2844 0.0321
Slide 3 0.0007* 0.0034* 0.0384 0.2947
Slide 4 0.0002* 0.0005* 0.081 0.0099*
Slide 5 0.2762 0.071 0.105 0.1660
BA/TA: Total Bone Area, SA/TA: residual Ceramic Area, FA/TA: Fibrous Tissue Area, CA/TA:
Cartilage Area
* Significant difference between the two groups (p<0.01)
Discussion.
In this study we compared the ability of ß-TCP scaffolds, either used in combination with autogenous blood or with autogenous bone marrow and cancellous bone, to support osteoregeneration after mandibular reconstruction in adult sheep. The chosen critical size defect model, described by Ayoub et al. (26), mimicked the clinical setting after sectional bone loss and permitted evaluation of the material in a functional area where it may be used in human and veterinary medicine (8, 19, 27).
Implant failure of the fixation devices, presumably caused by the rumination process, led to decreased mechanical stability at the defect site in four cases resulting in a high deviation of average estimates for BA/TA without statistically significant impact.
As anticipated, defects that were grafted with ß-TCPB showed significantly less bone formation than defects grafted with ß-TCPB/BM/CB, which yielded good results. Since no osteogenic cells or inductive substances were added in group A, osseointegration solely relied on new bone formation arising from local osteogenic cells. Specimens developed a bony collar around the osteotomized ends of the mandible and along the outer surface of the scaffold with an intervening soft tissue layer inhibiting direct bone apposition upon the ß-TCP surface. A ß-TCP core that was invaded by fibroblasts and vascular sprouts occupied the central part of the defect. In-growth of invading fibrovascular tissue within the porous structure could be accompanied by invasion of osteoprogenitor cells providing a source for new bone deposition within the scaffold (1, 28) but we had no possibility of evaluating the amount of osteoprogenitors within our graft. We consider it unlikely that any of the defects in group A would have
achieved homogeneous union after a longer healing period but since we have no comparable results the further bone development within the defects remains speculative.
In group B we introduced osteogenic cells and osteoinductive factors into the defect by the use of bone marrow and morselized bone delivered within the central passage of the scaffolds. Regardless of the individual contribution of osteogenic cells or natural growth factors, we could provoke a sufficient osseous healing response without the need of expensive in vitro techniques or addition of synthetic growth factors that might generate unexpected sequalae (12, 22). The required amount of bone marrow and cancellous bone was low and could be easily obtained prior to reconstruction. Cancellous bone chips that were milled and added to the marrow were solely contained from core biopsies that were taken to obtain the bone marrow.
In contrast to the considerable morbidity that is associated with harvesting bulk cortico-cancellous autografts in humans (5, 6) and less frequently seen in veterinary patients (19), this method does not require massive manipulation of soft tissues or bone in the iliac region. All animals that received cancellous bone marrow-ß-TCP composites presented substantial ossification of the defect with osteogenic activity and enchondral ossification of the remaining cartilagineous replacement tissue.
Minimal amounts of residual ß-TCP were present on the lateral and medial surfaces of the bony bridge within the defect and osseointegration of the residual material indicated further conversion. Ceramic degradation was uniform throughout the entire defect and higher compared to group A (after correction considering the initial
the added cancellous bone and bone marrow served as a source for osteogenic cells and osteoinductive factors (12, 29). The osteogenic potential of unfractioned bone marrow has been known for more than a century (14, 30), but the limited number of mesenchymal stem cells (MSC) contained in an bone marrow aspirate and individual variations led to the development of in vitro alternatives that focus purifying and expanding of MSC’s or osteoblasts prior to transplantation (9, 15, 17, 31, 32). In vitro manipulation of MSC’s relies on complex, time-consuming and expensive techniques (15, 33, 34). These limitations and the proven osteogenic potential of bone marrow led to the conclusion that bone marrow may be clinically underused (28). Our study strengthens this hypothesis and, in accordance with similar approaches, proved the good healing support of bone marrow when delivered in a ß-TCP carrier. In contrast to other substitution materials, ß-TCP scaffolds are biodegradable, thus no foreign material remains to interfere with normal bone remodeling and restoration of mechanical strength (34). Different authors have voiced concern that degradation may occur faster than osseointegration and inhibit healing (1, 35). We could neither confirm this concern nor identify the exact degradation mechanism of the used material. Our findings imply that ß-TCP degradation and new bone formation parallel each other. The amount of ceramic degradation in group A, which had minor bone formation, seemed to be lower than in group B. Furthermore it seemed as if the matrix did not only degrade but may have been incorporated during the mineralization process to serve as a substrate for bone matrix deposition. It is currently assumed that increased Ca2+ levels may lead to initiation of biomineralization, increased osteogenic phenotype commitment, stimulation of local osteoclasts or a combination of these mechanisms (28, 36, 37). We could not confirm
earlier observations that reported osteoclasts in close proximity to ß-TCP remnants (38, 39) but osteoclastic stimulation leading to increased osteoblast activation could explain the apparent synchronization of bone formation and matrix degradation.
Since we did not use tartrate-resistant acid phosphatase stain to identify osteoclasts it may be possible that multinucleated cells were present but could not be identified.
The ß-TCPB/BM/CB composite used in this study could sufficiently heal a mandibular critical size defect. Although our findings cannot be transferred to clinical situations where the host bed is frequently compromised by infection or irradiation, they strongly encourage additional investigation of the ß-TCPB/BM/CB composite to verify our results. The described method offers the advantage of manufacturing an efficient bone graft substitute table-side during surgery using the patient’s own cells, circumventing the need for cell culture, expansion, or preservation. Degradation of the material presumably supported matrix mineralization by means of a process that resembled physiologic bone remodeling. The osteoregenerative capabilities of the TCPB/BM/CB composite indicate a promising potential for various clinical indications including mandibular reconstruction.
Footnotes.
a Synthes Europe GmbH, Oberdorf, Switzerland
b Aesculap AG & CO. KG, Tuttlingen, Germany
c Leitz, Wetzlar, Germany
d Carl Zeiss AG, Obernkochen, Germany
e Olympus Soft Imaging Solutions, Münster, Germany
f SAS Institute, Cary, NC
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3 Manuskript II
Eingereicht am 20.08.2008 und akzeptiert zur Veröffentlichung am 24.0.2008 (siehe Seite xx) im ‘Journal of Cranio- and Maxillofacial Surgery, Impact Factor (2007).
Comparison of Computed Tomography and Microradiography for Graft Evaluation after Reconstruction of Critical Size Bone Defects using ß-Tricalcium- Phosphate.
M.C. Nolff 1,2,*, Dr. Dr. H. Kokemueller 1, Dr. G. Hauschild 2,3, Prof. Dr. M. Fehr 2, Dr. Dr. K.- H. Bormann 1, S. Spalthoff 1, Dr. K. Rohn 4 , Prof. Dr. Dr. M. Ruecker 1 , Prof. Dr. Dr. N.- C.
Gellrich 1
1 Department of Oral and Maxillofacial Surgery, Hannover Medical School, 30625 Hannover, Germany
2 Small Animal Clinic, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15,Germany
3 Department of Orthopedics, University of Münster, Münster, Germany
4 Institute for Biometry, Epidemiology and Information Processing; University of Veterinary Medicine Hannover; Foundation, Bünteweg 2, 30559 Hannover, Germany
*Corresponding author
Key words: Mandibular reconstruction, ß-TCP, bone graft substitute, radiological evaluation, Computed Tomography
Summary.
Introduction: The Aim of the study was to evaluate accuracy of CT for in-vivo patient follow up after mandibular reconstruction.
Material and Methods: Unilateral mandibular defects were surgically created in ten sheep and either reconstructed using blood soaked ß-TCP cylinders (group A, n=5) or blood soaked ß-TCP cylinders that were additionally loaded with autologous bone marrow (group B, n=5). The two graft designs resulted in different stages of graft ossification representative for different stages of healing. CT Datasets were fused with microradiographs and measurements of ceramic area based on both methods were compared.
Results: Two animals (group A (n=1) and B (n=1)) presented infection and graft dislocation that was visible on CT and were excluded from statistical evaluation.
Group A grafts underwent moderate degradation (53.55% ± 9.7) and incomplete bony incorporation representing an indermediate state of healing while ceramic grafts within group B developed a high grade of osseointegration and degradation (94.2% ± 3.3) consistent with progressed healing. Statistical comparison of measurements based on both methods revealed a significant bias (p<0.05) and a non-significant variance for group A and a significant variance (p<0.05) and non-significant bias for group B.
Conclusion: Our results indicate that conventional CT is not eligible to objectively evaluate ossification and degradation of a ß-TCP graft in vivo and further attempts to improve clinical visualization of ß-TCP need to be undertaken.
Introduction.
The core techniques of bone grafting established over a century ago have remained largely unchanged until today. Despite its known shortcomings, mainly limited availability and donor side morbidity, autologous cancellous bone is still considered the gold standard to fill bone defects and stimulate fracture healing in a variety of clinical settings covering indications in reconstructive-, orthopedic-, craniomandibulofacial- and spinal-surgery (Banwart et al., 1995, Wippermann et al., 1997, Rodriguez-Merchan and Forriol, 2004).
Among these indications, the reconstruction of mandibular defects remains to be a special challenge, even for experienced surgeons. Restoration of form and function is of paramount importance in this context since the mandible is crucial for fixation of the tongue and floor of the mouth-and by that for deglutition, speech formation and prevention of airway obstruction (Knoll et al., 2006, Chen et al., 2008). Due to this challenging need for optimized tissue reconstruction numerous approaches have been developed and published in the current literature (Kimura et al.,2006, Knoll et al., 2006, Chen et al., 2008, Li et al., 2008). Autogenous bone grafts as well as osteomyocutaneous flaps are still considered the gold standard, but unfortunately their harvest is associated with considerable donor side morbidity and their supply is limited. Modern biologic graft materials, such as allo- and xenografts, represent one alternative, especially since they were able to overcome some of the limitations of autologous bone, but healing remained difficult to anticipate and the safety of allo- and xenograft bone has been controversially discussed (Stevenson 1999, Wheeler and Ennekin 2005, Wenz et al., 2001). Consequently, an increasing demand for
synthetic bone graft substitutes free from the limitations of supply, consistency and disease has led to intense research during the past decades (Cornell 1999, Lane 1999, Bauer and Muschler, 2000, Den Boer et al., 2002). Among the variety of osteoconductive materials that have been investigated, calcium phosphate compounds have been considered especially effective (Tay et al., 1999, Bloemers et al., 2002, Mastrogiacomo et al., 2005). The main representatives of this group, Hydroxyapatite (HA; [Ca5(PO4)3OH]) and beta-Tricalcium Phosphate (ß-TCP;
[Ca3(PO4)2]), have been investigated in numerous experimental studies and clinical trials during the past 30 years (Hulbert et al., 1970, Hoogendorn et al., 1984, Jensen et al.,2006). Both substitutes yielded promising results in terms of osteoconduction, osseointegration and biocompatibility (Johnson et al., 1996, Wheeler et al. 2005) and modern manufacturing techniques allow prefabrication of ceramic scaffolds that meet the known demands regarding macro-pore size, interconnectivity, porosity, biocompatibility and biodegradability (Eggli et al., 1988, Lu et al., 1999, Blockhuis et al., 2000, Hutmacher 2001). Unfortunately the beneficial composition of ceramics also accounts for a major disadvantage - the radiodensity of the material overshadows bone healing and remodeling within the implant (Johnson et al., 1996, Rueger et al., 1998, Mankani et al,. 2004). Thus, the precision of in vivo radiologic evaluation remains questionable. As the clinical usage of ceramic materials increases, the need for reliable in vivo diagnostic evaluation becomes inevitable. This problem has been addressed by several authors and led to the conclusion that preclinical studies should include outcome measures similar to those anticipated to
reliability, in vivo diagnostic methods need to be correlated to established investigative methods for graft evaluation to determine their accuracy. The aim of this study was to objectively compare the results retrieved from CT scans and microradiographs for evaluation of a previously grafted bone defect in order to assess reliability of CT Data for clinical evaluation of ß-TCP grafted bone defects.
Material and Methods.
Graft Material.
The used material (ChronOs®a) has been described by the manufacturer as a radio- opaque porous beta-TCP ceramic, which will be degraded and replaced by bone in 6-18 months during the healing process. Material performance and biocompatibility have been well documented in preclinical and clinical trials and the manufacturer recommends usage as a bone-void-filler for trauma-, spinal- and cranio- mandibulofacial indications. For the purpose of this study, ten pre-shaped cylinders of standardized diameter and height (14mm x 24 mm) were provided by Synthes Europe GmbH. Five cylinders were unpacked, prepared with a through-bore-hole (7 mm diameter) along the central-line and sterilized by gamma-irradiation after repacking into surgical kits, while the other five cylinders were used as delivered.
Experimental Design.
Animal experiments were approved by the local governmental animal care committee and conducted in accordance with German legislation on protection of animals, which is in compliance with the NIH Guidelines for the Care and Use of Laboratory Animals (NIH Publication no. 85-23 Rev. 1985). Ten healthy adult female German Black- Headed Sheep with an average weight of 72.5 +/- 7.4 kg were either grafted with bulk blood soaked ß-TCP cylinders (Group A; n=5) or predrilled ceramics which were loaded with bone marrow aspirate and morselized cancellous bone received during surgery (Group B; n=5). Animals were housed in groups of two to four animals at the
sheep diet as well as water ad libitum at all times during the maintenance time. All sheep underwent partial mandibulectomy (25 mm segment) of the right mandible followed by reconstruction (Compact 2.4 UniLOCK Recostruction Plate) using a ß- TCP graft under general anesthesia. In animals that received graft type B, bone marrow was harvested from the iliac crest during surgery after removal of three standard core biopsies. The cancellous bone material contained in the biopsies was morselized, mixed with the attained amorphous marrow and loaded into the central passage of the predrilled cylinders. Finally, the cylinders were fitted into the basal aspect of the defect and secured to the distal plate in both groups. The soft tissue wounds were closed in layers using resorbable Vicryl 2.0 sutures. Pre-operative prophylactic penicillin (0.04 ml/kg, IM, q48h) as well as peri-operative analgesia (carprofen, initial dose: 4mg/kg followed by 2mg/kg, SC, q24h / buprenorphine 10 µg/kg, IM, q24h) was continued for four (carprofen) respectively three (buprenorphin) days. All sheep were allowed unlimited activity as well as access to food and water ad libitum immediately after surgery. All animals were euthanized twelve weeks post surgery after deep sedation with an overdose of pentobarbital (80 mg/kg IV). After decapitation, CT scans of the cranium were performed and following the scans, mandibular segments were retrieved for µ-radiological evaluation.
CT Scan.
After sacrifice and decapitation each head was scanned in a multi-slice spiral computer-tomographb. Reconstruction plates and screws remained in situ. Scanning was performed at 120 kV, 150 mAs with a table speed of 6.25 mm per rotation. Slice- thickness was set 0.6 mm with a reconstruction interval of 0.625 mm. The raw data
was processed by the use of a special bone algorithm that improved visualization of the bony structures for evaluation. The datasets were stored for further evaluation using Dicom 3.0 as a medical image file format.
Microradiography.
After retrieval, mandibular segments were fixated in 3.5% neutral buffered formalin for a week. After rinsing and dehydration all specimens were embedded in methylmethacrylate (MMA) under vacuum. Slow polymerization was completed in a drying-chamber at 37°C. The exact defect position was radiologically accessed to enable precise trimming of the MMA blocks and plastic embedded defects were then sectioned into serial sagittal slices using a modified inner hole diamond saw. Every fourth section (90 µm thickness) was selected for radiologic evaluation and fine-detail contact microradiography (15 kV, 7 sec) was performed in a Faxitronc cabinet using Kodak dental filmd. All radiographs were developed in an automated processor and digitalized.
Image Processing and Image Fusion.
To objectively verify subjective CT results ceramic area was measured at ten locations within the defect for each animal using both methods. To ensure comparison of corresponding areas the CT and microradiographic images were fused and aligned. To enable synchronization of the two different data sources, a scientific beta version of VoXim eosteo was provided by IVS Solutions. The
dicom dataset. The digitized microradiographs (7-11 images per mandibular segment) were imported, manually aligned and converted (Figure 1) into a serial dataset. In order to enable conversion into a serial dataset the distance between single images (dorsal plane, 1.17 cm) was manually set resulting from the number of slices between two specimens (3), slice thickness (90µm) and loss associated with processing of each slide (300µm).
Figure 1: Three-dimensional defect reconstruction after alignment and conversion ofmicroradiographic images. Blue: ChronOs® remnants; Red: Reconstruction plate; Green: tooth
Additionally the pixel size was adjusted in order to synchronize the voxel size of microradiographic and CT datasets. The adjusted serial microradiographic datasets were fused and manually aligned to the corresponding CT data using the Image Fusion module of the same software (Figure 2). The ceramic area was measured at ten different images of the CT dataset and re-estimated at the same locations after superimposing the microragiographs to determine the accuracy of the first measurement. Hounsfield-Units (HU) of ceramic and bone within the defect were measured on two random positions of each image per parameter within the defect. In order to ensure correct measurement this procedure was performed as well under superimposed microradiographic control.
Figure 2: Corresponding microradiographic (A) and CT images (B) after matching and fusion (C) of both datasets. Due to the low grade of mineralization fresh callus, which is clearly visible in the microradiographic image, is only partly visible in the CT image.
Finally the fused dataset was used to determine residual graft volume and to enable
determined by comparing initial graft volume of both groups with the residual volume.
We assumed that the more precise in vitro method, microradiography, presented the gold standard.
Statistical Analysis.
The data retrieved from eight animals by CT and microradiography were included in statistical analysis to determine comparability of the two diagnostic methods.
The objective of the study was to examine accuracy, reliability and validity of CT scan measured area to show agreement or disagreement between the two methods of measurement. The t-test for paired observations was used to detect significant bias between the two methods. Reliability of CT measurements was estimated by the chi- square test for variance. The tested null hypothesis assumed a variance of the difference of CT values against microradiography (gold standard) equal to a standard deviation from differences against the gold standard of one hundred percent.
Outcomes were considered statistically significant for p < 0.05.
Matched data of both methods were examined graphically with the Blant-Altmann- Diagram modified in abscissa (microradiography as reference value) and linear regression with calculating coefficient of determination. Because values of microradiography in x- coordinate were truly reference values, variance was only accounted for y-coordinate (ct-scan). The statistical software SAS version 9.1 was used for analysis of all data.
Results.
Two sheep (one of each group) presented inflammation of the graft side associated with graft displacement and sequestration as well as necrotic lysis of the mandibular bone at the time of sacrifice. Those two animals represented total graft failure.
Figure 3: Microradiographs show graft dislocation in two animals (A). In animals assigned to group A the remnants of the grafted ß-TCP cylinders were enveloped by an intervening layer of radiolucient tissue and newly deposited bone (B). Defects retrieved from animals assigned two group B presented bony healing of the defect with full integration of ceramic remnants (C).
Microradiography showed that the scaffold had rotated around the osteosyntheses plates leaving an unbridged defect with minimal callus formation around the cut ends.
The ceramic was surrounded by radiolucent tissue and gas. Bony contact did not occur and there were no signs indicating degradation of the ß-TCP scaffolds (Figure 3.A). CT images also revealed displacement of hyperdense ceramic material towards the reconstruction plate. The residual scaffold was delineated by hypodense
measured within the scaffold area (445-1142 HU) corresponded with estimates retrieved from bony structures (599-1108 HU). Thus identification of trabecular bone and ceramic based on the HU estimates proved inefficient. There were no signs of bridging ossification along the ceramic surface or within the defect (Figure 4) and major alterations of the three-dimensional graft shape were not visible. Since graft dislocation was evident irrespective of the mode of evaluation, image fusion and objective evaluation of ceramic area was considered non-relevant.
Figure 4: Coronar (A) and transversal (B) view of a specimen that developed graft dislocation. Bordering radiolucient tissue enables graft identification.
In a case like this representing graft failure the resuming ceramic volume is of secondary import. Additionally, a comparison of two different specimens would not allow satisfactory statistical comparison.
