scaffold guided mandibular reconstruction with axial ...€¦ · aus der plastisch-und hand...
TRANSCRIPT
Aus der
Plastisch-und Hand Chirurgischen Klinik
der
Friedrich-Alexander-Universität Erlangen-Nürnberg
Direktor: Prof. Dr. Raymund E. Horch
Scaffold Guided Mandibular Reconstruction With Axial
Vascularization Using The Arterio-Venous Loop Model
Inaugural-Dissertation
zur Erlangung der Doktorwürde
der
Medizinischen Fakultät
der Friedrich-Alexander-Universität
Erlangen-Nürnberg
vorgelegt von
Ahmad Eweida
aus
Ägypten
2
Gedruckt mit Erlaubnis der
Medizinischen Fakultät der Friedrich-‐Alexander-‐Universität Erlangen-‐Nürnberg
Dekan: Prof.Dr. Dr. Schüttler Referent: Prof. Dr. Raymund E. Horch Korreferent: Prof. Dr. Dr. Emeka Nkenke Tag der mündlichen Prüfung: 14.8.2012
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IN THE NAME OF GOD THE MOST GRACIOUS
TO;
MY MOM AND DAD WHOM I KNOW THEIR HOPE
WAS TO HOLD THIS BOOK TO;
MY BELOVED WIFE WHO HELPED ME BY EVERY MEANS
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Table of Contents Zusammenfassung .................................................................................................. 5
Hintergrund und Ziele ........................................................................................ 5 Material und Methoden ...................................................................................... 5 Ergebnisse und Beobachtungen ......................................................................... 5 Praktische Schlussfolgerung .............................................................................. 6
Summary ................................................................................................................ 7 Background and Objectives ............................................................................... 7 Materials and Methods ....................................................................................... 7 Results and Observations ................................................................................... 7 Conclusions ........................................................................................................ 8
Introduction ............................................................................................................ 9 Mandibular reconstruction ................................................................................. 9 1. Alloplastic materials .................................................................................... 9 2. Soft tissue coverage of mandibular reconstruction plates ......................... 10 3. Nonvascularized bone grafts (NVBG) ...................................................... 10 4. Free vascularized bone flaps (VBFs) ........................................................ 10 5. Synthetic biomaterials (scaffold guided mandibular regeneration) ............. 11 Vascularization of scaffolds ............................................................................. 12 Vascularization concerns in the mandible ........................................................ 14
Aim of the work ................................................................................................... 16
Materials and Methods ......................................................................................... 17 Study design ..................................................................................................... 17 Scaffold preparation ......................................................................................... 17 Animal surgery ................................................................................................. 19 Animal Harvest and Radiological evaluation .................................................. 21 Preparation and Explantation of the mandible ................................................. 22 Biomechanical evaluation ................................................................................ 23 Histological evaluation ..................................................................................... 23
Results .................................................................................................................. 28 Long term follow up ......................................................................................... 28 Radiological ..................................................................................................... 28 Macroscopic and Biomechanical ..................................................................... 29 Histological ...................................................................................................... 30
Discussion ............................................................................................................ 36
References ............................................................................................................ 43 List of Abbreviations ............................................................................................ 52
List of Pre-publications ........................................................................................ 50 Acknowledgment ................................................................................................. 51
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Zusammenfassung
Hintergrund und Ziele
Die Rekonstruktion großer und komplexer Knochendefekte ist eine der
anspruchsvollsten Fragestellungen in der heutigen klinischen Praxis. Um das
Problem der Hebemorbidität eines autogenen Knochenersatz zu umgehen,
werden heutzutage synthetische Biomaterialien verwendet. Die meisten der
heutigen Ansätze basieren dabei auf einer extrinsischen Blutversorgung. Dieses
Verfahren ist jedoch bei der Rekonstruktion nach Tumorentfernung nicht immer
anwendbar.
Ziel unserer Arbeit war die Evaluation eines axial und somit intrinsisch
vaskularisierten synthetischen Knochenersatzes an einem Großtier-modell. Der
Erfolg dieses Konzeptes erlaubt der Wiederherstellung von ausgedehnten,
mandibulären Knochendefekten nach onkologischer Resektion.
Material und Methoden Bei der vorliegenden Pilot-Studie wurde das Konzept des axial vaskularisierten
synthetischen Knochenersatzes an einem Großtier-Modell (Ziege) untersucht.
Hierbei wurde eine arterio-venöse Schleife basierend auf die Arterie und die
Vene Fazialis zentral in ein biphasisches Keramik-konstrukt eingebracht,
welches durch Zugabe von thrombozytenreichem Plasma (PRP) und
Knochenwachstumsproteinen (BMP) zur Osteogenese angeregt wurde. Nach
einer Beobachtungsintervall von 6 Monaten erfolgte die Charakterisierung der
Knochenneubildung und-vaskularisation mit Hilfe von Computertomographie
(CT) sowie durch biomechanische und histomorphologische Verfahren.
Ergebnisse und Beobachtungen Die technische Eignung der axialen Vaskularisation eines synthetischen
Knochenersatzes mittels einer arterio-venöser Schleife konnte beim Großtier-
Modell bestätigt werden. Hierbei wurde ca. 80% des Knochenvolumens
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regeneriert. Die biomechanische Untersuchung hat einen Brechpunkt von
1662.19 Newton und somit eine gute Stabilität nachgewiesen. Die
histomorphologische Untersuchung bestätigte die Wiederherstellung des
Knochens sowie auch die reichende Vaskularisation des Konstrukts.
Praktische Schlussfolgerung In der vorliegenden Arbeit wurde die Eignung das axial vaskulariserten,
synthetischen Knochenersatzes bei der Wiederherstellung eines kritischen
mandibulären Defektes erstmalig nachgewiesen. Der wiederhergestellte Knochen
war voll entwickelt, ausreichend vaskularisiert, und funktionsfähig. Erweiterung
dieses Modells durch Vergrößern bzw. Verstrahlen des mandibulären Defektes
kann den klinischen Szenarios ganz gut ähneln.
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Summary
Background and Objectives Reconstruction of large and complex bone segments is one of the most
challenging problems facing modern clinical practice. In order to diminish the
donor site morbidity associated with autogenous bone transfer, synthetic
biomaterials are being used nowadays to regenerate lost bone due to disease or
trauma. The majority of currently applied regenerative medicine approaches rely
on extrinsic vascularization, which could not be applied to reconstruction after
cancer ablation.
Our objective was to investigate the feasibility of regenerating a critical
size mandibular defect in a goat using an axially vascularized synthetic bone
substitute. Confirming the feasibility would help introducing regenerative
medicine to reconstruction after cancer surgery.
Materials and Methods This study is an experimental pilot study introducing the concept of axial
vascularization of bone substitutes to regenerate a critical size mandibular defect
in a large animal model (goat). In this study we used the facial vessels to create
an arterio-venous loop in order to vascularize a biphasic ceramic scaffold. The
scaffold was charged with platelet rich plasma and bone morphogenic proteins in
order to augment osteogenesis. After a 6 months- follow up period, the new bone
formation and vascularization were assessed through radiological (CT),
biomechanical (3 points bending), and histological studies.
Results and Observations We were able to demonstrate the technical feasibility of creating a local
vascular axis through arterio-venous anastomosis within a synthetic bone
substitute to regenerate a critical size mandibular defect. About 80% of the
volume of the resected segment was regenerated. The biomechanical test showed
8
that the mandible broke at a force of 1662.19 Newton. The histological study
confirmed bone regeneration and adequate vascularization of the scaffold.
Conclusions
We were able to demonstrate for the first time through long-term follow
up, radiological, histological, and biomechanical studies the feasibility of
regenerating a critical size mandibular defect in a large animal using an axially
vascularized bone substitute. The regenerated bone was mature, adequately
vascularized and functionally competent.
Further upgrading of this model by inducing a large segmental and
possibly irradiated mandibular defect would be helpful to put the model in a real
challenge and a very similar condition to clinical scenarios.
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Introduction
From the beginning of the 8th century, the work of Middle Eastern
Muslim physicians such as Avicenna, Albucasis, and Rhazes was of paramount
importance in guarding knowledge, particularly the contributions of Greek and
Roman scholars, until the 13th century. Many contributions from that period in
the field of craniofacial and neurosurgery created a solid ground for modern
medicine in this field (74).
In reconstructive surgery, the reconstruction of large and complex bone
segments remains one of the most challenging problems in modern clinical
practice. Worldwide, an estimated 2.2 million grafting procedures are performed
annually to repair bone defects in orthopaedics, neurosurgery, and dentistry (36).
Craniofacial bone grafting represents about 6% of all bone grafting procedures
(24). These procedures aim to replace bone lost due to trauma or disease.
Mandibular reconstruction
Conventional methods for mandibular reconstruction involve the use of
alloplastic materials, soft tissue coverage of mandibular reconstruction plates,
nonvascularized bone grafts (NVBGs), free vascularized bone flaps (VBFs), and
recently a variety of synthetic biomaterials.
1. Alloplastic materials
The potential for aesthetic reconstruction without donor site morbidity
has led many on the search for suitable alloplastic materials. Although these
implants offered some restoration of continuity and bulk, overall success has
been disappointing, especially when these devices are applied primarily in
previously irradiated areas of the head and the neck (55). For these reasons, these
devices are not currently favoured and should be avoided, if possible.
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2. Soft tissue coverage of mandibular reconstruction plates
An option for mandibular reconstruction includes the use of mandible
reconstruction plates covered with soft tissue. The pedicled pectoralis major
myocutaneous flap was widely used to cover titanium plates to prevent extrusion.
Soft tissue free flaps were also used to cover the reconstruction plates. Although
free flap coverage of the reconstruction plates showed better results than pedicled
flaps, the most common complication was still plate exposure (16, 17, 89). Also
failure to deliver autogenous bone to reconstruct the mandible will prevent later
dental rehabilitation and will eventually lead to reconstruction plate fatigue as the
contralateral molar loading exerts a torsional force which is more likely to cause
plate fracture (22). That is why soft tissue coverage of mandibular reconstruction
plates represents an alternative in patients who have lateral mandibular
continuity defects with a poor prognosis, in whom dental rehabilitation is not
desired or planned.
3. Nonvascularized bone grafts (NVBG)
These are suitable for smaller defects usually not subjected to
radiotherapy and in patients medically too compromised to tolerate free flap
surgeries. The rates of bony union and implant success with NVBG is less than
that with vascularized bone flaps (VBF) even in comparative studies where the
patients receiving VBF were older, had larger defects, and were treated primarily
for malignant disease with an associated higher incidence of radiation therapy
(32).
4. Free vascularized bone flaps (VBFs)
VBFs have revolutionized mandibular reconstruction. Even when these bone
flaps are transferred from distant sites into areas of irradiation, compromised
blood flow, and salivary contamination, the union of bone segments and the
support of functional loads are the usual result. Osseointegrated implants also
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can be successfully placed within vascularized bone free flaps, contributing to
rehabilitation and a stable dental arch (64).
An ideal VBFs provides adequate shape, width, and length of vascular bone,
but unfortunately, the ideal vascularized bone graft for all oromandibular
reconstructions does not exist; therefore, each patient and defect must be
evaluated separately to determine the best surgical approach. Fibular
osteocutaneous free flaps, scapular osteocutaneous free flaps, iliac crest
osteocutaneous free flaps, radial forearm osteocutaneous free flaps, and the
Latissimus-Serratus-rib free flap are all available options for mandibular
reconstruction.
For reconstructing critical size bone defects in clinical practice, vascularized
free flaps may be regarded as the “gold standard”. However, the use of these
bone grafts in the clinical practice presents several major inconveniences. The
harvesting of autologous bone often results in a significant donor site morbidity,
the extent of which may vary, according to the location of the site and possibly to
the intervention technique (10, 12, 14, 82). The problems include bleeding, pain,
infections, donor site fractures and prolonged hospital stay (39, 77).
5. Synthetic biomaterials (scaffold guided mandibular
regeneration)
Trying to reduce or even abolish donor site morbidity was a major trigger
that made researchers try to harness the regenerative capacity of the human body
to repair itself. In the last few decades new strategies started to emerge aiming at
mimicking the normal healing process in regenerating lost or damaged tissues.
The term "tissue engineering" was officially coined at a National Science
Foundation workshop in 1988 to mean "the application of principles and
methods of engineering and life sciences toward fundamental understanding of
structure-function relationships in normal and pathological mammalian tissues
and the development of biological substitutes to restore, maintain or improve
tissue function"(83). Tissue engineering and Regenerative medicine depend on
the presence of a biomaterial promoting cell growth and proliferation. In order to
regenerate the damaged or missing tissues, such biomaterials must effectively
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interact with the surrounding tissue and incite the host to populate the graft with
new tissue. This necessitates the establishment of an early and robust angiogenic
response leading to the development of a blood supply for the restoration of
structure and function (8, 41). Tissue regeneration could be reinforced by adding
cells or growth factors to the biomaterials (69) but the vascularization of
biomaterials is considered a determining issue in the success of critical size
defect regeneration (33).
Vascularization of scaffolds
The majority of currently applied regenerative medicine approaches rely
on the so-called extrinsic mode of vascularization. In this case the neovascular
bed originates from the periphery of the scaffold, and thus should be implanted
into a site of high vascularization potential. The pattern of vascularization in this
context is a ‘’random’’ pattern of vascularization where the construct is not
depending on a definite vascular axis for its supply (46).
This type of vascularization requires an optimal implantation site so that
the construct can be able to get its adequate blood supply. This is actually not the
case in most of the clinical scenarios such as cases of secondary reconstruction or
post radiotherapy. Furthermore, diffusion limits oxygen and nutrition supply to
cells to a maximum range of 200 µ into a given matrix (37) so that suboptimal
initial vascularization will definitely limit survival of cells in the centre of large
constructs.
These issues of vascularization implemented the need for novel
angiogenic approaches and new in vivo models evolved with the aim to generate
constructs with a dedicated neovascular network not under the immediate
influence of the local environment, i.e. an intrinsic mode of vascularization
(71).
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Intrinsic vascularization
This intrinsic vascularization approach is based on a concept that an
artery or vein can serve as a source of new blood vessels for prefabrication of
tissue for transplantation. Prefabrication is a technique of re-vascularization of a
tissue graft by implanting an arterio-venous loop (AVL) or a vascular pedicle
underneath or within a tissue graft, resulting in spontaneous angiogenic
development from the loop or pedicle and subsequent revascularization of the
tissue graft (27, 38, 62). This type of vascularization is not randomised and the
construct depends on a defined vascular axis for its nourishment. That is why it is
some times called ‘’intrinsic axial vascularization’’ (47).
It is important here to mention that the prefabrication depends on the
intrinsic mode of vascularization while ‘‘prelamination‘‘, a term introduced by
Pribaz and Fine in 1994 (73), depends on the extrinsic mode of vascularization of
the tissue or construct. Prefabrication basically means implanting a vascular
pedicle into a new territory, while prelamination refers to implanting tissues or
constructs into a flap to create a customized structure. The end result of both
techniques is an axially vascularized construct that could be transferred to the
recipient site as a pedicled or free flap. The prelamination technique, however,
could not be used to vascularize constructs at the defect site (28).
Intrinsic axial vascularization via the AV loop
Recently, the superiority of the AVL as a vascular carrier for intrinsic
axial vascularization has been clearly demonstrated. The AVL develops a
perfused capillary network that remodels to generate arterioles, post-capillary
venules, and venules (52). Three mechanisms are held responsible for this
phenomenon: a local inflammation due to the surgical trauma on the vessels, a
rise in mechanical stress on the vascular walls of the graft and the vein due to
arterializations and finally gradients in oxygenation along the matrix. A local
inflammatory response secondary to the surgical trauma induces a surge of
angiogenic substances. For example, pro-inflammatory chemokines are known to
induce up regulation of VEGF from platelets and endothelial cells (57, 65, 78). A
14
rise in pulsatile pressure and shear stress is another factor leading to enhanced
neovascularization. Insertion of a vascular graft into the arterial circulation is
known to generate a rise in VEGF production from the affected endothelium
both due to mechanical stimulation as well as sustained injury (9, 13, 44). The
combination of shear stress with turbulent flow present at the microvascular
anastomoses is known to be a major activator of endothelial cells (20). Gradients
in partial pressure of oxygen or hypoxia within the matrix may also play a role in
induction of the marked angiogenic phenomena (3, 40).
In general, this approach represents a significant step forward in
vascularization of tissues and development of axially vascularized bone
substitutes (AVBS). It was thoroughly investigated and has been applied in a
variety of tissues including bone, liver, cardiac and skeletal muscle tissue (2, 5,
30, 45, 49, 63). Further work beyond the small animal model of the rat and into
larger animal models such as goat and sheep affords the ability to monitor
vascularization in real-time using angiography techniques and complex 3D
reconstructions demonstrating the power of this model system (7).
Vascularization concerns in the mandible
Vascular pattern
Within the craniofacial bones, the vascular supply is more consistent with
that of the cancellous bone where the blood, in contrast to compact bone, reaches
its anatomical destinations more directly without significant branching. Together
with a relatively large surface area to bone volume; these bones are less prone to
vascular compromise. It is to be noted also that most of the mid-facial bones are
covered by mucosa over large areas of their surfaces. Thus, every part of these
bones retains its periosteal blood supply. The blood supply of the mandible,
however, is a mixture of that of the compact and cancellous bones and is
therefore more susceptible to compromise (35, 54).
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Radiotherapy
Although the craniofacial region has this abundant blood supply, it is
commonly compromised after treatment with radiotherapy following cancer
surgery (42). Radiotherapy causes damage to normal epithelial, dermal, and
endothelial cells. The resulting hypocellularity and hypoxic environment leads to
scarring and fibrosis that make secondary reconstruction of the surgical site
difficult (76).
Defect size
Bone regeneration is principally a part of the fracture healing process.
The majority of fractures heal well under standard conservative or surgical
therapy. However, extended bone defects following trauma or cancer resection
require more sophisticated treatment, as spontaneous bone healing is unexpected.
In a similar way, bone regeneration at the central region of large constructs
usually fails due to absence of adequate extrinsic vascularization (71). That is the
reason why all the clinical trials for craniofacial reconstruction using
regenerative medicine modalities without axial vascularization have never
addressed reconstruction following cancer. The trials were confined to
reconstruction post-infection, trauma, benign tumours, or congenital anomalies
(15, 25, 81, 85).
We are presenting in this study a new model for scaffold guided
mandibular reconstruction applying the techniques of intrinsic axial
vascularization to regenerate a critical size mandibular defect. We are using the
facial vessels as local vascular axes to create an AV loop which is used to
vascularize a bone construct at the same site of the defect abolishing the need for
any tissue transfer or donor site harvest. The study tries to mimic the clinical
scenario after cancer surgery in the head and neck region where the vascular bed
is not optimal for extrinsic random-type regeneration due to extensive tissue loss
and radiotherapy.
16
Aim of the work
The study aims at investigating the feasibility of regenerating a critical
size mandibular defect in a goat model using an axially vascularized synthetic
bone substitute.
Confirming the feasibility will help upgrading the model into a clinically
relevant size to investigate its efficiency in irradiated mandibular defects where
extrinsic vascularization is not efficient.
17
Materials and Methods
Study design
This study represents an experimental pilot study introducing the concept
of axial vascularization of bone substitutes to regenerate a critical size
mandibular defect in a large animal model (goat). In this study we used the local
vascular axes (facial vessels) to create an arterio-venous loop in order to
vascularize a biphasic ceramic (HA-ßTCP) scaffold. The scaffold was charged
with platelet rich plasma and bone morphogenic proteins (BMP) in order to
augment osteogenesis.
Charging of the scaffolds, the animal surgery, and the postoperative care
was performed in the Tissue Engineering Laboratories, University of Alexandria,
Egypt. The Radiological study was performed in the Faculty of Medicine,
University of Alexandria, Egypt. The Biomechanical study was performed in the
City for Scientific Research, Borg El-Arab, Egypt. The histological analysis was
performed in the Tissue Engineering laboratories, Department of Plastic and
Hand surgery, University of Erlangen-Nürnberg, Germany.
Scaffold preparation
Scaffold material
The scaffold was composed of 60% Hydroxyapatite (HA) and 40% ß Tri-
calcium phosphate (ßTCP), has 75% porosity, with an average pore size of 150
micron, and compressive strength of 3.83MPa (BioGraft Dental Bone Granules
and Blocks, India).
The scaffold was manufactured as a 3x2x1 cm cube with 2 holes (1.5 mm
in diameter each) traversing the scaffold to allow mounting the scaffold to the
titanium plate. The scaffold had a groove on one lateral surface to accommodate
the anastomosed loop (Figure 1). The scaffold was mounted to a titanium
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miniplate (L- Plate 10 holes 2.0 mm, G.P.C. Medical Ltd., India) using a
stainless steel wire.
PRP preparation
Under aseptic conditions, 10 ml of the goat’s blood was collected from
the right internal jugular vein shortly before induction of anaesthesia. The blood
was dispersed in a 15 ml falcon tube containing Sodium citrate 3.2 % as
anticoagulant in a proportion of 1 Na citrate to 9 Blood (1:9). The blood was
centrifuged at 200 g for 30 minutes with the lowest acceleration & lowest brake
(Eppendorf Centrifuge 5810R, Germany). This was called the first spin. Three
phases resulted from the first spin namely from top to bottom: plasma, platelets-
leucocytes, & RBCs. The RBCs were discarded and the supernatant was re-
centrifuged (2nd spin) at 2000 g for 5 minutes to pellet the platelets. The upper
half of the supernatant resulting from the 2nd spin (now considered the platelet
poor plasma - PPP) is collected and re-centrifuged to pellet any remaining
platelets which were then added to the first pellet to form the platelet rich plasma
(PRP). The 10ml of blood yielded 2.5 ml PRP. The rest of the PPP was
discarded. The concentration of the platelets was not measured and we relied on
a pre-estimated number of platelets in the sheep's blood (�350,000/µl). Counting
under the light microscope was done only in preliminary experiments in order to
fix and standardize the protocol.
Charging the scaffold with PRP & BMP2
1. Preparation of diluted Fibrinogen and mixing with PRP:
Fibrin (TISSUCOL®-Kit 5.0 Immuno, Baxter, Germany) was used as a slow
release system for charging the scaffold with BMP. 5ml of Aprotinin
solution was added to the powdered fibrinogen and mixed for 3 minutes. The
mixture was diluted by adding 17.5 ml of Fibrinogen diluting solution
(TISSUCOL®-dilution buffer). 3.1ml of the diluted fibrinogen solution was
added to 2.5 ml of autogenous fresh prepared PRP to form the mixture A.
2. Preparation of Thrombin and mixing with BMP2.
5ml of Cacl2 solution was added to the powdered thrombin and mixed in a
warm water bath for about 10 minutes. 0.123 ml of the thrombin solution
19
was added to 0.3 ml of the recombinant human BMP2 solution (InductOs®,
Wyeth USA) to form the mixture B.
3. Final mixing within the scaffolds:
Both A and B solutions are added onto the scaffold using a totally aseptic
technique. The mixture is forced into the scaffold through a vacuum
technique using a 50 ml tummy syringe. This technique allows even
distribution and solidification of the fibrin and BMP2 within the scaffold
material. A plastic tube was left inside the groove during preparation to
avoid filling the groove with the charging materials during vacuum charging.
The scaffold is then transferred to the operating room to be implanted in the
mandible.
Animal surgery The animal used in this study is an adult 3-year-old male goat (Genus:
Capra, Species: C. aegagrus, Subspecies: C. a. hircus, Breed: Egyptian Barki).
All the animal care and operative procedures were done according to the NIH
guidelines for animal surgery and were approved by the ethics committee of the
University of Alexandria and the local governmental authorities (84).
Premedication and Anaesthesia
Food was withdrawn from the animal 24 hours before the operation and
the water was withdrawn 2 hours before the operation.
Premedication
1. Xylaxzine HCl 20mg/ml (Xyla-ject) 1 ml IM.
2. Atropine sulphate 10mg/ml: 1ml IM.
3. Thiopental Na 50mg/ml: 2ml IV (on endotracheal intubation)
Maintenance
Inhalational anesthesia using O2 and Isoflorane with mechanical
vetillation (Penlon Sigma Delta sevoflurane vaporizer, Penlon Nuffield 200
Ventilator, USA).
20
Fluids
1500-2000ml IV fluids were introduced throughout the operation (0.9%
saline, Glucose 5%).
Antibiotics
Pen&Strep (Benzylpenicellin 200 mg/ml + dihydrostreptomycin sulphate
250mg/ml) in a dose of 0.04 ml/Kg. First dose was given on induction of
anaesthesia.
Operative procedures
The animal was laid in a right lateral decubitus on the operating table.
The left mandibular and submandibular regions were disinfected with Povidine
Iodine solution (Betadine®). Sterile draping of the surgical field was performed.
A 10 cm long skin incision was made in the left submandibular region opening
the skin and subcutaneous tissues. Dissection was continued using bipolar
diathermy with creation of subcutaneous flaps till reaching the masseter muscle.
The facial vessels were identified and retracted under surgical loup
magnification. The masseter muscle was detached from the mandible using
bipolar diathermy. The rest of tissues and periosteum were removed from the
angle of the mandible using periosteal elevator. Using the Oscillating Saw (5400-
031 Stryker TPS, USA) a 3 x 2 cm full-thickness marginal defect was created at
the angle of the mandible as previously designed (29). Continuous irrigation with
normal saline 0.9% was done throughout the sawing procedure. The bone
segment was dissected from the underlying medial pterygoid muscle using
bipolar diathermy. Haemostasis was performed using bipolar diathermy.
The BMP2/PRP charged scaffold being already mounted to the titanium
miniplate, was fixed to the mandible using three cortical screws (2.0 mm width,
length 6mm) (Figure 2). The facial artery and vein were further skeletonised and
drawn into the groove. The microanastomsis was done under the surgical
microscope (SHIN NIPPON OP-2, Japan) using 9/0 prolene sutures. The AV
loop was further secured in place by application of 2 strips of Type I bovine
collagen 5x1cm each (Wyeth, USA) (Figure 3).
21
The Masseter muscle was sutured back to the deep fascia of the lower flap
using Vicryl 3/0 continuous sutures except in the place of entry of the facial
vessels where it was sutured to the platysma muscle. Closure of the skin was
done through simple sutures using Prolene-0 sutures without drain. The wound
was then sprayed with a local antibiotic spray (oxytetracycline 2.5mg +crystal
violet 190mg/100ml) and covered with betadine-impregnated gauze sutured to
the wound margins with prolene 0 sutures. The whole procedures took about 5
hours with an approximate blood loss of about 100 ml.
Postoperative care
Analgin; metamizole Na 0.5 g/ml, a dose of 1ml was given daily for 10
days for analgesia. The same antibiotic used on induction of anaesthesia was
continued for 10 days postoperative. Only water was allowed on 1st day
postoperative. Semisolid diet was allowed from the 2nd day to the end of the first
week. Normal solid diet was allowed after 1 week postoperative. Sutures were
removed after 3 weeks. The wound was sprayed twice per week during the first
postoperative month using the antibiotic spray. Patency of the AV loop was
monitored using a hand-held Doppler throughout the first postoperative week.
The animal was monitored through the 6-month follow up period for the general
health, wound condition, and mandibular performance.
Animal Harvest and Radiological evaluation
10 minutes before sacrificing the animal, 5000 IU Heparin was injected
direct intravenously in the right jugular vein. The animal was slaughtered and
decapitated allowing blood to flow profusely from the neck vessels. After
decapitation, the whole vascular system of the head was flushed with warm
(40°C) Ringer-heparin (100 IU/ml) solution injected into both carotids till clear
fluid output was reached from the jugular veins. At least 500 ml Ringer-Heparin
was used for each side. The head was then sent fresh to perform the CTA
(Computerised Tomographic Angiography).
22
The CTA was performed using a 16 multi-slice CT scanner (PHILIPS
MX 16 slice, Philips Medical Systems; Netherlands) with automatic contrast
injection under the following settings: 120kV peak voltage, 2.400 mA tube
charge, 1.5mm slices thickness with 0.75mm incrimination. Metal artefact
reduction option was applied. An 18-gauge cannula was fixed to the left carotid
artery of the goat head and connected to the injector. 60 ml contrast injection
(Ultravist-300, Bayer; Germany) in a dilution of 1:1 with Ringer’s solution was
used at a rate of 3ml/seconds with 8 seconds acquisition delay. The contrast was
left to flow freely out of the veins into a special container mounted at a level
below the goat’s head in order not to induce artefacts. The head was scanned first
as plain CT then scanning under contrast injection was performed. A flush dose
of Ringer’s solution was injected thereafter to clear the vascular system of the
rest of the contrast. The results were saved as DICOM files and further
processed, analysed and 3D reconstructed using OsiriX v.4.0 32-bit program for
Apple Macintosh.
Preparation and Explantation of the mandible
Before excision of the mandible, the vascular system of the head was
injected with India ink to facilitate vessel identification later on through
histological evaluation (46). 48 g Mannitol 4% (D-Mannit, Carl Roth GmbH &
Co.KG) was mixed with 60g gelatine (Carl Roth GmbH & Co.KG) and 75ml
Ringer’s solution in a warm water bath (45°C). 75ml India ink was added to the
previous mixture and the whole mixture was kept warm at 45 degrees Celsius.
The vascular system of the goat’s head was once again washed by injecting about
200ml of warm Ringer’s solution in the carotid vessels of both sides. 150ml of
the India ink mixture was then injected in the left carotid artery of the goat’s
head under manual pressure. The head was kept for at least one hour after
injection in 4 degrees Celsius in order to let the mixture solidify inside the
vessels. The mandible was then sharply dissected from the head and the soft
tissues were removed in order to perform the biomechanical study. The titanium
screws were removed and the stainless steel wire was cut in order to remove any
23
artificial connection between the titanium plate and the mandible. The titanium
plate could be rocked within the regenerated bone.
Biomechanical evaluation
The Equipment used is the 3 points bending apparatus; Autograph AG-IS
100 KN, SHIMADZU. In room temperature, the apparatus was adjusted so that
the 2 resting points where 4 cm apart. The mandible was placed horizontally on
the 2 resting points so that the medial side is facing upwards (towards the
pressing blade) and in a tilt so that the pressing blade will apply the load on a line
overlying the anterior boundary of the proposed defect (29). The rate of
application was adjusted to 1 mm/minute (Figure 4).
The study was aborted immediately after reaching the break point to avoid
destroying the specimen, which will be further studied histologically. The result
was plotted in the form of a graph where the Force (N) is plotted against the
stroke (mm).
Histological evaluation
After performing the biomechanical testing, the specimen was cut to
include the scaffold and a 5mm margin of the native mandibular bone all around.
The specimen was put in formaldehyde 4% solution for 24 hours. The specimen
was then decalcified along 8 weeks by impregnation in
Ethylenediaminetetraacetic acid (EDTA) solution with continuous shaking. After
adequate decalcification, the specimen was sharply cut so that 5 regions of
interest would be examined (Figure 5). The specimens were dehydrated by
passing them through increasing strength of alcohol. The specimens were then
cleared with Xylol and embedded in paraffin. Sectioning was performed using
the microtome (Microm HM 355 S, Thermo Fisher scientific Inc., PA, USA) and
the slides were cut 3 µ thick.
The sections were then stained with Haematoxylin & Eosin stain and with elastic
van Gieson stain (EVG) and examined under light microscope (Olympus IX-2,
24
Tokyo, Japan) for vascularity and new bone formation. Staining with elastic van
Gieson stain was used to be able to adequately differentiate between the
remnants of the scaffold material and the newly formed collagen (53).
25
Figure 1: Grooved scaffold, the red arrow shows entry of the artery, the
blue arrow shows entry of vein, the black arrow points to the site of anastomosis.
Figure 2: Scaffold fixed to the mandible, vessels anastomosed inside the groove.
26
Figure 3: vessels covered by collagen occluding the groove.
Figure 4: 3 points- bending mechanical loading.
27
Figure 5: Schematic illustration of the scaffold; the dotted blue line shows the
level of cutting off the specimen, dotted red lines show the regions of interest to
be studied, dotted white line shows the place of the groove (AV loop).
28
Results
Long term follow up
General health of the animal
The animal tolerated the surgical procedure. The goat returned to full
activity after 24 hours of the operation. Throughout the 6 months follow up
period, no considerble changes in body weight or activity was noticed.
Wound status
The left mandibular region showed slight swelling early postoperatively
with no tenderness, overt signs of infection or dehiscence. The swelling was
conservatively managed and resolved completely within one month.
Mandibular performance
The animal tolerated normal diet after one week of the operations. No
deviations or defective chewing was noticed.
Radiological
The CT images showed the detailed anatomy of the head and neck region
of the goat with minimal artefacts due to the metal parts. The CTA imaging
technique has adequately showed the vascular pattern of the head and neck
region of the goat. Differences in tissue perfusion were evident pre and post
injection. The scaffold was found in place. No evidence of tilt or break within the
mandible, the plate or the scaffold was noticed. The scaffold preserved its overall
3D shape. The scaffold could still be identified from the surrounding bone
through its anatomical site and the difference in the Hounsfield unit where the
scaffold material ranged from 946 HU to 1174 HU while the surrounding
calcified bone ranged from 1214 HU to 1391 HU. The new calcified bone
formation was evident more at the upper, anterior, medial and lateral aspects of
29
the defect and was continuous with the mandibular bone density (Figure 6). The
new bone was crossing the mid zone of the scaffold and crossing over the
titanium plate. Contrast enhanced areas of cavitation within the newly formed
bone were detected and were continuous with marrow cavity of the mandible.
(Figure 7)
3D reconstruction allowed measuring the volume of the newly formed bone. It
measured 2.0993 cm3 representing about 80.15% of the volume of the resected
segment (2.6192 cm3). The defect area covered by new bone was 2.928 cm2
representing 51.8% of the original defect area (5.648 cm2). The newly
regenerated tissue volume showed enhancement with IV contrast injection where
the average HU increased from 832.03±481.17 HU to 858.34±441.04 HU. The
venous end of the loop could be identified on contrast injection as it came out of
the groove of the scaffold (Figure 8). However, neither the artery nor the AV
loop itself could be identified as a definite contrast enhanced structure within the
scaffold groove.
Macroscopic and Biomechanical
The naked eye examination after explantation confirmed the CT findings.
New bone growth was evident creeping from the defect edges and crossing the
mid-zone of the scaffold. The bone was covering parts of the titanium plate and
screws. The new bone seemed mature with evident ridges on its surface. Small
India ink-stained vessels were seen sprouting from of the groove where the AV
loop lied. The sprouting vessels were more evident on the venous side of the loop
(Figure 9). The outer most parts of the scaffold far from the defect margins were
still preserving their shape and structure.
The Biomechanical three points bending test showed that the mandible
broke at a force of 1662.19 Newton (Figure 10). The exact line of break could
not be identified as the test was immediately aborted after reaching the maximum
point to prevent disturbing the specimen before histological examination.
30
Histological
Histological examination confirmed mature bone formation along the
medial, lateral, upper and anterior parts of the scaffold, previously detected by
CT and naked eye examination. These areas revealed lamellar bone formation
with osteoblasts, osteocytes and osteoclasts confirming an on-going remodelling
procedure (Figure 11). Marrow-like spaces were also detected in these regions
filled with loose connective tissue. The newly formed bone was highly
vascularized as detected by the India ink filled vessels (Figure 12). In the central
parts of the scaffold immature woven bone formation was detected with collagen
deposited around the scaffold material by the osteoblasts. Remnants of the
scaffold material were still clearly detected. The scaffold remnants could be
identified clearly from the new collagen by the elastic van Gieson stain where the
scaffold appeared brick red in contrast to the bright pink collagen (Figure 13).
India ink-filled vessels were detected in the central parts of the scaffold about
5mm away from the outer surfaces of the scaffold (Figure 14).
31
Figure 6: CT image showing the integrated scaffold to the mandible
Figure 7: CT image showing integration between the marrow spaces and the
scaffold spaces.
32
Figure 8 3D reconstruction image of the goat head (view from inferior). The blue
arrow shows the contract enhanced facial vein. The upper image is before
contrast injection.
33
Figure 9: The explanted mandible, red arrow shows the sprouting vessels from
the venous side of the groove.
Figure 10: Biomechanical results showing break point.
34
Figure 11: H&E section showing mature bone, V: India ink filled vessel, N:
Mature new bone
Figure 12: EVG section of the scaffold edge, M: adjacent medial pterygoid
muscle, B: mature bone with osteoclasts. V: India ink filled vessel
35
Figure 13: EVG stained section showing the differentiation between scaffold
material (brick red) and collagen (pink).
Figure 14: H&E stained section showing India ink filled vessels at the central
part of the scaffold.
36
Discussion
We present a novel model for scaffold guided mandibular regeneration
with axial vascularization using the AV loop. In our design of this new model,
we tried to mimic as much as possible the clinical scenarios associated with head
and neck cancer ablation. In such a clinical scenario the vascular bed is not
optimal for extrinsic random-type regeneration due to extensive tissue loss and
radiotherapy. This experimental model was previously characterized through
anatomical, mechanical, and pilot surgical studies (29).
The defect design preserved the mandibular continuity and did not breach
the oral mucosa in order not to add to the complexity of the procedure, which
involves micro-anastomosis inside the construct. The animal tolerated the
relatively long operation and the technically demanding procedure. The animal
rapidly returned to normal diet and the observations through the long follow up
duration did not reveal any significant added morbidity to the animal. Though
simple, the defect was critical size (88), and significantly affected the mechanical
properties of the mandible (29). The defect could be considered practically as a
one-wall defect, which makes bone regeneration through osteoconduction from
the native mandible in this context a real challenge.
We used the facial vessels as a local vascular axis to create the AV loop.
In a clinical setting of cancer ablation and post-resection irradiation, local vessel
availability and reliability in creating an AV loop could be questionable.
Regarding the availability of the vessel per se, it is practically feasible to
preserve a medium-sized vascular axis in the region of cancer ablation. This is
done routinely in conventional surgical practice when preparing the recipient
vessels for free flaps. Being medium sized, the facial vessels can be used even
after exposure to irradiation as recipient vessels for free flaps (91) and thus can
be technically suitable to axially vascularize a bone substitute. However, the
problem would be in finding a vascular axis with an adequate length and arc of
rotation to be able to axially vascularize a bone construct. This point could be
overcome by using an interposition vein graft between the artery and the vein
used to create the AV loop (72). Regarding the local vessel reliability for
37
induction of adequate angiogenesis in a previously irradiated field, there is no
solid data throughout the literature quantifying the effect of radiotherapy on
neoangiogenesis in the AV loop model. This point needs further investigations to
detect the effect of dose, timing, and fractionation of irradiation on the AV loop
model. This can affect further standardization of the model to be used either for
primary or for secondary reconstruction after cancer ablation.
We used a scaffold composed of 60 % HA and 40 % ß- TCP. Throughout
the literature and among the calcium phosphate ceramics, the biphasic calcium
phosphates, composed of different concentrations of the stable phase,
hydroxyapatite (HA), and the more soluble phase, usually composed of ß-
tricalcium phosphate (ß-TCP), have presented significant advantages due to their
controlled bioactivity and balance between resorption/solubilization, which
guarantees the stability of the biomaterial while promoting bone ingrowth. It has
been shown that hydroxyapatite crystals are very slowly degradable, with
resorption rate of only 5-15 % per year (31, 87). This could explain the
preservation of the main 3D shape of our scaffold after 6 months. Depending
upon the concentration of the more stable and soluble phases, it is possible to
obtain a ceramic that can be applied to large bone defects, in load-bearing areas,
and as customized pieces which will maintain their shape over long periods of
time (18, 19, 21, 51).
The results showed that bone formation was more prominent at the
peripheral parts of the scaffold than the central parts. This could be explained by
the more extensive vascularity noticed at the outer zones of the scaffold than the
central parts. It could be explained also by the relatively limited
osteoconductivity of the scaffold due to the small pore size where the average
pore size of our scaffold was about 150 µ. The porosity and pore size are
important parameters for the possibility of tissue ingrowth into the substitute
material and to what extent the material is osseointegrated or transformed into
vital bone tissue. Size, interspace and connection of pores (interconnecting or
blind pores) determine nutrient diffusion as well as cell migration and adhesion.
Normal cortical bone has a pore size of 1–100 µ, while cancellous bone has a
pore size of 200–400 µ. A pore size of 100–500 µ is regarded as the ideal
38
precondition for the ingrowth of surrounding bone tissue into the implanted
material, and pores smaller than 100 µ can lead to fibrovascular encapsulation of
the implant (48). However, the more the porosity, the less the mechanical
stability of the scaffold, which is an important factor in load bearing areas such
as the mandible. Pore size also influences vessel ingrowth into the scaffolds,
which was most pronounced in the scaffolds with the largest pores (23, 45, 59).
Equilibrium between increasing the porosity and pore size, on one hand, and
maintaining acceptable compressive strength for mandibular reconstruction, on
the other hand, was crucial for the successful regeneration in our model.
We charged the scaffold with BMP2 and PRP in fibrin glue. Because we
used no cells, the use of BMP in our model was mandatory in order to get
significant bone formation. Previous studies indicate that, for most applications,
the union rates with BMPs are comparable or possibly better than with the use of
autografts, supporting that using BMPs could avoid the need for cell
transplantation. After the first publication on bone induction via growth factors
reported by Urist in 1965 (86), a number of animal models and clinical studies on
bone regeneration with BMPs have been carried out. Since 2002, rhBMP-2 and
rhBMP-7 have been available as therapeutics for use in humans and are now in
clinical use in orthopedics and spine surgery for nearly a decade. BMPs support
proliferation and differentiation of mesenchymal cells into chondroblasts and
osteoblasts, production and maturation of cartilage and bone matrix, and
differentiation of circulating osteoclast precursor cells into osteoclasts. This
effect, especially osteoclast differentiation, was evident in our histological
sections of the newly formed bone. Raida et al. (75) have also proved that BMP-
2 promotes vascularization.
Other than high cost, one concern regarding the use of BMP in the
clinical setting is the danger of heterotopic bone formation. In our model there
was slight new bone formation crossing over the titanium blade and screws. It
was reported that BMP when used to induce or augment spinal arthrodesis,
heterotopic bone formation could potentially result in compression of the thecal
sac or exiting nerve roots, calcification of the spinal cord or nerve roots, or
unintended fusion of adjacent spinal segments (60).
39
While overzealous bone formation constitutes a localized complication of
BMP use, antibody formation against implanted BMP may represent a potential
systemic complication (58). Although no adverse effects have yet been reported
with antibody formation to either rhBMP-2 or rhBMP-7, such antibody
formation may be worrisome. At a minimum, antibody formation may limit
future treatments with the same BMP subtype in patients in whom antibodies are
detected. Subsequent exposure to the same antigen may induce a significant
immune response and thus routine postoperative serological evaluation may be
indicated. Although these potential complications may be regarded as
‘‘theoretical’’, the clinician should be aware of them in a clinical setting. Also,
the patient should be educated of these possibilities in the process of obtaining
informed consent.
Another theoretical disadvantage of BMPs is the simultaneous induction
of osteoblasts and osteoclasts. This means that a potentially contrary
development to the main target is also initiated. This negative effect can be partly
counteracted by combining the BMPs with PRP. Cenni et al. (11) have proven
the inhibition of osteoclast activation using PRP. Park et al. (68) further
supported the combination of both and presented an in vitro study using BMP-2
and PRP pointing out that PRP with suboptimal doses of BMP-2 improved bone
formation and enhanced bone density. The combination of PRP and 1.2 mg
rhBMP-2 (1.5 mg/mL) with osteoinductive scaffolds in clinical case reports has
also shown excellent results (80, 81).
Platelet-rich plasma is defined as a portion of the plasma fraction of
blood having a platelet concentration above baseline (56). Supporters of platelet-
rich plasma technology suggest that the benefits include an increase in hard and
soft tissue wound healing and a decrease in postoperative infection, pain, and
blood loss (26). Although there have been numerous publications on the use of
platelet-rich plasma for several clinical applications, including maxillofacial
surgery (90), there is still controversial discussion regarding the use of PRP and
whether or not it favors bone regeneration (34, 66, 70). Hu et al. (43) highlighted
the enhancement of not only osteogenesis, but also angiogenesis. They
concluded that PRP possibly started the process of angiogenesis, recruiting the
40
endothelial cells lining the blood vessels and beginning the initiation of bone
regeneration.
Using Multislice CT technology, we were able to calculate the calcified
regenerated bone volume and area. It was concluded from previous studies that
the conventional CT is not efficient in differentiating the scaffold from the newly
formed bone (67). Those studies investigated a scaffold completely formed of
ßTCP that rendered a radiodensity of 445-1142 HU due to its low calcium
content in comparison to HA-containing scaffolds. Furthermore, the CT studies
were performed only after 12 weeks where complete ossification of the newly
formed bone is unlikely. The metal parts in our model did not cause significant
artifact effect. The cause of this was the adjustment of the CT settings during
image acquisition. The use of a high peak voltage, high tube charge, and thin
sections helps reduce metal-related artifacts (50).
The newly formed bone volume represented about 80.15% of the volume
of the resected segment. However 51.8% of the defect area was covered by new
bone. This was logic as the maximum regions of bone regeneration were those
near to the defect margins representing the thick part of the mandible, while the
non-regenerated distal regions (away from the defect margins) represent only the
thin non-bulky region of the normal goat mandible. With such a challenging
defect design, with no cells used, and with this dose of BMP2, similar results
were not reported previously in literature regarding mandibular regeneration of
large animal models. Previous studies used cells (93), bone marrow extract (67),
or extensive doses of BMPs (1, 4).
Through our novel protocol for post-mortem CTA in goats, we were able
to adequately show the vascular pattern of the head and neck region of the goat.
The newly regenerated tissue volume showed enhancement with IV contrast
injection. Although it is not a precise objective method for quantitative
assessment of vascularization, it gives a crude idea about the vascularity of the
region, as the change in attenuation after contrast injection (measured by HU
units) is directly proportional to the concentration of the contrast material. This is
actually the basic idea for the protocols and software packages currently
41
available for the perfusion CT technology (61). This vascularization was also
confirmed in our model through the histological sections.
The venous end of the loop could be identified on contrast injection as it
came out of the groove of the scaffold. However, the artery could not be
identified as a definite contrast enhanced structure entering the scaffold groove.
This could be attributed to technical adjustments regarding the acquisition delay
time. Another possibility would be the eventual blockade of the arterial side of
the loop before or after animal sacrifice with retrograde filling of the venous side
of the loop. The loop itself was not expected to be visualised inside the scaffold
after a 6 months follow up period. The Long-term studies have shown that
although initial shunting has the form of an arteriovenous fistula, arteriovenous
exchange after 6 weeks is partially taken over by the newly formed vascular
network. The pattern of blood flow within the construct resembles that of an
organoid with afferent artery, an efferent vein and a well vascularized
parenchyma in between and the main axis of the loop would be no longer
available (71). A better visualization technique would be the micro CT
technology, with a slice thickness less than 100 µ. The vasculature could be also
directly visualized after injecting a special contrast material (e.g. Microfil). The
micro-CT is however expensive, not suitable for in vivo assays and is not
suitable for large specimens (6, 92).
The Biomechanical three points bending test showed that the mandible
broke at a force of 1662.19 Newton. Our previous studies showed that the
average force needed to break a mandible with a similar defect was 210.63 ±
108.92 N while the average force used to break the normal mandible was 640.2 ±
137.89N. Although the biomechanical 3 point bending test does not mimic the
normal physiological stresses on the goat mandible, which is very difficult to
analyze and simulate (79), it showed that the newly formed bone added to the
mechanical properties of the mandible indicating a well-functioning new bone
formation.
The maturity of the regenerated bone was also confirmed by histological
examination. The presence of osteocytes in lacunae and maturation of the woven
42
into lamellar bone with bone remodeling evidenced by osteoclastic activity are
all signs of a structurally mature bone formation in our model. The histologically
mature bone was more evident at the peripheral parts of the scaffold in contact
with the defect margins, indicating a conductive pattern of bone growth from the
defect margins. The histological sections revealed adequate vascularization of
most of the scaffold regions. The vascularization was more evident at the
periphery.
Direct vascular sprouting from the loop was not expected to be clearly
evident after a 6 months follow up period (71), however, India ink filled vessels
deep inside the scaffold could be seen and could never be attributed to extrinsic
vascularization. Further quantitative analysis of the precise role of the AV loop
in vascularizing the scaffold in this model needs to be performed.
To the best of our knowledge, successful trials for mandibular
reconstruction using intrinsic axial vascularization have not been reported yet.
Moreover, the concept of induction of axial vascularization of synthetic tissue
engineering constructs at the very same site of reconstruction without the need
for tissue transfer has also not been introduced.
We are presenting a clinically relevant model for axially vascularized
mandibular regeneration. The model had the advantage of similarity to the
clinical scenarios, technical feasibility, usage of already clinically approved
products, and functionally irrelevant donor site morbidity. Also, the presented
approach does not necessitate sophisticated and, under GMP (Good
Manufacturing Practice) conditions, expensive cell culture techniques that might
pose regulatory problems in clinical practice. Further development of this model
may include segmental resection of the mandible accompanied by radiation
therapy and final assessment of the vascularization potential of the AV loop. We
conclude that a model like the one presented in this thesis may well be
considered a typical preclinical model.
43
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List of Abbreviations AV: Arterio-venous
AVBS: Axially Vascularized Bone
Substitutes
AVL: Arterio-Venous Loop
BMP: Bone Morphogenic Protein
Co.KG: Compagnie
Kommanditgesellschaft
CT: Computerized Tomography
CTA: Computerized Tomography
Angiography
DICOM: Digital Imaging and
Communications in Medicine
EDTA: Ethylene-Diamine-
Tetraacetic Acid
EVG: Elastic Van Gieson
g: Gram
GmbH: Gesellschaft mit
beschränkter Haftung
GMP: Good Manufacturing Practice
GPC: Global Products Corporation
HA: Hydroxyapatite
HU: Hounsfield Unit
IM: Intra Muscular
IU: International Unit
IV: Intra Venous
Kg: Kilo gram
Kv: Kilo volt
Ltd: Limited.
mA: Milli-Ampere
ml: Millilitre
mm: Millimetre
N: Newton
Na: Natrium (Sodium)
NVBG: Non Vascularized Bone
Graft
O2: Oxygen
OP-1: Osteogenic protein-1
PA: Pennsylvania
PPP: Platelet Poor Plasma
PRP: Platelet Rich Plasma
rh: Recombinant Human
USA: United States of America
VBF: Vascularized Bone Flaps
VEGF: Vascular Endothelial
Growth Factor
3D: Three Dimensional
ßTCP: Beta Tri-calcium Phosphate
µ : Micron
µ l: Micro litre
50
List of Pre-publications
• Eweida AM, Nabawi AS, Marei MK, Khalil MR, Elhammady HA.
Mandibular reconstruction using an axially vascularized tissue-
engineered construct. Ann Surg Innov Res. 2011;5:2.
• Eweida AM, Nabawi AS, Elhammady HA, Marei MK, Khalil MR,
Shawky MS, Arkudas A, Beier JP, Unglaub F, Kneser U, Horch RE.
Axially vascularized bone substitutes: a systematic review of literature
and presentation of a novel model. Arch Orthop Trauma Surg. 2012;
Epub ahead of print.
51
Acknowledgment
I would like gratefully to thank Prof. Dr. Raymund E. Horch, who kindly
guided me through my research work, for the great help and support he gave me
to complete this peace of work.
I would like also to thank Prof. Horch’s team, namely Dr. Ulrich Kneser,
for helping me with my research.
I would like to acknowledge the help of Mrs Arnold, Mr. Fleischer, and
Mrs Weigand for their valuable technical assistance and Dr. Mohamed Kaed for
his assistance with CT imaging.
The study was partially funded by a research grant from the University
of Alexandria, Egypt (Alexandria University Research Enhancement Program;
Alex REP, code HLTH-09).