Mandibular Reconstruction With Axially Vascularised Bone Substitutes
Mandibular Reconstruction With Axially Vascularised Bone Substitutes
Mandibular reconstruction is necessary following trauma, tumour resections and extensive
 infections resulting in severe defects of the mandibular arch. For reconstructing large and
 recurrent defects, the vascularized free flaps are currently regarded as the gold standard.
 The use of these flaps, however, presents several major inconveniences. Although regenerative
 medicine in the field of cranio-maxillofacial reconstruction has now become a common
 practice, the main technical challenge is still related to vascularization of the regenerated
 tissue in large defects. Axial vascularization of constructs using a microvascular
 arteriovenous fistula/loop (AV loop) aims at providing the construct with blood supply
 through a defined and dedicated vascular axis. This technique was successfully demonstrated
 in some case reports, but was never applied in the craniofacial region. The current study
 aims to apply and assess the technique of axial vascularization using the AV loop of a bone
 substitute to reconstruct mandibular defects.
Background:

 Mandibular reconstruction is necessary following tumour resections, infections or trauma
 resulting in severe defects of mandibular arch continuity and sacrifice of teeth. Basic
 reconstruction involves the use of non-vascularized bone grafts together with restoration of
 lost teeth by means of dental implants and implant-supported prostheses. Smaller bony defects
 (<6 cm) are commonly treated with nonvascularized corticocancellous grafts harvested from the
 anterior or posterior iliac crest. (Goh et al. 2008) For reconstructing larger and recurrent
 defects, currently the vascularized free flaps are regarded as the "gold standard". The use
 of these flaps, however, presents several major inconveniences. Harvesting of autologous
 tissue may result in a significant donor site morbidity, the extent of which may vary,
 according to the donor site and possibly according to the intervention technique. The
 problems include bleeding, pain, infections, donor site fractures and prolonged hospital
 stay. (Hartman et al. 2002; Rogers et al. 2003) The field of Regenerative Medicine promises
 new alternatives to surgical reconstruction through harnessing the regenerative capacity of
 the human body to repair itself. In the last few decades the rapid expansion of knowledge
 about the biological basis of wound healing and the role of cells, signals, and biological
 scaffolds has drawn the attention from ''tissue reconstruction'' to ''tissue regeneration''.
 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".

 Tissue engineering and Regenerative medicine depend on the presence of a biomaterial
 promoting cell growth and proliferation. In order to populate this biomaterial (scaffold)
 with new tissue, the body must effectively interact with this biomaterial. This necessitates
 the establishment of an early and reliable angiogenic response leading to the development of
 an adequate blood supply for the restoration of structure and function. (Hodde 2002) The
 three main components required for regeneration are the cells, scaffolds, and induction
 molecules. When growing tissues in vitro (Tissue Engineering), all three components should
 exist, however, when referring to Regenerative Medicine, any of these can be provided to the
 body in an attempt to optimize its capacity for regenerating its own tissues. Adding cells or
 growth factors to the biomaterials can reinforce tissue regeneration (Pellegrini et al.
 2009), but the vascularization, and thus the integration, of theses biomaterials is still
 considered the determining issue in the success of any critical size defect regeneration.
 (Novosel et al. 2011) Applying principles of regenerative medicine in the field of
 cranio-maxillofacial reconstruction has now become a daily practice. The wide spectrum of
 applications ranges from simple addition of bioactive bone fillers to much more sophisticated
 techniques for bone replacement and reconstruction. The indications included reconstruction
 after minor developmental defects, trauma, infections, benign cysts or tumours but seldom
 following malignant tumour excision. (Clokie and Sandor 2008; Schuckert et al. 2009;
 Trautvetter et al. 2011) Warnke et al (Warnke et al. 2004), who used a completely different
 technique than those used in the previous case reports, reported the only case of
 regeneration after cancer ablation. The main technical difference was related to
 vascularization of the regenerated tissue. While all the reported cases for mandibular
 regeneration used the conventional extrinsic vascularization strategy, where the constructs
 were left to acquire a parasitic blood supply form the recipient site of implantation, Warnke
 et al (Warnke et al. 2004) used an axial vascularization strategy through a prelamination
 procedure in the Latissimus dorsi (LD) muscle followed by free tissue transfer of the
 regenerated mandible. Although this technique avoided bony donor site morbidities, the need
 to harvest the LD muscle represented a major drawback of this prelamination technique. This
 single case report highlighted the need for an efficiently vascularized construct if the
 regenerative therapy is to be applied for larger and recurrent defects.

 Axial vascularization of scaffolds aims at providing the construct with blood supply through
 a defined and dedicated vascular axis. In this context, the blood supply of the construct is
 not randomly acquired from the implantation site, and thus the implantation in an area of low
 vascularization potential, as in irradiated or fibrosed surgical sites could be possible
 (Kneser et al. 2006). The two major techniques for axial vascularization are prelamination
 and prefabrication.

 Prefabrication of a tissue construct is simply done by implanting an arterio-venous fistula
 or loop (AVL) or a vascular pedicle underneath or within the construct. This results in
 spontaneous sprouting of vessels from the loop or pedicle and subsequent revascularization of
 the whole tissue construct (Erol and Spira 1979; Guo and Pribaz 2009). Prelamination is
 another technique introduced by Pribaz and Fine (Pribaz and Fine 1994) in 1994 where the
 implantation of a construct into a vascularized territory (flap) is performed to create a
 customized vascularized unit. The end result of both techniques is an axially vascularized
 unit that depends for its nourishment upon a defined vascular axis.

 Two more important terms to mention in this context are ''intrinsic'' and ''extrinsic''
 vascularization modes. The extrinsic vascularization of a construct denotes acquiring its
 blood supply from the periphery towards the centre, while the intrinsic vascularization mode
 denotes that the core region of the construct is being vascularized first (Lokmic and
 Mitchell 2008). Accordingly, prefabrication is considered an intrinsic axial vascularization
 strategy. The construct in prelamination, however, is extrinsically vascularized within an
 intrinsically vascularized territory (Eweida et al. 2012).

 As the reconstruction of challenging or irradiated bone defects requires an axially
 vascularized tissue bulk, applying the prelamination strategy will always end up in a
 remarkable donor site morbidity where the whole vascularized territory (mostly a muscle flap)
 should be transferred to the recipient site (Mesimaki et al. 2009; Warnke et al. 2004). The
 prefabrication technique, however, when applied to a tissue construct, entails only the
 transfer of this construct with its pedicle, thus diminishing the donor site morbidity to the
 minimal. Moreover, the prefabrication technique could be applied at the recipient site as a
 primary reconstruction technique, abolishing the donor site morbidity completely (Eweida et
 al. 2014; Horch et al. 2014).

 One of the most extensively investigated techniques to induce axial vascularization in the
 tissue constructs is the arterio-venous loop or fistula (Arkudas et al. 2013; Horch et al.
 2012) and its superiority over the vascular bundle in terms of vascular density and tissue
 regeneration potential was clearly demonstrated (Tanaka et al. 2003).

 Preclinical data:

 The first documented idea for axial vascularization using the AV loop was described by Erol
 and Spira (Erol and Spira 1979) in 1979 in a rat model. Morrison et al further developed the
 model and inserted the loop into isolation chambers (Hofer et al. 2003). They successfully
 demonstrated the induction of vascularization in polymer and gel matrices (Cassell et al.
 2001). Since 2006, the design and characterization of the isolation chambers and the inset of
 the AV loop were further developed by the work of Horch et al (Kneser et al. 2006) where the
 engineering of vascularized transplantable bone was first successfully demonstrated by this
 research group. After successful evaluation of axial vascularisation in different bone
 substitutes, the concept was translated from the rat model to a large animal model (sheep)
 (Beier et al. 2011; Boos et al. 2012). Based on that model, axial vascularization was induced
 in constructs made of processed bovine cancellous bone substitutes and ß-TCP/HA
 (Tricalcium-phosphate/ hydroxyapatite) scaffolds. The sheep AV loop showed similar
 vascularization patterns like that of the rat but the optimum vascularization density was
 reached after a longer duration (8-12 weeks versus 4 weeks respectively) (Boos et al. 2011).
 Induction of new bone formation in the axially vascularized sheep isolation chamber was then
 demonstrated through implantation of MSC (Mesenchymal Stem Cells) in combination with
 osteogenic growth factor BMP based on a clinically applicable ß-TCP/HA bone substitute (Boos
 et al. 2013).

 In 2011 the investigators introduced the AV Loop model for the first time for mandibular
 reconstruction in goats (Eweida et al. 2012; Eweida et al. 2011). The investigators could
 demonstrate successful regeneration of critical size marginal mandibular defects through
 axial vascularization of ßTCP/HA scaffolds charged with BMP (Bone morphogenic protein). The
 technique was discussed previously in details (Eweida et al. 2014; Eweida et al. 2012; Eweida
 et al. 2011). Briefly, a critical size (3 x 2 cm) marginal defect was created at the angle of
 the goat mandible. An equivalent sized scaffold made of ßTCP/HA was grooved to accommodate
 the AV Loop created by direct anastomosis of locally available vessels under the operative
 microscope. The scaffold was then mounted on a titanium plate and fixed to the mandible.
 Through our comparative studies between the AV Loop and the non-AV loop constructs, the
 axially vascularized constructs showed significantly more central vascularization and
 markedly enhanced central bone formation. The biomechanical characteristics were remarkably
 enhanced as well. The safety and efficacy of the model at a preclinical level was
 successfully demonstrated along a 6 months follow-up period (Eweida et al. 2014).

 Clinical data to date:

 Only two previous studies represented case reports of craniofacial bone regeneration using
 axially vascularized bone substitutes (non-randomised vascularization). The first study was
 published in 2004 by Warnke et al (Warnke et al. 2004) and further evaluated in 2006 (Warnke
 et al. 2006). The authors used a prelamination technique in the LD muscle of bovine mineral
 blocks (BioOss) to reconstruct of a large mandibular defect. The defect was reconstructed 8
 years following subtotal mandibulectomy and irradiation. The construct was charged with BMP
 and autogenous bone marrow aspirate from the iliac crest. Although this prelamination
 strategy for mandibular regeneration has shown some promising initial results, the long-term
 results were not free of complications.

 The second report was published in 2009 by Mesimaki et al (Mesimaki et al. 2009) who reported
 a similar prelamination technique for vascularizing a bone substitute made of ß-TCP (beta
 Tricalcium phosphate) in the rectus abdominis muscle. The vascularized construct was used to
 reconstruct a complex maxillary defect following hemi-maxillectomy due to a large keratocyst.
 An extensive review of literature (up to 06.2019) has shown that the prefabrication using the
 AV loop was never used for bone regeneration within the craniofacial region.

 The introduced technique of axial vascularization of bone substitutes, however, was
 successfully demonstrated in humans. A case report was presented by Horch et al who have
 demonstrated successful bone regeneration and in situ axial vascularization using the AV loop
 model in two patients with bony defects in the radius and the tibia (Horch et al. 2014).
 Horch et al have demonstrated a safe and successful technique, encouraging results, and a
 complication-frees follow up period of 72 months.

 Aim:

 Applying and assessment of the technique of axial vascularization of a bone substitute using
 the arteriovenous loop (AVL) to reconstruct a mandibular defect

 Patients and Methods:

 Ten patients will be included in this prospective study.

 Informed consent:

 The patients will be fully oriented through a documented written informed consent in Arabic
 language. The informed written consent will include the following points:

 -  The procedure is related to research (Application of this technique in the craniofacial
 region)

 -  The purpose of this research.

 -  Alternative methods for mandibular reconstruction.

 -  The technical details of the procedure.

 -  The advantages and disadvantages of the technique.

 -  The risks of the procedure.

 -  The possible peri- and postoperative side effects.

 -  The management plan for the possible side effects.

 -  A statement that participation is voluntary, refusal to participate will involve no
 penalty or loss of benefits to which the patient is otherwise entitled, and the patient
 may discontinue participation at any time without penalty or loss of benefits, to which
 the subject is otherwise entitled

 -  An explanation of whom to contact for answers to questions about the procedure and
 patient's rights, and whom to contact in the event of research-related injury to the
 patient.

 Preoperative assessment:

 The patient will be subject to thorough history taking and full medical examinations.

 Investigations will include:

 -  Routine laboratory work-up (Coagulation profile, fasting blood sugar, serum creatinine,
 blood urea, Complete Blood Count)

 -  Panoramic x-ray view of the mandible

 -  Computerized tomographic angiography (CTA) of the head and neck region.

 -  Three-dimensional printing of the mandible and the defect to facilitate preoperative
 orientation, plate and mesh pre-bending

 Surgical procedures:

 The procedure will be done under general anaesthesia in a supine position. Through a
 submandibular skin incision, the mandible will be exposed. The defect will be refreshed
 through bone curettage. The preformed reconstruction plate will be fixed to the mandible and
 traversing the defect. A preformed U-shaped titanium mesh will be mounted to the mandible and
 fixed with screws. The ipsilateral facial artery and vein (or other available vascular axes
 in case of lack of the facial vascular axis) will be anastomosed using the operative
 microscope via a reversed vein graft to create an AV loop harvested from the forearm. The AV
 loop will be laid within the defect. The titanium mesh will be filled with a mixture of:

 1. Silicated Hydroxylapatite granules (NanoBone® vial 1.2 ml, ARTOSS, Rostock, Germany).

 2. Autogenous bone marrow aspirate from the iliac crest.

 3. Bone morphogenic protein 2 (BMP 2- InductOs®, Medtronic BioPharma B.V., Tolochenaz,
 Switzerland) The submandibular wound closed in layers.

 Follow up and Evaluation:

 Close monitoring of the patient for the vital signs, patency of the AV loop (via hand
 Doppler) and possible perioperative complications. The patient will be discharged on the 3rd
 day postoperative. The patient will be followed up regularly every week for wound healing,
 patency of the AVL and possible postoperative complications. Serial X-ray (panoramic view)
 will be done monthly to monitor the new bone formation. CTA will be performed after 6 months.

 According to the clinical and radiological findings further dental rehabilitation will be
 planned after complete healing of the defect (6-9 months postoperative). Bone biopsies from
 dental rehabilitation (bone drilling for implants) will be studied for bone quality and
 vascularization using the standard H&E staining after decalcification.

 Qualitative and quantitative data from Panoramic X-rays, CTA, and histological analysis will
 be evaluated for:

 1. Bone regeneration and density.

 2. Vascularization of bone substitutes. Results will be tabulated and compared with
 literature.

 Risk analysis:

 After successful demonstration of the safety and efficacy of the ''AV loop vascularization
 technique'' at the preclinical and clinical levels, we believe it is time to start applying
 the technique for mandibular regeneration. This new application, however, will not be without
 challenges. A major concern would be the hemodynamic changes resulting from the AV fistula.
 Although a similar model was never reported in literature, reports of the use of a distal
 arteriovenous fistula provide evidence that a controlled fistula placed in a peripheral
 artery is well-tolerated, providing that the size of the fistula is less than 1 cm and the
 fistula accepts less than 20 percent of the cardiac output (Blaisdell et al. 1966; Eweida et
 al. 2013; Horch et al. 2014; Tukiainen et al. 2006). Regarding bone density and
 mineralization distal to an AV fistula, contradictory results are shown in literature ranging
 from an increase of bone growth (Vanderhoef et al. 1963) to decreased mineralization and
 augmented osteopenia (Muxi et al. 2009). These studies focused on the hemodynamics and its
 impact on bone growth distal to the fistula. The long-term consequences of an intraosseous
 fistula, however, were not adequately studied in literature.

 An early throbbing sensation or hum would be expected due to the initial high flow. The
 long-term studies at the preclinical level, however, have shown that the vascular bed matures
 with time leading to dumping down of the direct arteriovenous jet (Polykandriotis et al.
 2007; Polykandriotis et al. 2009).
Inclusion Criteria:

 -  Patients requiring mandibular reconstruction for further dental rehabilitation

 -  Mandibular defect (marginal/segmental) equals or more than 6 cm in largest dimension

 -  Middle age adult (18-65 years)

 -  Radiologically and pathologically documented tumour free mandibular defect

 Exclusion Criteria:

 -  Extremes of age (<18 or > 65 years)

 -  Associated uncontrolled chronic illness (Diabetes mellitus, Hypertension, Rheumatoid
 arthritis, collagen disease, Chronic obstructive pulmonary disease)

 -  Primary reconstruction of a mandibular defect after tumour excision