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1.  Mandibular reconstruction using an axially vascularized tissue-engineered construct 
Background
Current reconstructive techniques for continuity defects of the mandible include the use of free flaps, bone grafts, and alloplastic materials. New methods of regenerative medicine designed to restore tissues depend mainly on the so-called extrinsic neovascularization, where the neovascular bed originates from the periphery of the construct. This method is not applicable for large defects in irradiated fields.
Methods
We are introducing a new animal model for mandibular reconstruction using intrinsic axial vascularization by the Arterio-Venous (AV) loop. In order to test this model, we made cadaveric, mechanical loading, and surgical pilot studies on adult male goats. The cadaveric study aimed at defining the best vascular axis to be used in creating the AV loop in the mandibular region. Mechanical loading studies (3 points bending test) were done to ensure that the mechanical properties of the mandible were significantly affected by the designed defect, and to put a base line for further mechanical testing after bone regeneration. A pilot surgical study was done to ensure smooth operative and post operative procedures.
Results
The best vascular axis to reconstruct defects in the posterior half of the mandible is the facial artery (average length 32.5 ± 1.9 mm, caliber 2.5 mm), and facial vein (average length 33.3 ± 1.8 mm, caliber 2.6 mm). Defects in the anterior half require an additional venous graft. The defect was shown to be significantly affecting the mechanical properties of the mandible (P value 0.0204). The animal was able to feed on soft diet from the 3rd postoperative day and returned to normal diet within a week. The mandible did not break during the period of follow up (2 months).
Conclusions
Our model introduces the concept of axial vascularization of mandibular constructs. This model can be used to assess bone regeneration for large bony defects in irradiated fields. This is the first study to introduce the concept of axial vascularization using the AV loop for angiogenesis in the mandibular region. Moreover, this is the first study aiming at axial vascularization of synthetic tissue engineering constructs at the site of the defect without any need for tissue transfer (in contrast to what was done previously in prefabricated flaps).
doi:10.1186/1750-1164-5-2
PMCID: PMC3069948  PMID: 21418603
2.  GMP-level adipose stem cells combined with computer-aided manufacturing to reconstruct mandibular ameloblastoma resection defects: Experience with three cases 
Annals of Maxillofacial Surgery  2013;3(2):114-125.
Background:
The current management of large mandibular resection defects involves harvesting of autogenous bone grafts and repeated bending of generic reconstruction plates. However, the major disadvantage of harvesting large autogenous bone grafts is donor site morbidity and the major drawback of repeated reconstruction plate bending is plate fracture and difficulty in reproducing complex facial contours. The aim of this study was to describe reconstruction of three mandibular ameloblastoma resection defects using tissue engineered constructs of beta-tricalcium phosphate (β-TCP) granules, recombinant human bone morphogenetic protein-2 (rhBMP-2), and Good Manufacturing Practice (GMP) level autologous adipose stem cells (ASCs) with progressively increasing usage of computer-aided manufacturing (CAM) technology.
Materials and Methods:
Patients’ three-dimensional (3D) images were used in three consecutive patients to plan and reverse-engineer patient-specific saw guides and reconstruction plates using computer-aided additive manufacturing. Adipose tissue was harvested from the anterior abdominal walls of three patients before resection. ASCs were expanded ex vivo over 3 weeks and seeded onto a β-TCP scaffold with rhBMP-2. Constructs were implanted into patient resection defects together with rapid prototyped reconstruction plates.
Results:
All three cases used one step in situ bone formation without the need for an ectopic bone formation step or vascularized flaps. In two of the three patients, dental implants were placed 10 and 14 months following reconstruction, allowing harvesting of bone cores from the regenerated mandibular defects. Histological examination and in vitro analysis of cell viability and cell surface markers were performed and prosthodontic rehabilitation was completed.
Discussion:
Constructs with ASCs, β-TCP scaffolds, and rhBMP-2 can be used to reconstruct a variety of large mandibular defects, together with rapid prototyped reconstruction hardware which supports placement of dental implants.
doi:10.4103/2231-0746.119216
PMCID: PMC3814659  PMID: 24205470
Adipose-derived stem cells; beta-tricalcium phosphate; bone morphogenetic protein; computer-aided design
3.  Tissue engineering of bone: the reconstructive surgeon's point of view 
Bone defects represent a medical and socioeconomic challenge. Different types of biomaterials are applied for reconstructive indications and receive rising interest. However, autologous bone grafts are still considered as the gold standard for reconstruction of extended bone defects. The generation of bioartificial bone tissues may help to overcome the problems related to donor site morbidity and size limitations. Tissue engineering is, according to its historic definition, an “interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function”. It is based on the understanding of tissue formation and regeneration and aims to rather grow new functional tissues than to build new spare parts. While reconstruction of small to moderate sized bone defects using engineered bone tissues is technically feasible, and some of the currently developed concepts may represent alternatives to autologous bone grafts for certain clinical conditions, the reconstruction of largevolume defects remains challenging. Therefore vascularization concepts gain on interest and the combination of tissue engineering approaches with flap prefabrication techniques may eventually allow application of bone-tissue substitutes grown in vivo with the advantage of minimal donor site morbidity as compared to conventional vascularized bone grafts. The scope of this review is the introduction of basic principles and different components of engineered bioartificial bone tissues with a strong focus on clinical applications in reconstructive surgery. Concepts for the induction of axial vascularization in engineered bone tissues as well as potential clinical applications are discussed in detail.
doi:10.1111/j.1582-4934.2006.tb00287.x
PMCID: PMC3933098  PMID: 16563218
tissue engineering; bone replacement; vascularization; flap prefabrication; microsurgery; AV loop
4.  Anatomic basis for an algorithmic approach for free fibula flap donor side selection in composite oro-mandibular defects 
Introduction:
Head and neck oncological resections may result in composite oro-mandibular defects involving the oral mucosa (lining), mandibular bone and the skin (cover). Reconstructive options for such defects have evolved over a period. Free fibula flap reconstruction is currently accepted the world over as the gold standard for oro-mandibular defect reconstruction. Existing literature provides conflicting views about the use of a particular side and orientation of the fibula flap for achieving the optimal outcome. The purpose of this study is to confirm anatomically the effect of bone, soft tissue and vessel orientation on the ease of doing reconstruction.
Materials and Methods:
This is a cadaveric study. A mandibular model with a defect was used. This was pre plated to maintain continuity. Composite fibula flaps of the same dimension were harvested from both legs of a fresh cadaver. The harvested flaps were used to reconstruct the mandibular defect in different orientations and the best configuration for each reconstructive requirement was assessed.
Results:
Keeping the peroneal surface for plating, that is, facing outwards, four different configurations of the fibula flap are possible for a given mandibular defect. With a posterior vascular pedicle ipsilateral fibula is suitable for skin cover and contralateral for mucosal lining and the reverse for an anteriorly placed pedicle.
Conclusion:
The algorithm based selection of appropriate sided fibula flap facilitates complex mandibular reconstruction by placing the right kind of tissue at the right place and helps in reducing the donor site morbidity by allowing the surgeon to harvest only the required amount of skin.
doi:10.4103/0970-0358.155268
PMCID: PMC4413489  PMID: 25991885
Free fibula flap; ideal side of fibula; mandibular reconstruction
5.  Engineering of pre-vascularized urethral patch with muscle flaps and hypoxia-activated hUCMSCs improves its therapeutic outcome 
Tissue engineering has brought new hopes for urethral reconstruction. However, the absence of pre-vascularization and the subsequent degradation of materials often lead to the failure of in vivo application. In this study, with the assistance of hypoxia-activated human umbilical cord mesenchymal stem cells (hUCMSCs), pedicled muscle flaps were used as materials and pre-incubated in ventral penile subcutaneous cavity of rabbit for 3 weeks to prepare a pre-vascularized urethral construct. We found that small vessels and muscle fibres were scattered in the construct after 3 weeks' pre-incubation. The construct presented a fibrous reticular structure, which was similar to that of the corpus spongiosum under microscope examination. The produced constructs were then used as a patch graft for reconstruction of the defective rabbit urethra (experimental group), natural muscular patch was used as control (control group). Twelve weeks after the reconstructive surgery, urethrography and urethroscope inspections showed wide calibres of the reconstructed urethra in the experimental group. Histopathological studies revealed that fibrous connective tissues and abundant muscle fibres constituted the main body of the patch-grafted urethra. In contrast, in the control group, only adipose tissue was found in the stenosis-reconstructed urethra, replacing the originally grafted muscular tissue. To our knowledge, this is the first report that successfully constructed a pre-vascularized urethral construct by using hypoxia-activated hUCMSC and pedicled muscle flaps. More importantly, the pre-vascularized construct showed a good performance in urethral reconstruction when applied in vivo. The study provided a novel strategy for tissue engineering of pre-vascularized urethral construct for the defective urethra, representing a further advancement in urethral reconstruction.
doi:10.1111/jcmm.12157
PMCID: PMC3955150  PMID: 24460735
hUCMSC; tissue engineering; urethral reconstruction; pedicled muscle flaps
6.  Loss of apical vertebral derotation in adolescent idiopathic scoliosis: 2-year follow-up using multi-planar reconstruction computed tomography 
European Spine Journal  2012;21(6):1111-1120.
Purpose
The objective of this study was to evaluate 2 years post-surgical loss of three-dimensional correction in adolescent idiopathic scoliosis (AIS) patients using multi-planar reconstruction computed tomography (CT).
Methods
Twenty-seven AIS patients treated by segmental pedicle screw (PS) constructs were included in this study. Correction in the axial plane was evaluated using the “relative apical vertebral rotation angle” (rAVR), defined as the difference between the axial rotation angles of the upper instrumented vertebra and the apical vertebra on reconstructed axial CT images. The Cobb angle of the main curve and apical vertebral translation was measured to evaluate the coronal correction. Thoracic kyphosis was also measured for the evaluation of sagittal profile. Measurements were performed before surgery, and 1 week and 2 years after surgery. The relationships between the correction losses and skeletal maturity, and variety of spinal constructs were also evaluated.
Results
The mean preoperative Cobb angle of the major curve was 59.1° ± 11.2° before and 13.0° ± 7.2° immediately after surgery. Two years later, the mean Cobb angle had increased significantly, to 15.5° ± 7.8°, with a mean correction loss of 2.5° ± 1.5° (p < 0.001). The mean preoperative rAVR of 28.5° ± 8.4° was corrected to 15.8° ± 7.8° after surgery. It had increased significantly to 18.5 ± 8.4 by 2 years after surgery, with a mean correction loss of 2.7° ± 1.0° (p < 0.001). The mean correction losses for both the Cobb angle and rAVR were significantly greater in the skeletally immature patients. The significant correlations were recognized between the correction losses and the proportion of multi-axial screws, and the materials of constructs.
Conclusions
Statistically significant loss of correction in the Cobb angle and apical vertebral axial rotation angle (AVR) were recognized 2 years after surgery using PS constructs. The correction losses, especially AVR, were more evident in the skeletally immature patients, and in patients treated with more multi-axial screws and with titanium constructs rather than with stainless constructs.
doi:10.1007/s00586-012-2274-3
PMCID: PMC3366141  PMID: 22438165
Adolescent idiopathic scoliosis; Apical vertebral rotation; Correction loss; Coronal correction
7.  The Nature and Role of Periosteum in Bone and Cartilage Regeneration 
Cells, Tissues, Organs  2011;194(2-4):320-325.
This study was undertaken to determine whether periosteum from different bone sources in a donor results in the same formation of bone and cartilage. In this case, periosteum obtained from the cranium and mandible (examples of tissue supporting intramembranous ossification) and the radius and ilium (examples of tissues supporting endochondral ossification) of individual calves was used to produce tissue-engineered constructs that were implanted in nude mice and then retrieved after 10 and 20 weeks. Specimens were compared in terms of their osteogenic and chondrogenic potential by radiography, histology, and gene expression levels. By 10 weeks of implantation and more so by 20 weeks, constructs with cranial periosteum had developed to the greatest extent, followed in order by ilium, radius, and mandible periosteum. All constructs, particularly with cranial tissue although minimally with mandibular periosteum, had mineralized by 10 weeks on radiography and stained for proteoglycans with safranin-O red (cranial tissue most intensely and mandibular tissue least intensely). Gene expression of type I collagen, type II collagen, runx2, and bone sialoprotein (BSP) was detectable on QRT-PCR for all specimens at 10 and 20 weeks. By 20 weeks, the relative gene levels were: type I collagen, ilium >> radial ≥ cranial ≥ mandibular; type II collagen, radial > ilium > cranial ≥ mandibular; runx2, cranial >>> radial > mandibular ≥ ilium; and BSP, ilium ≥ radial > cranial > mandibular. These data demonstrate that the osteogenic and chondrogenic capacity of the various constructs is not identical and depends on the periosteal source regardless of intramembranous or endochondral ossification. Based on these results, cranial and mandibular periosteal tissues appear to enhance bone formation most and least prominently, respectively. The appropriate periosteal choice for bone and cartilage tissue engineering and regeneration should be a function of its immediate application as well as other factors besides growth rate.
doi:10.1159/000324642
PMCID: PMC3178095  PMID: 21597269
Periosteum; Bone; Cartilage; Regeneration; Tissue engineering
8.  A two-year audit of non-vascularized iliac crest bone graft for mandibular reconstruction: technique, experience and challenges 
Objectives
Non-vascularized iliac crest bone graft (NVIBG) is a known treatment option in mandibular reconstruction following jaw resection, but no documented review of patients treated with NVIBG exists for northern Nigeria. The experience and technique from a Nigerian tertiary hospital may serve as baseline data for comparison and improvement of practice for other institutions.
Materials and Methods
A retrospective review of medical records and patient case files from January 2012 to December 2013 was undertaken. All case files and other medical records of patients who had reconstruction with NVIBG for benign or malignant lesions with immediate or delayed reconstruction were selected for review.
Results
Twenty patients had mandibular reconstruction with NVIBG during the study period. Two patients were excluded because of incomplete medical records. Eighteen patients' (male=14, female=4) records were reviewed. Their ages ranged from 13 to 62 years (mean 26.0±10.6 years). Indications for NVIBG included jaw tumors (n=16; 88.3%), jaw cyst (n=1; 5.6%) and gunshot injury (n=1; 5.6%). Jaw tumors seen were ameloblastoma (n=15; 83.3%) and osteosarcoma (n=1; 5.6%). Treatments done were mandibular resection with condylar resection (n=7; 38.9%), mandibular segmental resection (n=10; 55.6%) and subtotal mandibulectomy (n=1; 5.6%). Patients' postoperative reviews and radiographs revealed good facial profile and continued bone stability up to 1 year following NVIBG.
Conclusion
NVIBGs provide an acceptable alternative to vascularized bone grafts, genetically engineered bone, and distraction osteogenesis for mandibular reconstruction in resource-limited centers.
doi:10.5125/jkaoms.2014.40.6.272
PMCID: PMC4279977  PMID: 25551091
Mandibular resection; Mandibular reconstruction; Non-vascularized iliac crest bone graft
9.  Prefabrication of 3D Cartilage Contructs: Towards a Tissue Engineered Auricle – A Model Tested in Rabbits 
PLoS ONE  2013;8(8):e71667.
The reconstruction of an auricle for congenital deformity or following trauma remains one of the greatest challenges in reconstructive surgery. Tissue-engineered (TE) three-dimensional (3D) cartilage constructs have proven to be a promising option, but problems remain with regard to cell vitality in large cell constructs. The supply of nutrients and oxygen is limited because cultured cartilage is not vascular integrated due to missing perichondrium. The consequence is necrosis and thus a loss of form stability. The micro-surgical implantation of an arteriovenous loop represents a reliable technology for neovascularization, and thus vascular integration, of three-dimensional (3D) cultivated cell constructs. Auricular cartilage biopsies were obtained from 15 rabbits and seeded in 3D scaffolds made from polycaprolactone-based polyurethane in the shape and size of a human auricle. These cartilage cell constructs were implanted subcutaneously into a skin flap (15×8 cm) and neovascularized by means of vascular loops implanted micro-surgically. They were then totally enhanced as 3D tissue and freely re-implanted in-situ through microsurgery. Neovascularization in the prefabricated flap and cultured cartilage construct was analyzed by microangiography. After explantation, the specimens were examined by histological and immunohistochemical methods. Cultivated 3D cartilage cell constructs with implanted vascular pedicle promoted the formation of engineered cartilaginous tissue within the scaffold in vivo. The auricles contained cartilage-specific extracellular matrix (ECM) components, such as GAGs and collagen even in the center oft the constructs. In contrast, in cultivated 3D cartilage cell constructs without vascular pedicle, ECM distribution was only detectable on the surface compared to constructs with vascular pedicle. We demonstrated, that the 3D flaps could be freely transplanted. On a microangiographic level it was evident that all the skin flaps and the implanted cultivated constructs were well neovascularized. The presented method is suggested as a promising alternative towards clinical application of engineered cartilaginous tissue for plastic and reconstructive surgery.
doi:10.1371/journal.pone.0071667
PMCID: PMC3739741  PMID: 23951215
10.  Defining the critical-sized defect in a rat segmental mandibulectomy model 
Importance
Advances in tissue engineering offer potential alternatives to current mandibular reconstructive techniques; however, prior to clinical translation of this technology, a relevant animal model must be used to validate possible interventions.
Objective
This study aims to establish the critical-sized segmental mandibular defect that does not heal spontaneously in the rat mandible.
Design
Prospective study using an animal model.
Setting
Animal laboratory.
Participants
Sprague-Dawley rats.
Interventions
Twenty-nine Sprague-Dawley rats underwent creation of one of four segmental mandibular defects: 0-mm, 1-mm, 3-mm and 5-mm. All mandibular wounds were internally fixated with 1-mm microplates and screws and allowed to heal for 12-weeks.
Main Outcomes and Measures
Mandibles were analyzed with micro-computed tomography (microCT) and bony healing was graded on a semi-quantitative scale.
Results
Seven animals were utilized in each experimental group. No 5-mm segmental defects successfully developed bony union, whereas all 0-mm and 1-mm defects had continuous bony growth across the original defect on micro-CT. Three of the 3-mm defects had bony continuity, and three had no healing of the bony wound. Bony union scores were significantly lower in the 5-mm defects compared to 0-mm, 1-mm and 3-mm defects (all p < 0.01).
Conclusion and Relevance
The rat segmental mandible model cannot heal a 5-mm segmental mandibular defect. Successful healing of 0-, 1- and 3-mm defects confirms adequate stabilization of bony wounds with internal fixation with 1-mm microplates. The rat segmental mandibular critical-sized defect provides a clinically relevant testing ground for translatable mandibular tissue engineering efforts.
doi:10.1001/jamaoto.2013.5669
PMCID: PMC4343284  PMID: 24232293
Critical-sized defect; Craniofacial; Mandible; Tissue Engineering
11.  Development of Scaffold-Free Elastic Cartilaginous Constructs with Structural Similarities to Auricular Cartilage 
Tissue Engineering. Part A  2014;20(5-6):1012-1026.
External ear reconstruction with autologous cartilage still remains one of the most difficult problems in the fields of plastic and reconstructive surgery. As the absence of tissue vascularization limits the ability to stimulate new tissue growth, relatively few surgical approaches are currently available (alloplastic implants or sculpted autologous cartilage grafts) to repair or reconstruct the auricle (or pinna) as a result of traumatic loss or congenital absence (e.g., microtia). Alternatively, tissue engineering can offer the potential to grow autogenous cartilage suitable for implantation. While tissue-engineered auricle cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for reconstruction. Similarly, as routine cell expansion can elicit negative effects on chondrocyte function, we have developed an approach to generate large-sized engineered auricle constructs (≥3 cm2) directly from a small population of donor cells (20,000–40,000 cells/construct). Using rabbit donor cells, the developed bioreactor-cultivated constructs adopted structural-like characteristics similar to native auricular cartilage, including the development of distinct cartilaginous and perichondrium-like regions. Both alterations in media composition and seeding density had profound effects on the formation of engineered elastic tissue constructs in terms of cellularity, extracellular matrix accumulation, and tissue structure. Higher seeding densities and media containing sodium bicarbonate produced tissue constructs that were closer to the native tissue in terms of structure and composition. Future studies will be aimed at improving the accumulation of specific tissue constituents and determining the clinical effectiveness of this approach using a reconstructive animal model.
doi:10.1089/ten.tea.2013.0159
PMCID: PMC3938945  PMID: 24124666
12.  Guanylate-binding protein 1 expression from embryonal endothelial progenitor cells reduces blood vessel density and cellular apoptosis in an axially vascularised tissue-engineered construct 
BMC Biotechnology  2012;12:94.
Background
Guanylate binding protein-1 (GBP-1) is a large GTPase which is actively secreted by endothelial cells. It is a marker and intracellular inhibitor of endothelial cell proliferation, migration, and invasion. We previously demonstrated that stable expression of GBP-1 in murine endothelial progenitor cells (EPC) induces their premature differentiation and decreases their migration capacity in vitro and in vivo. The goal of the present study was to assess the antiangiogenic capacity of EPC expressing GBP-1 (GBP-1-EPC) and their impact on blood vessel formation in an axially vascularized 3-D bioartificial construct in vivo.
Results
Functional in vitro testing demonstrated a significant increase in VEGF secretion by GBP-1-EPC after induction of cell differentiation. Undifferentiated GBP-1-EPC, however, did not secrete increased levels of VEGF compared to undifferentiated control EPC expressing an empty vector (EV-EPC). In our In vivo experiments, we generated axially vascularized tissue-engineered 3-D constructs. The new vascular network arises from an arterio-venous loop (AVL) embedded in a fibrin matrix inside a separation chamber. Total surface area of the construct as calculated from cross sections was larger after transplantation of GBP-1-EPC compared to control EV-EPC. This indicated reduced formation of fibrovascular tissue and less resorption of fibrin matrix compared to constructs containing EV-EPC. Most notably, the ratio of blood vessel surface area over total construct surface area in construct cross sections was significantly reduced in the presence of GBP-1-EPC. This indicates a significant reduction of blood vessel density and thereby inhibition of blood vessel formation from the AVL constructs caused by GBP-1. In addition, GBP-1 expressed from EPC significantly reduced cell apoptosis compared to GBP-1-negative controls.
Conclusion
Transgenic EPC expressing the proinflammatory antiangiogenic GTPase GBP-1 can reduce blood vessel density and inhibit apoptosis in a developing bioartificial vascular network and may become a new powerful tool to manipulate angiogenetic processes in tissue engineering and other pathological conditions such as tumour angiogenesis.
doi:10.1186/1472-6750-12-94
PMCID: PMC3610105  PMID: 23217187
Angiogenesis; Endothelial progenitor cells; Guanylate-binding protein 1; In vivo tissue engineering
13.  Skin paddle vascularity of free fibula flap – A study of 386 cases and a classification based on contribution from axial vessels of the leg 
Objective:
The skin paddle of the free fibula flap receives its vascular supply from septocutaneous perforators, musculocutaneous perforators or from both, and these perforators might originate from the peroneal or posterior tibial vessels or from both. The objective of this study was to classify the skin paddles based on the dominance of vascular contribution by these axial vessels through their different perforator systems.
Materials and Methods:
A retrospective analysis of 5-year data of 386 free fibula flaps used in oro-mandibular reconstruction was done and the skin paddle vascularity was studied. While majority of the skin paddles received their blood supply from the peroneal septocutaneous perforators, a few had their dominant supply from the soleus musculocutaneous perforators in addition to peroneal septocutaneous perforators. In few cases, the soleus musculocutaneous perforators were the sole source of blood supply to the skin paddle. The limitation in this study was the inability to augment the clinical observation with cadaveric study.
Results:
The skin paddle of the free fibula flap was classified into four different types (a–d) based on the dominance of vascular contribution by axial vessels of the leg.
Conclusion:
The skin paddle of the free fibula flap has reliable blood supply, but a thorough knowledge of the variations in vascular pattern of the skin paddle is required especially to salvage the larger paddles used in the reconstruction complex oro-mandibular defects.
doi:10.4103/0970-0358.96586
PMCID: PMC3385400  PMID: 22754154
Free fibula flap; musculocutaneous perforators; septo cutaneous perforators; skin paddle; vascular supply
14.  Regenerating mandibular bone using rhBMP-2: part 2 – treatment of chronic, defect non-union fractures 
Veterinary surgery : VS  2014;44(4):410-416.
Objective
To describe a surgical technique utilizing a regenerative approach and internal fixation for reconstruction of critical size bone defect non-union mandibular fractures.
Study design
Case series
Animals
Dogs (n = 6) that had internal fixation of defect non-union mandibular fracture.
Methods
In 5 of the 6 cases the repair was staged and extraction of teeth performed during the first procedure. After 21-98 days (mean 27 days) a pharyngotomy intubation and temporary maxillomandibular fixation were performed. Using an extraoral approach, a locking titanium miniplate plate was contoured and secured. A compression resistant matrix (CRM) infused with rhBMP-2, was implanted in the defect. The implant was then covered with a soft tissue envelope followed by routine closure.
Results
All dogs had healed with intact gingival covering over the mandibular fracture site defect and had immediate return to normal function and correct occlusion. Hard-tissue formation was observed clinically within 2 weeks and solid cortical bone formation within 3 months. Computed tomographic findings in one case at 3 months postoperatively demonstrated that the newly regenerated mandibular bone had 92% of the bone density and porosity compared to the contralateral side. Long-term follow-up revealed excellent outcome.
Conclusion
Mandibular reconstruction using internal fixation and CRM infused with rhBMP-2 is an excellent solution for the treatment of critical size defect non-union fractures in dogs.
Clinical Relevance
In dogs with a mandibular critical size defect non-union fractures, reconstruction using rhBMP-2 and a CRM should be considered as a viable surgical option.
doi:10.1111/j.1532-950X.2014.12122.x
PMCID: PMC4451182  PMID: 24410723
15.  Tissue-Engineered Mandibular Bone Reconstruction for Continuity Defects: A Systematic Approach to the Literature 
Background: Despite significant surgical advances over the last decades, segmental mandibular bone repair remains a challenge. In light of this, tissue engineering might offer a next step in the evolution of mandibular reconstruction.
Purpose: The purpose of the present report was to (1) systematically review preclinical in vivo as well as clinical literature regarding bone tissue engineering for mandibular continuity defects, and (2) to analyze their effectiveness.
Materials and Methods: An electronic search in the databases of the National Library of Medicine and ISI Web of Knowledge was carried out. Only publications in English were considered, and the search was broadened to animals and humans. Furthermore, the reference lists of related review articles and publications selected for inclusion in this review were systematically screened. Results of histology data and amount of bone bridging were chosen as primary outcome variables. However, for human reports, clinical radiographic evidence was accepted for defined primary outcome variable. The biomechanical properties, scaffold degradation, and clinical wound healing were selected as co-outcome variables.
Results: The electronic search in the databases of the National Library of Medicine and ISI Web of Knowledge resulted in the identification of 6727 and 5017 titles, respectively. Thereafter, title assessment and hand search resulted in 128 abstracts, 101 full-text articles, and 29 scientific papers reporting on animal experiments as well as 11 papers presenting human data on the subject of tissue-engineered reconstruction of mandibular continuity defects that could be included in the present review.
Conclusions: It was concluded that (1) published preclinical in vivo as well as clinical data are limited, and (2) tissue-engineered approaches demonstrate some clinical potential as an alternative to autogenous bone grafting.
doi:10.1089/ten.teb.2013.0131
PMCID: PMC3967381  PMID: 23865639
16.  Efficacy of engineered FVIII-producing skeletal muscle enhanced by growth factor-releasing co-axial electrospun fibers 
Biomaterials  2010;32(6):1669-1677.
Co-axial electrospun fibers can offer both topographical and biochemical cues for tissue engineering applications. In this study, we demonstrate the sustained treatment of hemophilia through a non-viral, tissue engineering approach facilitated by growth factor-releasing co-axial electrospun fibers. FVIII-producing skeletal myotubes were first engineered on aligned electrospun fibers in vitro, followed by implantation in hemophilic mice with or without a layer of core-shell electrospun fibers designed to provide sustained delivery of angiogenic or lymphangiogenic growth factors, which serves to stimulate the lymphatic or vascular systems to enhance the FVIII transport from the implant site into systemic circulation. Upon subcutaneous implantation into hemophilic mice, the construct seamlessly integrated with the host tissue within one month, and specifically induced either vascular or lymphatic network infiltration in accordance with the growth factors released from the electrospun fibers. Engineered constructs that induced angiogenesis resulted in sustained elevation of plasma FVIII and significantly reduced blood coagulation time for at least two months. Biomaterials-assisted functional tissue engineering was shown in this study to offer protein replacement therapy for a genetic disorder such as hemophilia.
doi:10.1016/j.biomaterials.2010.10.049
PMCID: PMC3053058  PMID: 21084118
Hemophilia; skeletal muscle engineering; angiogenesis; lymphangiogenesis; electrospinning; protein replacement therapy
17.  Mandibular Repair in Rats with Premineralized Silk Scaffolds and BMP-2-modified bMSCs 
Biomaterials  2009;30(27):4522-4532.
Premineralized silk fibroin protein scaffolds (mSS) were prepared to combine the osteoconductive properties of biological apatite with aqueous-derived silk scaffold (SS) as a composite scaffold for bone regeneration. The aim of present study was to evaluate the effect of premineralized silk scaffolds combined with bone morphogenetic protein-2 (BMP-2) modified bone marrow stromal cells (bMSCs) to repair mandibular bony defects in a rat model. bMSCs were expanded and transduced with adenovirus AdBMP-2, AdLacZ gene in vitro. These genetically modified bMSCs were then combined with premineralized silk scaffolds to form tissue engineered bone. Mandibular repairs with AdBMP-2 transduced bMSCs/mSS constructs were compared with those treated with AdLacZ transduced bMSCs/mSS constructs, native (nontransduced) bMSCs/mSS constructs and mSS alone. Eight weeks post-operation, the mandibles were explanted and evaluated by radiographic observation, micro-CT, histological analysis and immunohistochemistry. The presence of BMP-2 gene enhanced tissue engineered bone in terms of the most new bone formed and the highest local bone mineral densities (BMD) found. These results demonstrated that premineralized silk scaffold could serve as a potential substrate for bMSCs to construct tissue engineered bone for mandibular bony defects. BMP-2 gene therapy and tissue engineering techniques could be used in mandibular repair and bone regeneration.
doi:10.1016/j.biomaterials.2009.05.021
PMCID: PMC2871698  PMID: 19501905
18.  Fabrication of a Neotrachea Using Engineered Cartilage 
The Laryngoscope  2008;118(4):593-598.
Objectives
Surgical management of long-segment tracheal stenosis is an ongoing problem. Many types of tracheal prostheses have been tried but with limited success because of immune rejection, graft ischemia, or restenosis. Tissue engineered cartilage may offer a solution to this problem, although scaffolds, which are currently often used for support, can lead to biocompatibility problems. This study investigated the feasibility of scaffold-free cartilage to tissue engineer a vascularized neotrachea in rabbits.
Study Design
Animal study.
Methods
Autologous neotracheal constructs were implanted in the abdomen of six New Zealand white rabbits. Auricular chondrocytes were used to engineer scaffold-free cartilage sheets. A muscle flap raised from the external abdominal oblique muscle and the engineered cartilage were wrapped around a silicone stent to fabricate a vascularized neotrachea in vivo. In two of the six rabbits, a full thickness skin graft was used to create an epithelial lining. The constructs were harvested after either 6 or 10 weeks.
Results
All neotracheal constructs were healthy with well-vascularized and integrated layers. The implanted engineered cartilage underwent a remodeling process, forming a solid tracheal framework. Constructs harvested after 10 weeks proved to have significantly better mechanical properties than after 6 weeks and were comparable with the rabbit's native trachea.
Conclusion
Scaffold-free engineered cartilage can successfully fabricate a well-vascularized, autologous neotrachea with excellent mechanical properties. The results suggest that this approach can be used to reconstruct tracheal defects in rabbits.
doi:10.1097/MLG.0b013e318161f9f8
PMCID: PMC2504720  PMID: 18197138
Scaffold-free cartilage; cartilage tissue engineering; tracheal reconstruction; tracheal substitute; engineered trachea
19.  Myogenic Differentiation of Mesenchymal Stem Cells in a Newly Developed Neurotised AV-Loop Model 
BioMed Research International  2013;2013:935046.
Generation of axially vascularized muscle tissue constitutes a promising new approach to restoration of damaged muscle tissue. Mesenchymal stemcells (MSC), with their ability to be expanded to large cell numbers without losing their differentiation capacity into the myogenic lineage, could offer a promising cell source to generate neomuscle tissue. In vitro experiments showed that cocultures of primary myoblasts and MSC undergo myogenic differentiation by stimulation with bFGF and dexamethasone. A newly developed AV-Loop model with neurotization was established in this study. It encompasses axial vascularization and the additional implantation of a motor nerve serving as myogenic stimulator. Myoblasts and MSCs were coimplantated in a prevascularized isolation chamber. Cells were differentiated by addition of bFGF and dexamethasone plus implantation of a motor nerve. After 8 weeks, we could observe areas of myogenic differentiation with α-sarcomeric actin and MHC expression in the constructs. Quantitative PCR analysis showed an expression of myogenic markers in all specimens. Thus, neurotization and addition of bFGF and dexamethasone allow myogenic differentiation of MSC in an axially vascularized in vivo model for the first time. These findings are a new step towards clinical applicability of skeletal muscle tissue engineering and display its potential for regenerative medicine.
doi:10.1155/2013/935046
PMCID: PMC3782807  PMID: 24106724
20.  Growth Factor Stimulation Improves the Structure and Properties of Scaffold-Free Engineered Auricular Cartilage Constructs 
PLoS ONE  2014;9(8):e105170.
The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-β1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation.
doi:10.1371/journal.pone.0105170
PMCID: PMC4134285  PMID: 25126941
21.  Total disc replacement using a tissue-engineered intervertebral disc in vivo: new animal model and initial results  
Study type: Basic science
Introduction: Chronic back pain due to degenerative disc disease (DDD) is among the most important medical conditions causing morbidity and significant health care costs. Surgical treatment options include disc replacement or fusion surgery, but are associated with significant short- and long-term risks.1 Biological tissue-engineering of human intervertebral discs (IVD) could offer an important alternative.2 Recent in vitro data from our group have shown successful engineering and growth of ovine intervertebral disc composites with circumferentially aligned collagen fibrils in the annulus fibrosus (AF) (Figure 1).3
Tissue-engineered composite disc a Experimental steps to generate composite tissue-engineered IVDs3 b Example of different AF formulations on collagen alignment in the AF. Second harmonic generation and two-photon excited fluorescence images of seeded collagen gels (for AF) of 1 and 2.5 mg/ml over time. At seeding, cells and collagen were homogenously distributed in the gels. Over time, AF cells elongated and collagen aligned parallel to cells. Less contraction and less alignment is noted after 3 days in the 2.5 mg/mL gel. c Imaging-based creation of a virtual disc model that will serve as template for the engineered disc. Total disc dimensions (AF and NP) were retrieved from micro-computer tomography (CT) (left images), and nucleus pulposus (NP) dimensions alone were retrieved from T2-weighted MRI images (right images). Merging of MRI and micro-CT models revealed a composite disc model (middle image)—Software: Microview, GE Healthcare Inc., Princeton, NJ; and slicOmatic v4.3, TomoVision, Montreal, Canada. d Flow chart describing the process for generating multi-lamellar tissue engineered IVDs. IVDs are produced by allowing cell-seeded collagen layers to contract around a cell-seeded alginate core (NP) over time
Objective: The next step is to investigate if biological disc implants survive, integrate, and restore function to the spine in vivo. A model will be developed that allows efficient in vivo testing of tissue-engineered discs of various compositions and characteristics.
Methods: Athymic rats were anesthetized and a dorsal approach was chosen to perform a microsurgical discectomy in the rat caudal spine (Fig. 2,Fig. 3). Control group I (n = 6) underwent discectomy only, Control group II (n = 6) underwent discectomy, followed by reimplantation of the autologous disc. Two treatment groups (group III, n = 6, 1 month survival; group IV, n = 6, 6 months survival) received a tissue-engineered composite disc implant. The rodents were followed clinically for signs of infection, pain level and wound healing. X-rays and magnetic resonance imaging (MRI) were assessed postoperatively and up to 6 months after surgery (Fig. 6,Fig. 7). A 7 Tesla MRI (Bruker) was implemented for assessment of the operated level as well as the adjacent disc (hydration). T2-weighted sequences were interpreted by a semiquantitative score (0 = no signal, 1 = weak signal, 2 = strong signal and anatomical features of a normal disc). Histology was performed with staining for proteoglycans (Alcian blue) and collagen (Picrosirius red) (Fig. 4,Fig. 5).
Disc replacement surgery a Operative situs with native disc that has been disassociated from both adjacent vertebrae b Native disc (left) and tissue-engineered implant (right) c Implant in situ before wound closureAF: Annulus fi brosus, nP: nucleus pulposus, eP: endplate, M: Muscle, T: Tendon, s: skin, art: artery, GP: Growth plate, B: Bone
Disc replacement surgery. Anatomy of the rat caudal disc space a Pircrosirius red stained axial cut of native disc space b Saffranin-O stained sagittal cut of native disc space
Histologies of three separate motion segments from three different rats. Animal one = native IVD, Animal two = status after discectomy, Animal three = tissue-engineered implant (1 month) a–c H&E (overall tissue staining for light micrsocopy) d–f Alcian blue (proteoglycans) g–i Picrosirius red (collagen I and II)
Histology from one motion segment four months after implantation of a bio-engineered disc construct a Picrosirius red staining (collagen) b Polarized light microscopy showing collagen staining and collagen organization in AF region c Increased Safranin-O staining (proteoglycans) in NP region of the disc implant d Higher magnification of figure 5c: Integration between implanted tissue-engineered total disc replacement and vertebral body bone
MRI a Disc space height measurements in flash/T1 sequence (top: implant (714.0 micrometer), bottom: native disc (823.5 micrometer) b T2 sequence, red circle surrounding the implant NP
7 Tesla MRI imaging of rat tail IVDs showing axial images (preliminary pilot data) a Diffusion tensor imaging (DTI) on two explanted rat tail discs in Formalin b Higher magnification of a, showing directional alignment of collagen fibers (red and green) when compared to the color ball on top which maps fibers' directional alignment (eg, fibers directing from left to right: red, from top to bottom: blue) c Native IVD in vivo (successful imaging of top and bottom of the IVD (red) d Gradient echo sequence (GE) showing differentiation between NP (light grey) and AF (dark margin) e GE of reimplanted tail IVD at the explantation level f T1Rho sequence demonstrating the NP (grey) within the AF (dark margin), containing the yellow marked region of interest for value acquisition (preliminary data are consistent with values reported in the literature). g T2 image of native IVD in vivo for monitoring of hydration (white: NP)
Results: The model allowed reproducible and complete discectomies as well as disc implantation in the rat tail spine without any surgical or postoperative complications. Discectomy resulted in immediate collapse of the disc space. Preliminary results indicate that disc space height was maintained after disc implantation in groups II, III and IV over time. MRI revealed high resolution images of normal intervertebral discs in vivo. Eight out of twelve animals (groups III and IV) showed a positive signal in T2-weighted images after 1 month (grade 0 = 4, grade 1 = 4, grade 2 = 4). Positive staining was seen for collagen as well as proteoglycans at the site of disc implantation after 1 month in each of the six animals with engineered implants (group III). Analysis of group IV showed positive T2 signal in five out of six animals and disc-height preservation in all animals after 6 months.
Conclusions: This study demonstrates for the first time that tissue-engineered composite IVDs with circumferentially aligned collagen fibrils survive and integrate with surrounding vertebral bodies when placed in the rat spine for up to 6 months. Tissue-engineered composite IVDs restored function to the rat spine as indicated by maintenance of disc height and vertebral alignment. A significant finding was that maintenance of the composite structure in group III was observed, with increased proteoglycan staining in the nucleus pulposus region (Figure 4d–f). Proteoglycan and collagen matrix as well as disc height preservation and positive T2 signals in MRI are promising parameters and indicate functionality of the implants.
doi:10.1055/s-0028-1100918
PMCID: PMC3623095  PMID: 23637671
22.  Successful human long-term application of in situ bone tissue engineering 
Tissue Engineering (TE) and Regenerative Medicine (RM) have gained much popularity because of the tremendous prospects for the care of patients with tissue and organ defects. To overcome the common problem of donor-site morbidity of standard autologous bone grafts, we successfully combined tissue engineering techniques for the first time with the arteriovenous loop model to generate vascularized large bone grafts. We present two cases of large bone defects after debridement of an osteomyelitis. One of the defects was localized in the radius and one in the tibia. For osseus reconstruction, arteriovenous loops were created as vascular axis, which were placed in the bony defects. In case 1, the bone generation was achieved using cancellous bone from the iliac crest and fibrin glue and in case 2 using a clinically approved β-tricalciumphosphate/hydroxyapatite (HA), fibrin glue and directly auto-transplanted bone marrow aspirate from the iliac crest. The following post-operative courses were uneventful. The final examinations took place after 36 and 72 months after the initial operations. Computer tomogrphy (CT), membrane resonance imaging (MRI) and doppler ultrasound revealed patent arterio-venous (AV) loops in the bone grafts as well as completely healed bone defects. The patients were pain-free with normal ranges of motion. This is the first study demonstrating successfully axially vascularized in situ tissue engineered bone generation in large bone defects in a clinical scenario using the arteriovenous loop model without creation of a significant donor-site defect utilizing TE and RM techniques in human patients with long-term stability.
doi:10.1111/jcmm.12296
PMCID: PMC4124030  PMID: 24801710
tissue engineering; human application; long term success; large bone defect; arteriovenous loop; clinical translation; regenerative medicine
23.  Bilateral Mandibular Condylysis from Systemic Sclerosis: Case Report of Surgical Correction with Bilateral Total Temporomandibular Joint Replacement 
Systemic sclerosis (SSc) is a multisystem connective tissue disease of unknown etiology. The hallmark of SSc is scleroderma, referring to the presence of thickened, hardened skin. Oral and maxillofacial manifestations of the disease are numerous including masklike appearance, trismus, muscular atrophy, thin atrophied lips, secondary microstomia, xerostomia, rigidity of tongue and lips, widening of the periodontal ligament space, trigeminal neuralgia, and resorption of the mandible. A 35-year-old woman with limited cutaneous SSc presented with bilateral mandibular condylysis, severe class II mandibular deficiency, and large anterior open bite and limited range of mandibular opening at 27 mm. Surgical correction consisted of bilateral total temporomandibular joint reconstruction with stock prostheses combined with Le Fort I maxillary impaction and functional advancement genioplasty. This resulted in a functional occlusion with elimination of her open bite and a more esthetic profile. Her occlusion has remained stable at 7 months. The incidence of mandibular resorption in SSc has been found to be 20% to 33%. The mandibular angles are most commonly involved (37.6%), followed by the condyle (20.8%), coronoid process (20.0%), and the posterior border of the ascending ramus (14.4%). Bilateral condylysis is present in 13.7% of the cases. Very few cases of surgical correction of malocclusion induced by SSc-related condylysis have been reported in the literature. To the best of our knowledge, this is the first case report of bilateral condylysis from SSc where surgical replacement of the resorbed condyles was attempted. Bilateral total temporomandibular joint replacement can give these patients a functional occlusion, improved facial balance, and improved quality of life.
doi:10.1055/s-0031-1272904
PMCID: PMC3208339  PMID: 22379502
Scleroderma; systemic; temporomandibular joint; arthroplasty; replacement; osteolysis
24.  Adipose-Derived Stem Cells and BMP-2 Delivery in Chitosan-Based 3D Constructs to Enhance Bone Regeneration in a Rat Mandibular Defect Model 
Tissue Engineering. Part A  2014;20(15-16):2169-2179.
Reconstructing segmental mandiblular defects remains a challenge in the clinic. Tissue engineering strategies provide an alternative option to resolve this problem. The objective of the present study was to determine the effects of adipose-derived stem cells (ASCs) and bone morphogenetic proteins-2 (BMP-2) in three-dimensional (3D) scaffolds on mandibular repair in a small animal model. Noggin expression levels in ASCs were downregulated by a lentiviral short hairpin RNA strategy to enhance ASC osteogenesis (ASCsNog−). Chitosan (CH) and chondroitin sulfate (CS), natural polysaccharides, were fabricated into 3D porous scaffolds, which were further modified with apatite coatings for enhanced cellular responses and efficient delivery of BMP-2. The efficacy of 3D apatite-coated CH/CS scaffolds supplemented with ASCsNog− and BMP-2 were evaluated in a rat critical-sized mandibular defect model. After 8 weeks postimplantation, the scaffolds treated with ASCsNog− and BMP-2 significantly promoted rat mandibular regeneration as demonstrated by micro-computerized tomography, histology, and immunohistochemistry, compared with the groups treated with ASCsNog− or BMP-2 alone. These results suggest that our combinatorial strategy of ASCsNog−+BMP-2 in 3D apatite microenvironments can significantly promote mandibular regeneration, and these may provide a potential tissue engineering approach to repair large bony defects.
doi:10.1089/ten.tea.2013.0523
PMCID: PMC4137352  PMID: 24524819
25.  Regenerative medicine as applied to solid organ transplantation: current status and future challenges 
Summary
In the last two decades, regenerative medicine has shown the potential for “bench-to-bedside” translational research in specific clinical settings. Progress made in cell and stem cell biology, material sciences and tissue engineering enabled researchers to develop cutting-edge technology which has lead to the creation of nonmodular tissue constructs such as skin, bladders, vessels and upper airways. In all cases, autologous cells were seeded on either artificial or natural supporting scaffolds. However, such constructs were implanted without the reconstruction of the vascular supply, and the nutrients and oxygen were supplied by diffusion from adjacent tissues. Engineering of modular organs (namely, organs organized in functioning units referred to as modules and requiring the reconstruction of the vascular supply) is more complex and challenging. Models of functioning hearts and livers have been engineered using “natural tissue” scaffolds and efforts are underway to produce kidneys, pancreata and small intestine. Creation of custom-made bioengineered organs, where the cellular component is exquisitely autologous and have an internal vascular network, will theoretically overcome the two major hurdles in transplantation, namely the shortage of organs and the toxicity deriving from lifelong immuno-suppression. This review describes recent advances in the engineering of several key tissues and organs.
doi:10.1111/j.1432-2277.2010.01182.x
PMCID: PMC3817209  PMID: 21062367
decellularization; extracellular matrix; regenerative medicine; scaffold; solid organ transplantation; stem cells; tissue engineering

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