Search tips
Search criteria

Results 1-25 (311438)

Clipboard (0)

Related Articles

1.  Mandibular reconstruction using an axially vascularized tissue-engineered construct 
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.
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.
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).
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).
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.
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.
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.
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.
PMCID: PMC3814659  PMID: 24205470
Adipose-derived stem cells; beta-tricalcium phosphate; bone morphogenetic protein; computer-aided design
3.  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.
PMCID: PMC3955150  PMID: 24460735
hUCMSC; tissue engineering; urethral reconstruction; pedicled muscle flaps
4.  A two-year audit of non-vascularized iliac crest bone graft for mandibular reconstruction: technique, experience and challenges 
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.
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.
NVIBGs provide an acceptable alternative to vascularized bone grafts, genetically engineered bone, and distraction osteogenesis for mandibular reconstruction in resource-limited centers.
PMCID: PMC4279977  PMID: 25551091
Mandibular resection; Mandibular reconstruction; Non-vascularized iliac crest bone graft
5.  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.
PMCID: PMC3178095  PMID: 21597269
Periosteum; Bone; Cartilage; Regeneration; Tissue engineering
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.
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).
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.
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.
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.
PMCID: PMC3366141  PMID: 22438165
Adolescent idiopathic scoliosis; Apical vertebral rotation; Correction loss; Coronal correction
7.  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.
PMCID: PMC3739741  PMID: 23951215
8.  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.
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.
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.
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.
PMCID: PMC3610105  PMID: 23217187
Angiogenesis; Endothelial progenitor cells; Guanylate-binding protein 1; In vivo tissue engineering
9.  Skin paddle vascularity of free fibula flap – A study of 386 cases and a classification based on contribution from axial vessels of the leg 
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.
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.
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.
PMCID: PMC3385400  PMID: 22754154
Free fibula flap; musculocutaneous perforators; septo cutaneous perforators; skin paddle; vascular supply
10.  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.
PMCID: PMC3053058  PMID: 21084118
Hemophilia; skeletal muscle engineering; angiogenesis; lymphangiogenesis; electrospinning; protein replacement therapy
11.  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.
PMCID: PMC2871698  PMID: 19501905
12.  Fabrication of a Neotrachea Using Engineered Cartilage 
The Laryngoscope  2008;118(4):593-598.
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.
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.
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.
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.
PMCID: PMC2504720  PMID: 18197138
Scaffold-free cartilage; cartilage tissue engineering; tracheal reconstruction; tracheal substitute; engineered trachea
13.  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.
PMCID: PMC3782807  PMID: 24106724
14.  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.
PMCID: PMC4134285  PMID: 25126941
15.  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.
PMCID: PMC3623095  PMID: 23637671
16.  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.
PMCID: PMC4124030  PMID: 24801710
tissue engineering; human application; long term success; large bone defect; arteriovenous loop; clinical translation; regenerative medicine
17.  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.
PMCID: PMC3208339  PMID: 22379502
Scleroderma; systemic; temporomandibular joint; arthroplasty; replacement; osteolysis
18.  Reconstruction of mandibular defects - clinical retrospective research over a 10-year period - 
Head & Neck Oncology  2011;3:23.
Functional and cosmetic defects in the maxillofacial region are caused by various ailments and these defects are addressed according to their need. Simplicity of procedure, intact facial function and esthetic outcome with the least possible donor site morbidity are the minimum requirements of a good reconstruction. Oro-mandibular reconstruction, although a challenge for the head and neck reconstructive surgeon, is now reliable and highly successful with excellent long-term functional and aesthetic outcomes with the use of autogenous bone grafts. Reconstruction of trauma- or mandibular oncologic defects with bony free flaps is considered the gold standard. However the the optimal reconstruction of mandibular defects is still controversial in regards to reconstructive options which include the donor site selection and the timing of surgery. The purpose of this study was to determine the outcome of different osseous reconstruction options using autogenous bone grafts for mandibular reconstructions.
This study was carried out on 178 patients with mandibular bone defects. They were reconstructed with autogenous bone grafts from different donor sites. At post operative visits they were evaluated for functional and cosmetic results.
The success rate found in this study was around 90%. Only 7.6% of the cases showed poor results regarding facial contours and mouth opening. All other patients were satisfied with their cosmesis and mouth opening at the recipient sites was in the normal range during last follow-up visits. Donor sites were primarily closed in all cases and there was no hypertrophic scar.
Based on this study, autogenous bone grafts are a reliable treatment modality for the reconstruction of mandibular bone defects with predictable aesthetic and functional outcomes. As the free vascularized fibular flap has the least resorption and failure rate, it should be the first choice for most cases of mandiblular reconstruction.
PMCID: PMC3098818  PMID: 21527038
19.  Regenerative medicine as applied to solid organ transplantation: current status and future challenges 
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.
PMCID: PMC3817209  PMID: 21062367
decellularization; extracellular matrix; regenerative medicine; scaffold; solid organ transplantation; stem cells; tissue engineering
20.  The promotion of mandibular defect healing by the targeting of S1P receptors and the recruitment of alternatively activated macrophages 
Biomaterials  2013;34(38):9853-9862.
Endogenous signals originating at the site of injury are involved in the paracrine recruitment, proliferation, and differentiation of circulating progenitor and diverse inflammatory cell types. Here, we investigate a strategy to exploit endogenous cell recruitment mechanisms to regenerate injured bone by local targeting and activation of sphingosine-1-phosphate (S1P) receptors. A mandibular defect model was selected for evaluating regeneration of bone following trauma or congenital disease. The particular challenges of mandibular reconstruction are inherent in the complex anatomy and function of the bone given that the area is highly vascularized and in close proximity to muscle. Nanofibers composed of poly(DL-lactide-co-glycolide) (PLAGA) and polycaprolactone (PCL) were used to delivery FTY720, a targeted agonist of S1P receptors 1 and 3. In vitro culture of bone progenitor cells on drug loaded constructs significantly enhanced SDF1α mediated chemotaxis of bone marrow mononuclear cells. In vivo results show that local delivery of FTY720 from composite nanofibers enhanced blood vessel ingrowth and increased recruitment of M2 alternatively activated macrophages, leading to significant osseous tissue ingrowth into critical sized defects after 12 weeks of treatment. These results demonstrate that local activation of S1P receptors is a regenerative cue resulting in recruitment of wound healing or anti-inflammatory macrophages and bone healing. Use of such small molecule therapy can provide an alternative to biological factors for the clinical treatment of critical size craniofacial defects.
PMCID: PMC3797185  PMID: 24064148
Craniofacial Reconstruction; S1P; FTY720; Neovascularization; Bone Healing
21.  Apatite-coated Silk Fibroin Scaffolds to Healing Mandibular Border Defects in Canines 
Bone  2009;45(3):517-527.
Tissue engineering has become a new approach for repairing bony defects. Highly porous osteoconductive scaffolds perform the important role for the success of bone regeneration. By biomimetic strategy, apatite-coated porous biomaterial based on silk fibroin scaffolds (SS) might provide an enhanced osteogenic environment for bone-related outcomes. To assess the effects of apatite-coated silk fibroin (mSS) biomaterials for bone healing as a tissue engineered bony scaffold, we explored a tissue engineered bony graft using mSS seeded with osteogenically induced autologous bone marrow stromal cells (bMSCs) to repair inferior mandibular border defects in a canine model. The results were compared with those treated with bMSCs/SS constructs, mSS alone, SS alone, autologous mandibular grafts and untreated blank defects. According to radiographic and histological examination, new bone formation was observed from 4 weeks post-operation, and the defect site was completely repaired after 12 months for the bMSCs/mSS group. In the bMSCs/SS group, new bone formation was observed with more residual silk scaffold remaining at the center of the defect compared with the bMSCs/mSS group. The engineered bone with bMSCs/mSS achieved satisfactory bone mineral densities (BMD) at 12 months post-operation close to those of normal mandible (p>0.05). The quantities of newly formed bone area for the bMSCs/mSS group was higher than the bMSCs/SS group (p<0.01), but no significant differences were found when compared with the autograft group (p>0.05). In contrast, bony defects remained in the center with undegraded silk fibroin scaffold and fibrous connective tissue, and new bone only formed at the periphery in the groups treated with mSS or SS alone. The results suggested apatite-coated silk fibroin scaffolds combined with bMSCs could be successfully used to repair mandibular critical size border defects and the premineralization of these porous silk fibroin protein scaffolds provided an increased osteoconductive environment for bMSCs to regenerate sufficient new bone tissue.
PMCID: PMC2828815  PMID: 19505603
Tissue engineering; Silk; Apatite; Bone marrow stromal cells; Canine
22.  Repositioning template for mandibular reconstruction with fibular free flaps: an alternative technique to pre-plating and virtual surgical planning 
Oral malignancies involving the mandibular bone require a complex reconstructive plan. Mandibular reconstruction with a fibular free flap is currently considered the best choice for functional and aesthetic rehabilitation after oncological surgery. This flap can be modelled with multiple osteotomies and can provide bone, muscle and skin for composite reconstruction. One of the most delicate aspects of mandibular reconstruction is the technique of bone modelling; the risk of prolonging the period of ischaemia and not restoring the correct maxillomandibular and occlusal relationships can ultimately lead to a higher rate of complications as well as poor aesthetic and functional results. Recently, there has been rising interest in virtual surgical planning and computer-assisted mandibular reconstruction in pre-operative planning; however, this is not always possible because of the costs involved and the set-up time for the entire procedure. In this paper, we present a simple and inexpensive technique for fibular free flap modelling and repositioning after segmental resection of the mandible; the technique entails the pre-operative preparation of a resin repositioning template on a stereolithographic model. This technique has been successfully applied in four cases: two cases underwent resection involving only the mandibular body, one case involving the mandibular body and symphysis and one case in which a ramus to ramus resection was performed. In this preliminary report, we show that the resin repositioning template is an easy, safe and useful tool for mandibular reconstruction with a fibular free flap.
PMCID: PMC4157527  PMID: 25210223
Mandibulectomy; Mandibular reconstruction; Oral cavity reconstruction; Reconstruction plates
23.  A Novel Cylindrical Biaxial Computer-Controlled Bioreactor and Biomechanical Testing Device for Vascular Tissue Engineering 
Tissue Engineering. Part A  2009;15(11):3331-3340.
It is becoming evident that tissue-engineered constructs adapt to altered mechanical loading, and that specific combinations of multidirectional loads appear to have a synergistic effect on the remodeling. However, most studies of mechanical stimulation of engineered vascular tissue engineering employ only uniaxial stimulation. Here we present a novel computer-controlled bioreactor and biomechanical testing device designed to precisely and simultaneously control mean and cyclic values of transmural pressure (at rates up to 1 Hz and ranges of 40 mmHg), luminal flow rate, and axial length (or load) applied to gel-derived, scaffold-derived, and self-assembly-derived tissue-engineered blood vessels during culture, while monitoring vessel geometry with a resolution of 6.6 μm. Intermittent monitoring of the extracellular matrix and cells is accomplished on live tissues using multi-photon confocal microscopy under unloaded and loaded conditions at multiple time-points in culture (on the same vessel) to quantify changes in cell and extracellular matrix content and organization. This same device is capable of performing intermittent cylindrical biaxial biomechanical testing at multiple time-points in culture (on the same vessel) to quantify changes in the mechanical behavior during culture. Here we demonstrate the capabilities of this new device on self-assembly-derived and collagen-gel-derived tissue-engineered blood vessels.
PMCID: PMC2792052  PMID: 19385725
24.  Stem cells: Update and impact on craniofacial surgery 
With the rapidly expanding field of tissue engineering, surgeons have been eager to apply these principles to craniofacial surgery. Tissue engineering strategies combine the use of a cell type placed on a scaffold and subsequently implanted in vivo to address a tissue defect or tissue dysfunction. In this review we will discuss the current clinical need for skeletal and soft tissue engineering faced by craniofacial surgeons and subsequently we will explore cell types and scaffold designs being employed for tissue engineering treatment options. We will conclude by discussing ways to enhance the vascularity of tissue engineered constructs as this will ultimately allow for a definitive repair. Current “stem cell” options include pluripotent stem cells as well as multipotent mesenchymal cells. Biomimetic scaffolds can function to protect and enhance differentiation of stem cells by providing inductive cues. Larger scale studies including prospective, randomized control trials must be performed to determine the optimum cell delivery method and cytokine stimuli for tissue engineering driven strategies to address the clinical needs in skeletal and soft tissue reconstruction.
PMCID: PMC3282019  PMID: 22337434
Stem Cells; Craniofacial Surgery; adipose derived mesenchymal cells; bone marrow derived mesenchymal cells; scaffolds; vascularization
25.  Bifocal Distraction to Regenerate Segmental Mandibular Defects Using a Custom Made Device: A Report of Two Cases 
Background and objective: Mandibular reconstruction following tumor ablative surgery had been a challenge. It has gone through days of temporary stabilization with k-wire, stainless steel reconstruction plate to avascularized and vascularized bone grafts with varying degree of success and failure. Reconstruction with vascularized bone grafts, though most definitive, requires special expertise, expensive equipment, and long operative time. It also produces donor site morbidity and requires a significant learning curve. With the development of transport distraction techniques there has been a paradigm shift from “reconstructive” to “regenerative” surgery. The objective of this study was to identify the feasibility of an internal device for reconstruction of extensive mandibular defects. Methods and material: This article is to highlight a process of attempted mandibular regeneration in two cases using an indigenously designed distractor device. In individuals with extensive post ablative mandibular defects who were unwilling to have a secondary surgical site or were medically unfit for a long reconstructive procedure, the distraction technique can be used to reconstruct the lost mandibular structures. Conclusion: A satisfactory amount of regenerate was achieved using a bifocal distraction osteogenesis technique for extensive defects of the mandible. The custom made device which was used for this purpose was moderately satisfactory and requires further evaluation and refinement.
PMCID: PMC3052676  PMID: 22110823
Transport distraction; extensive mandibular defects; single unit distractor device; mandibular reconstruction

Results 1-25 (311438)