Join us in Nashville for
"To Graft or Not to Graft" - February 15-16, 2019 Event Info
Osteo Science Foundation is proud to award research grants that support the advancement of hard and soft tissue regeneration in Oral and CranioMaxillofacial Surgery. Our researchers are making significant strides forward and their work enlightens the future of our field. We are proud to be their partners and to help lead the way by funding initiatives that are making a true difference.
The following are grant recipients of the Peter Geistlich Rewarch Award.
Steven I. Goudy, MD and Shelly Abramowicz, DMD, MPH - Emory University
Bone Regenerative Strategies Targeting the Notch Pathway
Dr. Steven Goudy is the director of pediatric otolaryngology at Emory University where he has a laboratory focused on craniofacial bone development and regeneration. Working closely with Dr. Shelly Abramowicz, Associate Professor of Oral Surgery at Emory, they focus on developing regenerative approaches to pediatric craniofacial bone loss using animal models.
Using in vitro and in vivo approaches, Drs. Goudy and Abramowicz are developing innovative approaches for drug and cell delivery, leveraging their collaborations with the Georgia Institute of Technology. Dr. Bauer is a graduate of the University of Pittsburgh Schools of Dental Medicine and Medicine.
The current options for bone grafting in adults include autologous bone graft harvest, donated autogenous bone, and bone regeneration using growth factor delivery, primarily using Bone Morphogenetic Protein 2 (BMP2). Options for bone grafting in children are limited due to concerns about use of donor bone and lack of FDA approval of BM
P2 use in children. The development of a regenerative strategy to replace and/or repair bone loss in children is critically needed to reduce cost and morbidity.
Delivery of Bone Morphogenetic Protein 2 (BMP2) using a collagen sponge has successfully regenerated bone in adults to treat facial bone loss and during cervical spine fusion. BMP2 is not FDA-approved for the treatment of children due to the off target effects of BMP2, including life-threatening swelling and inflammation. Delivery of growth factors and cells requires that the delivery vehicle has moderate term permanence, avoids significant host tissue inflammation, and is easily cleared from the host. Many investigators are pursuing multiple pathways to identify additional genes, growth factors and cells that can be targeted for bone regeneration.
This proposal focuses on the Notch signaling pathway as a potential target to regenerate bone without inducing significant inflammation. Our lab has demonstrated the requirement of Jagged1, a cell surface ligand in the Notch pathway, during craniofacial bone formation and we have created a mouse model of maxillary bone loss. Jagged1 is a cell surface ligand in the Notch gene family that is necessary in determining cell fate. Human mutation of JAGGED1 leads to Alagille syndrome, characterized by cardiac, and bony phenotypes. Taken together these findings indicate that Jagged1 plays an important role in bone development, and interruption of Jagged1 function is associated with obvious clinical manifestations of bony loss and targeted Jagged1-therapies may provide another therapeutic option for bone regeneration in children.
Volume-Stable Collagen Matrix for Intraoral Soft Tissue Reconstruction and Augmentation
Dr. Bauer is a graduate of the University of Pittsburgh Schools of Dental Medicine and Medicine. Dr. Bauer completed his residency training in Oral and Maxillofacial Surgery at the University of Pittsburgh Medical Center. Dr. Bauer is a full-time faculty member and Residency Program Director at the University of Pittsburgh in the department of Oral and Maxillofacial Surgery and his practice is focused on dental implants, corrective jaw surgery and surgical management of sleep apnea. He has been active in research with focuses on virtual applications for computer assisted surgery and tissue regeneration.
This study is designed as a longitudinal clinical evaluation of the effectiveness and stability of volume stable collagen matrix, Geistlich Fibro-Gide. Tissue integration of volume-stable collagen matrix (VCMX) will provide an alternative for intraoral reconstruction and augmentation. Thoma et al. (2016) has recently shown short term effectiveness of VCMX, Geistlich Fibro-Gide, at single site defects.
Preclinical and clinical evaluation of VCMX has been completed with great promise. It has been proven to be a safe and effective product in the short term and in isolated areas and has FDA approval for intraoral soft tissue regeneration and augmentation. It is my hypothesis that VCMX will provide a stable alternative to autogenous connective tissue grafting and acellular human dermal matrix in patients with deficiencies larger than single tooth sites, around recently placed dental implants and patients that are status post oral reconstruction with composite grafts seeking implant supported dental reconstructions.
The patients included in this study will be those undergoing dental implant therapy, including multiple consecutive sites or long span edentulous ridges. 2. Patients in need of soft tissue volume increase at dental implant sites will be consecutively recruited, informed, and screened for inclusion. VCMX will be used as connective tissue augmentation at the ridge crest and facial aspect of the dental implant site.
Jajit Dillon, MBBS, DDS, BDS, FDSRCS, FACS - University of Washington
Pentoxifylline and tocopherol (PENTO) in the treatment of MRONJ Formation Targeting the VEGFa Pathway
Jasjit Kaur Dillon is an Associate Clinical Professor and Program Director of Oral and Maxillofacial Surgery at the University of Washington, Seattle. She obtained her dental degrees from the University of Newcastle Upon Tyne (BDS), the University of California San Francisco (DDS) and her medical degree from St Bartholomew’s School of Medicine, University of London (MBBS). She is a member of the Royal College of Surgeons of England (FDSRCS), the American College of Surgeons (FACS) and is an examiner for the American Board of OMS. She has numerous peer reviewed scientific publications, book chapters and lectures nationally and internationally. She is a previous recipient of the OMS Foundation’s Stephen Milam award for best research grant.
Our phase II PENTO trial would follow a randomized, double-blind, placebo controlled, multi-center design. A stratified permuted- block randomization will be used to allocate patients to treatment. Allocations will be balanced by stage and type of drug exposed to. We recognize numerous medications have been linked to cause MRONJ and there are many newly proposed, non-operative treatments for MRONJ. However, our group will only study bisphosphonates and RANK-L inhibitor medications and the PENTO treatment regimen. With future clinical trials we hope to continue investigating other etiologies and treatments of MRONJ.
The primary predictor variable is the therapeutic intervention (PENTO). The experimental arm will be PENTO plus standard of care (SOC) as defined by the AAOMS Position Paper on MRONJ and the control arm will be SOC with a placebo. The primary outcome variable will be bone exposure area (mm2) of the primary bone lesion. Secondary outcomes will include patient pain perception measured by a visual analogue scale, radiographic changes on orthopantomogram and MRONJ stage. Outcomes will be collected at time of inclusion and then on a three-month cycle with the patient’s treatment ending at fifty-two weeks. Masked members of the data collection team include the clinicians measuring outcomes, the study coordinator and statistician. The primary analysis of interest is the association between treatment arms and area of exposed bone in mm2. The primary analysis will be completed using a mixed-effects model. A comparison will be completed for the area of the exposed bone between the two treatment arms. To power the study an alpha of 0.05, a beta of 0.10 and an anticipated dropout rate of 30% was assumed. To detect a relative change in primary outcome ≥20%, a minimum sample of forty-four patients in each study arm is required. Our desired sample size would have fifty patients per arm.
Alireza Moshaverinia, DDS, PhD - University of Pennsylvania
Engineering microscale delivery systems capable of precisely controlled growth factor release
Dr. Alireza Moshaverinia is a Diplomate of the American Board of Prosthodontics and a tenure track assistant professor at the UCLA School of Dentistry, Division of Advanced Prosthodontics. Alireza has received his DDS degree from Iran. He has an MS degree in Dental Biomaterials from the Ohio State University, College of Dentistry. He completed advanced clinical education in Prosthodontics and earned his PhD in Craniofacial Biology from USC School of Dentistry. He is the recipient of several awards in recognition of his scientific achievements including: GSK Prosthodontist Innovator Award, NIH Career Development Award, IADR Innovation in Oral Care Award, and IADR Academy of Osseointegration innovation in Implant Sciences Award.
Soft tissue reconstruction is still a challenging clinical task in reconstructive surgeries, regenerative medicine, and dentistry. Current modalities of treatment might lead to donor site morbidity and suboptimal outcomes. Mesenchymal stem cells (MSCs) encapsulated in hydrogel biomaterials have been extensively used for tissue engineering applications. Gingival mesenchymal stem cells (GMSCs) can be considered as an alternative therapeutic option for soft tissue reconstruction or augmentation. GMSCs are of special interest as they are easily accessible in the oral cavity and readily found in discarded tissue samples. Biomaterials are widely used as cell delivery vehicle to direct stem cell differentiation toward desired phenotypes. Adhesion and retention of the biomaterial at the application site as well as its regenerative properties are vital factors for successful tissue regeneration applications. However, the major drawbacks of the current cell-laden biomaterials for tissue engineering are weak adhesion to the tissue, poor mechanical strength, fast/uncontrolled degradation, and absence of regenerative properties. Another obstacle in soft tissue engineering is finding a way to precisely deliver specific growth factors (GFs) and have control over their presentation over time. In this proposal, we aim to address these limitations by engineering an adhesive hydrogel based on a visible light crosslinkable dopamine-modified alginate-Heparin hydrogel with the ability to have sustained release of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) for possible soft tissue regeneration applications in medicine and dentistry. This project will introduce a promising treatment approach for soft tissue regeneration. The result will be a novel injectable and biodegradable scaffold with the ability to have sustained and localized delivery of growth factors, presenting an innovative treatment modality for soft tissue regeneration.
Rabie Shanti, DMD, MD - UCLA School of Dentistry
Application of SIS-ECM Constructs Laden with Gingiva-derived MSCs in Tongue Myomucosal Regeneration
Rabie M. Shanti is currently an Assistant Professor in Oral & Maxillofacial Surgery and Otorhinolaryngology/Head and Neck Surgery of University of Pennsylvania. He received his DMD degree from Harvard School of Dental Medicine and later received his MD degree from New Jersey Medical School-Newark in 2011. Dr. Shanti’s current clinical practices mainly focus on the reconstruction of patients with post-ablative or post-traumatic defects of the oral and maxillofacial region. His current research activities involve stem cell biology and regenerative medicine to improve the understanding of the pathogenesis of ameloblastoma in order to discover new and less invasive treatments for this aggressive benign tumor.
Reconstruction of the tongue while preserving and/or restoring its critical vocal, chewing, swallowing, and tasting functions remains one of the major challenges in head and neck oncologic surgery. Through decades of evolution in reconstructive principles and techniques, microvascular soft tissue free flaps have evolved as the reconstructive technique of choice following extirpation of malignant neoplasms. However, these free flaps, made up of skin, subcutaneous adipose tissue and fascia, do not allow for the structural restoration of muscular-like tissues and functional restoration of the tongue (innervation, tasting). Additionally, harvesting these flaps will always result in donor site morbidity that will cause some degrees of local pain, edema, limb weakness, and scar formation. Therefore, novel approaches are imperatively needed to generate tissue engineering/regenerative medicine (TE/RM) product for tongue myomucosal regeneration following the surgical ablation of oral cancers. In recent years, there is growing enthusiasm for the use of mesenchymal stem cells (MSCs) for generation of TE/RM products to facilitate repair/regeneration of damaged muscles. We have recently isolated a subpopulation of MSCs from human gingival tissues (GMSCs), which exhibit potent multipotent differentiation and immunomodulatory/anti-inflammatory capabilities. This work will test the hypothesis that generation of GMSC-based TE/RM products represents a promising approach to promote myomucosal regeneration of the tongue and has the potential for clinical application. Specifically, we will generate GMSC-laden porcine small intestine submucosa extracellular matrix (SIS-ECM) constructs and optimize their regenerative potentials in a critical-sized tongue defect model in rats and further explore the potential mechanisms. Accomplishment of this study will provide substantial evidence for the potential clinical application of GMSC-laden SIS/ECM constructs for tongue reconstruction.
Three-Dimensional Printing of Osteogenic Engineered Networks (OGEN) for Craniomaxillofacial Defects.
Dr. Steven Alexander is a professor in Molecular & Cellular Physiology is a vascular cell biologist and tissue engineer working on 3-D models of soft tissue and bone reconstruction using placenta derived stem cells using innovative vascularizing scaffold composites. A graduate of Boston University, (BS/PhD) he trained as a biomedical engineering at Vanderbilt University (1990-4) until joining the faculty at LSUHSC-Shreveport in 1994. He has joint appointments in the departments of Medicine and Neurology and has been funded by the NIH, AHA and DOD and has authored/co-authored 257 reports.
Christen J. Boyer, PhD
Dr. Boyer is a postdoctoral fellow in the departments of Oral and Maxillofacial Surgery and Molecular and Cellular Physiology at LSUHSC-Shreveport. Dr. Boyer’s work at LSU Health includes the development of novel medical 3D printing technologies. He received his PhD in Molecular Sciences and Nanotechnology from of Louisiana Tech University and his previous degree was a B.S. with a concentration in chemistry, from Midland University in Nebraska. Dr. Boyer has previously been funded by NASA / LaSPACE and has filed numerous nanotechnology and 3D printing related invention disclosures and patents.
Jennifer Woerner, DMD, MD, FACS
Dr. Woerner is an assistant professor in the Department of Oral and Maxillofacial Surgery at LSU Health Shreveport Health Sciences Center. Dr. Woerner graduated from Florida Southern College before attending the University of Florida College of Dentistry, where she earned her doctorate of dental medicine. She began her residency training in oral and maxillofacial surgery at LSU Health Sciences Center and completed her medical degree in 2009. She completed her internship in general surgery in 2010. Following graduation from her residency, in 2013 she completed a fellowship in cleft and craniofacial surgery under Dr. G.E. Ghali. She currently works on the Ark-La-Tex Craniofacial and Cleft team and Shriners Hospital for Children Cleft Lip and Palate Team as a primary cleft and craniofacial surgeon. She is also the Fellowship Director of Craniofacial and Cleft Surgery and the Residency Program Director.
David K. Mills, PhD
Dr. Mills has been on the ‘road less traveled’ for over 40 years. He has met few fellow travelers during his academic odyssey. He holds a BA degree in Ancient History and a second BA in Classics from Indiana University. He received an MA degree in Biological Anthropology in 1984 and a PhD. in Anatomy and Cell Biology in 1999 both from the University of Illinois. He currently is a Professor of Biological Sciences and has a joint appointment in the Center for Biomedical Engineering and Rehabilitation Science at Louisiana Tech University. Mills’ has mentored over 94 graduate students (including 14 PhD students) towards their advanced degrees. He has received funding from DARPA, NIH, NSF, the Shell Foundation, the Biomedical Research Foundation, Louisiana Biomedical Research Network, and the Louisiana Board of Regents. His research is focused on the development bioactive therapeutics that prevents infection and advances tissue repair and regeneration, and customized drug-loaded 3D printed biomedical devices and bioprinted tissues.
Yuping Wang, MD, PhD
Dr. Yuping Wang is a professor in Department of Obstetrics and Gynecology, Louisiana State University Health Sciences Center – Shreveport (LSUHSC-Sh). Dr. Wang received her M.D. degree in Medicine from Harbin Medical University, China, in 1980 and her Ph.D. degree in Physiology from Medical College of Virginia, Virginia Commonwealth University, Virginia, USA, in 1994. In 1997, Dr. Wang jointed the faculty of LSUHSC-Shreveport, Department of Obstetrics and Gynecology. She is a placental biologist and her researches focus on placental trophoblasts, vascular cells, and mesenchymal stem cells and have been funded by NICHD and NHLBI, and is co-author over 120 publications.
While autologous or allograft sources for bone grafting remain a standard clinical platform for repair of cranio-maxillofacial defects, the availability of source material is limited, especially in pediatric patients, and harvesting tissues from secondary sites can be traumatic and cause complications. Synthetic bone grafts reduce risks in donor site morbidity and viral/prion transmission from allografts, but often the materials are strictly osteoconductive and need to be shaped to fit patient anatomies. Manufacturing patient specific synthetic bone grafts for craniomaxillofacial defects through three-dimensional (3-D) printing is not yet well established and the roles of nanotechnology and nanoparticles in regenerative medicine are not fully understood either. We have developed a 3-D printable, patient-specific synthetic bone graft platform impregnated with osteoconductive and osteoinductive nanomaterials. These osteogenic synthetic grafts are derivatized to covalently bind growth factors, guidance molecules and extracellular matrix proteins which allows for more sustained and extended release of growth factors to drive both graft mineralization and vascularization. The specific and controllable osteogenic features of this platform will permit enhanced programming of stem cells to facilitate and optimize mineral deposition, osteogeneis and bio-integration. These biomimetic hybrid chemistries will also provide a synthetic approach to create enhanced nanostructured coatings for existing devices and generate patient-customizable bioengineering materials for use in oral and maxillofacial regenerative medicine research.
VEGF Encapsulation Through a Novel Microfluidic Technique for Bone Tissue Regeneration and Repair
Lobat Tayebi is an Associate Professor and Director of Research at Marquette University School of Dentistry. She received her PhD from University of California-Davis in 2011. She is a researcher in tissue engineering and regenerative medicine with multiple patents in the field. Her publication list comprises of more than 115 peer-reviewed articles including papers in Nature Materials and Advanced Materials. Her current research activities cover projects in treatment of complex multi-tissue oral and craniomaxillofacial defects, growth factor delivery and interfacial hard/soft tissue expansion, growth factor delivery, vascularization and stem cell seeding in patient specific 3D-bioprinted scaffolds.
Using growth factors in tissue regeneration has generated great enthusiasm and an intensive research effort leading to recent clinical trials, many of which have yielded unsatisfactory outcomes. Interestingly, the trials with the most satisfactory results have shared a common denominator: the presence of a vehicle for controlled growth factor delivery.
Blood vessels provide oxygen and nutrients for tissues and remove waste products. A reduction or loss in vascularization can lead to tissue necrosis, tissue death and organ failure. Vascular endothelial growth factor (VEGF) is an important vasculogenic and angiogenic agent which regulates signaling, proliferation and migration of endothelial cells. The VEGF pathway is critical for bone regeneration by promoting activity of bone forming cells and mobilization of involved progenitor cells.
However, the delivery of VEGF is specifically sensitive and should be localized and transported to a specific target tissue which requires a prolonged and sustained exposure to a low dose of VEGF. A bolus injection, imprecise use (off targeting), or unsatisfactory drug delivery can increase the threat of unwanted side effects and often lead to tumorigenesis. Currently, delivery systems are imperfect and limited technologies exist for precise encapsulation of VEGF and its sustained and controlled release. The use of microparticles as vehicles can provide an efficient and directed means for VEGF delivery that can be controlled, localized and released in a sustained fashion.
In this project using a new microfluidic approach, we will develop a method that creates precise customized encapsulated VEGF particles with well-regulated release rates. These particles can be embedded in scaffolds and implants designed for critically sized defects and used along with other growth factors to promote tissue regeneration and repair in oral and craniomaxillofacial injuries.
Novel Bone Grafting Procedures for Oral and Maxillofacial Applications
Dr. Jeremy Mao is a clinician/scientist, and currently Edwin S. Robinson Professor of Dentistry at Columbia University. Dr. Mao has published over 200 scientific articles, proceedings, book chapters and books. Dr. Mao has delivered over 360 invited, keynote and plenary lectures worldwide. Dr. Mao has been active in the field of orthopedic research, plastic surgery research and dental/craniofacial research. Dr. Mao’s laboratory has trained dozens of scientists and clinicians that are in academia, industry and government. Dr. Mao’s research group is currently funded by NIH and other grants in the areas of stem cell biology, tissue engineering and wound healing.
One of the dire needs in Oral and Maxillofacial Surgery (OMS) is craniofacial bone defects following loss of natural teeth, trauma, tumor resection and infections. Approximately over 50% of dental implant patients require ridge augmentation of resorbed alveolar bone prior to or at the time of implant placement. In OMS, autogenous bone grafts have been considered the “gold standard” for regenerative procedures. However, due to the limitation of the supply and morbidity on the secondary surgical site, a substitute to autogenous bone grafts represents an acute medical need. Various allografts, xenografts and alloplast materials have been tested for decades. To date, no bone graft products in the market can meet clinical needs.
Development of a Compromised Maxillofacial Wound Healing Model for Bone Graft Evaluation
Dr. Young’s research interest includes the design of materials for the promotion of bone regeneration in the craniomaxillofacial complex. He has broad experience in the fields of biomaterials, growth factor delivery, in vivo models, and characterization of bone and neovascularization. As an oral & maxillofacial surgeon with experience treating traumatic defects and pathology, Dr. Young understands the unique challenges associated with the reconstruction of complex maxillofacial wounds. By designing a novel preclinical model of compromised wound healing, Drs. Young and Kasper hope to better understand the mechanisms which prevent successful bone grafting, and use these insights to design better therapies in the future.
Despite our understanding of bone regeneration in sites with an optimized underlying physiological environment, it is still poorly understood why bone grafting fails in the setting of the compromised wound (i.e. osteoradionecrosis, multiply-operated sites, etc.). Whether the defect lies in an inadequately vascularized environment, an adversely affected (or missing) progenitor cell population, the complicating presence of bacterial contamination, or a sub-optimal cytokine milieu, the relative contributions of these factors remains to be clearly elucidated. A clinically relevant, reproducible model of compromised wound healing would be invaluable not only to study these potential mechanisms of bone graft failure, but to inform future strategies to improve bone grafting in these situations.
The studies outlined in this proposal seek to build upon our established rabbit mandibular defect model to develop a new pre-clinical model of compromised maxillofacial wound healing for application as a clinically-relevant platform for the elucidation of key differences between compromised and non-compromised maxillofacial wound environments. The novel model of compromised maxillofacial wound healing will be characterized to determine if significant vascular, cellular, or cytokine expression differences are present between the compromised wound healing environment and non-irradiated controls. This model can then be utilized in future studies to characterize the efficacy of various standard bone grafting materials and aid in the fabrication of rationally-designed bone tissue engineering materials.
Application of NSC-like Progenitors Induced from Gingiva-derived MSCs in Facial Nerve Regeneration
Dr. Qunzhou Zhang received his PhD in Biochemistry and Molecular Biology from West China University of Medical Sciences in 2000. He is currently a senior investigator and the leading scientist in Dr. Anh Le’s laboratory at the Department of Oral & Maxillofacial Surgery, University of Pennsylvania School of Dental Medicine. The primary focus of Dr. Zhang’s current research is immunomodulatory and regenerative function of human gingiva-derived mesenchymal stem cells. His research is funded by Stephan B. Milam Research Support Grant from Oral & Maxillofacial Surgery Foundation, University of Pennsylvania Diabetes Research Center (DRC) and Schoenleber Pilot Grant from Penn School of Dental Medicine.
Fully functional recovery of facial nerve injury is a major challenge for oral surgeons. Autologous nerve grafts currently remain the gold standard for repairing injured peripheral nerves with a large gap. However, donor site morbidity, availability of donor nerve and danger of neuroma formation significantly impede their clinic application. Even though alternative allogenic grafts and bioengineered nerve conduits are used in clinic, their overall outcomes are still suboptimal. The combination of bioengineered nerve conduits and stem cells is emerging as a novel approach for peripheral nerve regeneration. Neural stem (NSC) or progenitor cells are considered an ideal candidate seed cell source for stem cell-based treatment of nerve injury, but it remains a challenge to get enough transplantable NSCs for clinical application. Expandable and multipotent neural progenitor cells (NPCs) can be induced from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), but the process is complicated and time-consuming and needs the introduction of exogenous genes, which raise concerns regarding the safety for their clinical use. Thus, generation of expandable NPC-like cells directly from somatic cells may represent an ideal approach for stem cell-based therapy of peripheral nerve injury. This work will test the hypothesis that human gingiva-derived mesenchymal stem cells (GMSCs) can be directly converted to multipotent NPC-like cells with therapeutic potentials for facial nerve defects. Specifically, we will optimize the culture conditions for induction of NPC-like cells from GMSCs and evaluate their multipotency both in vitro and in vivo. We will then use a rat model to test the therapeutic efficacy of GMSC-derived NPC-like cells on repair/regeneration of facial nerve defects. Accomplishment of this study will provide substantial evidence for the potential clinical application of GMSC-derived NPC-like cells for facial nerve regeneration.
1. Zhang Q, Nguyen P, Xu Q, Park W, Lee S, Furuhashi A, Le AD. Neural Progenitor-Like Cells Induced from Human Gingiva-Derived Mesenchymal Stem Cells Regulate Myelination of Schwann Cells in Rat Sciatic Nerve Regeneration. Stem Cells Transl Med. 2017 Feb;6(2):458-470. doi: 10.5966/sctm.2016-0177 PMID: 27604215 Full Text
2. Zhang Q, Nguyen PD, Shi S, Burrell JC, Xu Q, Cullen KD, Le AD. Neural Crest Stem-Like Cells Non-genetically Induced from Human Gingiva-Derived Mesenchymal Stem Cells Promote Facial Nerve Regeneration in Rats. Mol Neurobiol. 2018 Jan 25. doi: 10.1007/s12035-018-0913-3. [Epub ahead of print] PMID: 29372546 Full Text
3. Qunzhou Zhang, Phuong Nguyen, Shihong Shi, Justin Burrell, Kacy Cullen, Anh D. Le. 3D Bioprinted Scaffold-free Nerve Constructs with Human Gingiva-derived Mesenchymal Stem Cells as Bioink Promote Rat Facial Nerve Regeneration. Full Text
Abstracts, Posters & Oral Presentation:
1. Qin Mao, Qunzhou Zhang, Neeraj Panchal, Pasha Shakoori, Anh D. Le. Human Gingiva-Derived Mesenchymal Stem Cells Regulate Myelination of Schwann Cells via EGR2/Krox20 Pathway. 46th AADR/95th IADR, 2017 March 22-25, San Francisco, California., USA
A Biomimetic Heterogeneous Synthetic Matrices for TMJ Condylar Cartilage Repair
Dr. X. Lucas Lu received his PhD in Biomedical Engineering from Columbia University with distinction in 2007. He is currently an Assistant Professor of Mechanical Engineering at the University of Delaware with a joint appointment in the Biomedical Engineering program and the Biomechanics and Movement Science program. The primary focus of Dr. Lu’s current research is cartilage tissue engineering and the prevention, treatment and rehabilitation of osteoarthritis. His research is funded by the Department of Defense, National Institutes of Health, the National Science Foundation, and the Musculoskeletal Transplant Foundation.
Temporamandibular joint (TMJ) disorders affect over 10 million Americans and are often caused by osteoarthritis (OA) or “internal derangement” of the joint. TMJ disorders pose a significant challenge in maxillofacial surgery. Here, we propose a matrix-guided, tissue engineering approach for the repair and regeneration of TMJ condylar cartilage. Our approach relies on the development of a multilayered synthetic matrix that recapitulates the anisotropic feature and the tension-compression nonlinearity. We will create a composite matrix consisting of a bottom, hyaluronan (HA)-based gel layer with spatial gradient of biochemical cues, and a top fibrous layer, de novo designed to mimic the structure and function of type I collagen. Mesenchymal stem cells residing in the matrix will receive signals from the matrix to undergo programmed differentiation in a spatial fashion. The potential of the synthetic matrix in cartilage repair will be tested in an in vitro culture system, as a TMJ osteochondral explant with physiologically relevant mechanical loading, for the generation of new cartilage tissue at the lesion site. We expect that the engineered tissue will exhibit a defined layered and zonal structure and integrate with the host tissue to fulfill the mechanical requirements of daily TMJ activities. This project represents the first effort to fabricate a biomimetic scaffold for TMJ condylar cartilage repair by replicating the unique multilayered structure found in the native tissue. The new synthetic matrix also provides a powerful in vitro platform to study the mechnobiology of TMJ chondrocytes and the developmental biology of condylar cartilage.
1. Han, Z.; Trout, W. S.; Liu, S.; Andrade, G. A.; Hudson, D. A.; Scinto, S. L.; Dicker, K. T.; Li, Y.; Lazouski, N.; Rosenthal, J.; Thorpe, C; Jia, X.; Fox, J. M. "Rapid Bioorthogonal Chemistry Turn–on through Enzymatic or Long Wavelength Photocatalytic Activation of Tetrazine Ligation", J. Am. Chem. Soc. 2016, 38, 5978–5983. Full Text
2. Ravikrishnan, A.; Ozdemir, T.; Bah, M.; Baskerville, K. A.; Shah, S. I.; Ayyappan, R. K.; Jia, X. "Regulation of Epithelial–to–Mesenchymal Transition Using Biomimetic Fibrous Scaffolds" ACS Appl. Mater. Interfaces 2016, DOI: 10.1021/acsami.6b05646. Full Text
3. Ruggiero L, Zimmerman BK, Park M, Han L, Wang L, Burris DL, Lu XL. Roles of the Fibrous Superficial Zone in the Mechanical Behavior of TMJ Condylar Cartilage. Annals of biomedical engineering. 2015; PMID: 25893511 Abstract
1. Jia, X. “Modular and orthogonal approaches for the construction of functional biomaterials” Center for Neurosciences and Cell Biology, University of Coimbra, Coimbra, Portugal, July 13, 2015.
2. Jia, X. “Modular and orthogonal approaches for the construction of functional biomaterials” Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland, July 15, 2015.
3. Jia, X. “Modular and orthogonal approaches for the construction of functional biomaterials” CNRS et Institut Polytechnique de Grenoble, Université de Grenoble, Grenoble, France, July 17, 2015.
4. Jia, X. “Modular and orthogonal approaches for the construction of functional biomaterials” Gordon Research Conference, Biomaterials & Tissue Engineering, Girona, Spain, July 19-24, 2015.
5. Jia, X. “Modular and orthogonal approaches for the construction of functional biomaterials”
6. Department of Materials, Imperial College, London, United Kingdom, July 27, 2015.
7. Jia, X. “Modular and orthogonal approaches for the construction of functional biomaterials”
8. Department of Chemistry, University of Warwick, Warwick, United Kingdom, July 29, 2015.
9. Dicker, K. T.; Zhang, H.; Liu, S.; Fox, J. M.; Jia, X. “Modular and orthogonal approaches for the construction of functional biomaterials” 250th ACS National Meeting & Exposition, Boston, MA, August 16-20, 2015
10. Jia, X. “Engineering Cell-Instructive Environments for the Assembly of Functional Tissues” The James Buchanan Brady Urological Institute and Department of Urology, Johns Hopkins University School of Medicine, Baltimore, MD, July 1, 2015.
11. Jia, X. “Biomaterials-Based Approaches for the Engineering of Physiologically-Relevant Tissue Models” Department of Chemical Engineering, University of Illinois at Chicago, Chicago, IL, February 11, 2016.
Biphasic Scaffolds for Alveolar Bone and Tooth Regeneration
Research in Dr. Yelick's Laboratory works to define effective methods to regenerate bone and tooth structures for craniofacial repair and reconstruction. Her models include Tissue Engineering approaches that employ novel biocompatible scaffolds seeded with dental progenitor cells, which are uniquely designed to form craniofacial bone and tooth tissues. Her successful collaborative efforts in this field have resulted in numerous published reports documenting significant progress towards achieving these goals.
Previous studies have shown that tyrosine based E1001-1K scaffolds can promote mineralized tissue formation. Here we will test whether E1001-1K scaffolds can support the formation of alveolar bone, the specialized type of jaw bone that supports dentition. Our approach involves seeding E1001-1K scaffolds with cultured dental stem cells (DSCs) derived from extracted human wisdom teeth, followed by developmental in vitro and in vivo characterizations of alveolar bone, dentin, pulp, periodontal ligament, and enamel tissue formation. Our approach is unique in that we use neural crest cell (NCC) derived dental pulp stem cells that naturally form alveolar jaw bone and tooth tissues. In contrast, mesenchymal stem cells (MSCs), commonly used for craniofacial reconstructions, are derived from the embryonic mesoderm, and do not naturally form alveolar bone, whose specialized architecture can withstand the strong mechanical forces of mastication. The ability to successfully engineer functional, durable alveolar jaw bone would be a significant improvement over current craniofacial repair techniques using bone grafts from non-NCC derived bone (fibula, rib, etc.), which eventually resorb over time. To date we have performed in vitro characterizations of DSC-seeded E1001-1K scaffolds. To continue these promising studies, here we propose studies to validate the formation of alveolar jaw bone and tooth tissues in situ, in a rat mandible critical sized defect model. The successful completion of the proposed studies will allow us to move forward to a large animal mandibular defect model, prior to pre-clinical human trials.
Effects of Synthetic Graft Versus Autograft in the Generation of Autologous Free Tissue Flaps
Mark Wong is Professor and Chairman of the Department of Oral and Maxillofacial Surgery at The University of Texas School of Dentistry at Houston, where he also serves as the Director of Residency Training. Dr. Wong is currently the President of the American Academy of Craniomaxillofacial Surgeons and the President of the International Board for the Certification of Specialists in Oral and Maxillofacial Surgery. His clinical and research interests are focused on reconstructive surgery, tissue engineering of bone and the biomechanical characterization and regeneration of the temporomandibular joint. His research is funded by the NIH and the Department of Defense. Dr. Wong has served on a number of educational and research committees for AAOMS. He is a Past President of ABOMS, President-elect of the American Academy of Craniomaxillofacial Surgeons, and currently Chairs a Steering Committee for the development of an International Board for the Certification of Specialists in Oral and Maxillofacial Surgery.
Reconstruction of large mandibular defects following blast injury or the resection of advanced pathology is particularly challenging when both bone and soft tissue is missing. Current methods to address this problem include staged reconstruction techniques delaying hard tissue reconstruction until the soft tissue bed has been restored or the use of vascularized hard and soft tissue flaps. Donor site morbidity is significant with either approach and this concern forms the basis for a new method of regenerating bone with accompanying soft tissue.
This project leverages technology and expertise developed for a project initially funded by the Armed Forces Institute of Regenerative Medicine II initiative. Using a different site in the flap recipient as an in vivo bioreactor, synthetic materials placed into chambers will be used to generate bone flaps of customizable dimensions. These flaps are allowed to vascularize before they are harvested and transferred to the recipient defect. The Osteo Science Foundation award will provide resources to study the osteogenic capabilities of different materials implanted into a validated large animal (sheep) model as well as characterize the niche environment surrounding the bone chamber by analyzing the cell population, gene expression and other tests for osteogenic activity. Additional mechanical and histomorphometric tests will determine the load-bearing characteristics of the construct as well as morphology.
While the project is focused on mandibular defects, the same strategy could be adopted for other challenging defects in the maxillofacial region.
A.M. Tatara, S.R. Shah, N. Demian, T. Ho, J. Shum, J.J.J.P. van den Beucken, J.A. Jansen, M.E. Wong, and A.G. Mikos, “Reconstruction of Large Mandibular Defects Using Autologous Tissues Generated From In Vivo Bioreactors,” Acta Biomaterialia, 45, 72-84 (2016). DOI:10.1016/j.actbio.2016.09.013 Read Abstract
AM Tatara, SR Shah, E Watson, BT Smith, N Demian, IA Hanna, JC Melville, J Shum, T Ho, JJ van den Beucken, JA Jansen, ME Wong, AG Mikos “Ovine Model For Generation of Tissue-Engineered Vascularized Bone of Large Volume and Custom Geometry” Tissue Engineering Part A (22). 2016, Dec; S155-156 Read Abstract
A.M. Tatara, S.R. Shah, E. Watson, B.T. Smith, N. Demian, I.A. Hanna, J.C. Melville, T. Ho, J. Shum, J.J.J.P. van den Beucken, J.A. Jansen, M.E. Wong, and A.G. Mikos, “Leveraging Biomaterials for High Fidelity Mandibular Reconstruction,” Tissue Engineering and Regenerative Medicine International Society 2016 Americas Meeting, San Diego, California, December 11-14th, 2016.
A.M. Tatara, S.R. Shah, E. Watson, B.T. Smith, N. Demian, I.A. Hanna, J.C. Melville, T. Ho, J. Shum, J.J.J.P. van den Beucken, J.A. Jansen, M.E. Wong, and A.G. Mikos, “Ovine Model For Generation of Tissue-Engineered Vascularized Bone of Large Volume and Custom Geometry,” Tissue Engineering and Regenerative Medicine International Society 2016 Americas Meeting, San Diego, California, December 11-14th, 2016.
Hard and Soft Tissue Engineering to Regenerate Mandibular Segmental Defects
Tara Aghaloo is Associate Professor in Oral and Maxillofacial Surgery at the UCLA School of Dentistry. She completed dental school at UMKC, as well as Oral and Maxillofacial Surgery residency, a medical degree, and PhD in oral biology at UCLA. Her research is in bone biology and regeneration, while maintaining an active clinical practice focusing on implants and hard and soft tissue regeneration. She is also active in professional organizations where she is a board member of the Academy of Osseointegration, and section editor of the International Journal of Oral and Maxillofacial Implants.
Through-and-through mandibular defects can arise from the treatment of various pathological processes. Failure to successfully restore mandibular continuity results in devastating consequences for patients. Reconstruction usually involves major autogenous bone grafting with potential complications and morbidity. To explore tissue engineering alternatives to hard and soft tissue grafting for mandibular continuity defects, we created a clinically relevant rat segmental defect model. Though our long-term goal is to incorporate growth factor and collagen matrix-based technologies that can translate basic science research to solve important clinical problems, the short-term goal of this proposal is to explore bone and soft tissue regeneration in animals with mandibular continuity defects. Here, we will directly evaluate rhBMP-2 with Bio-oss +/- cross-linked collagen compared to autogenous bone in mandibular defect bone and soft tissue regeneration. Our rationale is that identifying effective tissue engineering constructs for segmental mandibular defects will improve outcomes and decrease morbidity of autologous-based treatment protocols.
1. Kim IA, Bezouglaia O, Sebastian C, Lee M, Grogan T, St John M, Aghaloo T. Bio-Oss® Successfully Induces Bone Healing in a Marginal Mandibular Defect. Annual American College of Surgeons meeting, Jan 18, 2015, oral abstract presentation.
Thrombopoietin in Cranial Regeneration
Dr. Tien-Min Chu received his DDS degree from Kaohsiung Medical College in Taiwan. He later received his PhD in materials science and engineering from the University of Michigan in 1999. He is currently an Associate Professor of Dental Biomaterials at the Indiana University School of Dentistry. Dr. Chu’s current research activities mainly focus on cranial bone tissue engineering and the in vivo dental implant evaluations.
When large bone loss in the craniofacial area occurs, oral surgeons are faced with a very challenging reconstruction task to address both the functional and the esthetical needs for the patient. To accomplish this, they often use a combination of biological factors and graft materials. In the past, tissue engineering approach of using a three-dimensional (3D) scaffold conforming to the shape of the missing bone, loaded with mesenchymal stem cells (MSCs) and bone morphogenetic protein (BMP) has shown great promise. However, several papers since 2011 revealed serious health risks in association with the use of BMP-2. Recently, we have shown that thrombopoietin (TPO) can indirectly promote the osteogenic differentiation of MSCs and stimulate osteoblast proliferation through its action on megakaryocytes (MKs). Others have shown that TPO can potentially promote angiogenesis and endochondral ossification indirectly through MSCs. In our pilot studies, we demonstrated that TPO can induce bridging callus formation in critical-size femoral defects and can induce bone formation in cranial defects. Combined with our prior success in fabricating 3D scaffolds to carry MSCs, we hypothesize that TPO can indirectly stimulate MSCs delivered by 3D scaffolds to induce complete regeneration in critical-size cranial defects. We will first study the effects of stem cell source (bone marrow versus dental pulp), seeding density and pre-culture condition combinations on bone regeneration from stem cells seeded on scaffold and stimulated by TPO. The best combinations will then be used to investigate the dose-response of TPO in vivo. Finally, the best dose from the dose-response study will be used to study the time-response of TPO and compare that to BMP-2 in vivo. The success of this project will provide preliminary data to secure funding to allow a more comprehensive evaluation on the potentials of using TPO in this challenging task of large cranial defect regeneration.
1. AM Emmakah1, 4, HE Arman2, JC Bragg3, T Greene3, MB Alvarez2, PJ Childress2, WS Goebel4,MA Kacena2, CC Lin1, 3, and TM Chu. A Fast-Degrading Thiol-Acrylate based Hydrogel for Cranial Regeneration, Journal of Biomaterials., at press: 2017. Abstract
1. Emmakah, A.; Alvarez, M.; Childress, P.; Goebel, S.; Kacena, M.; Lin, C.;Chu, T.G., “A Fast-degrading Thiol-acrylate Hydrogel as a Cell Carrier for Craniofacial Bone Regeneration”, Society for Biomaterials, Minneapolis, MN, April 5-8, 2017
2. Emmakah, A.; Greene, T.; Bragg, J.; Lin, C.; Alvarez, M.; Childress, P.; Goebel, S.; Kacena, M.; Chu, T.G., “Cranial regeneration using Stem cells encapsulated in fast-degrading thiol-acrylate hydrogel”, Annual Meeting of the International Association for Dental Research, San Francisco, CA, March 22-25, 2017
3. Emmakah, A.; Alvarez, M.; Childress, P.; Goebel, S.; Kacena, M.; Lin, C.;Chu, T.G., “A fast-degrading thiol-acrylate hydrogel shows promise as a cell carrier for stem-cell assisted cranial regeneration”, Annual Meeting of the Orthopedic Research Society, San Diego CA, March 19-22, 2017
4. Ballenger, B.; Emmakah, A.; Lin, C.; Chu, T.G., “Degradation and Cell Proliferation in a Fast-Degrading Thiol-Acrylate based Hydrogel for Cranial Regeneration”, Annual Meeting of the American Association for Dental Research, Los Angeles, CA, March 16-19, 2016
5. Emmakah, A.; Cheng, Y.; Alvare, B.; Kacena, M.; Chu, T.G., “Proliferation and Differentiation of Stem Cells Co-Cultured with Megakaryocytes”, Annual Meeting of the American Association for Dental Research, Los Angeles, CA, March 16-19, 2016
6. Emmakah, A.; Greene, T.; Bragg, J.; Lin, C.; Chu, T.G., “A Fast-Degrading Thiol-Acrylate based Hydrogel for Cranial Regeneration”, Annual Meeting of the International Association for Dental Research, Seoul Republic of Korea, June 22-25, 2016
7. Emmakah, A.; Cheng, Y.; Alvare, M.; Kacena, M.; Chu, T.G., “Megakaryocytes Enhances Proliferation but Delayed Differentiation in Stem Cells” Annual Meeting of the World Biomaterials Congress, Montreal, QC Canada, May 17-22, 2016
8. Emmakah, A.; Lin, C.; Chu, T.G., “Degradation and Cell Proliferation in a Fast-Degrading Thio-Acrylate based Hydrogel for Cranial Regeneration” Annual Meeting of the World Biomaterials Congress, Montreal, QC Canada, May 17-22, 2016
Tracking Cells and Biomaterial Remodeling in Tissue Engineered Bone Grafts
Dr. Eisig is Chief of Hospital Dental Service at New York-Presbyterian Hospital, and the William Carr Professor and Director of Oral and Maxillofacial Surgery at both the Hospital and Columbia University School of Dental and Oral Surgery. He is a diplomat of the American Board of Oral and Maxillofacial Surgery and volunteers abroad by treating patients with cleft lip and palate on Healing the Children missions to South American countries. He practices full-scope oral and maxillofacial surgery, with a particular interest in orthognathic, craniofacial and cleft palate surgery, maxillofacial pathology and reconstruction, and pediatric oral and maxillofacial surgery.
Tissue engineered bone grafts present an attractive solution to the complexity involved in oral and maxillofacial bone grafts. Despite the functional success with tissue engineered bone grafts for bone reconstruction, little is known concerning the tissue engineered bone graft method of action and the fate of the implanted cells. In the proposed study, the implanted cell localization and contribution to bone regeneration will be investigated in a rat calvarial defect model. Utilizing genetically altered rat adipose derived stem cells (ADSCs) to fabricate our scientifically proven tissue engineered bone grafts, the objectives of the study will provide key information about the implanted stem cells concerning their function in reconstruction and site-localized safety. These results will not only benefit the clinical translation of tissue engineered bone grafts, but will provide important information to educate decisions regarding cell activity in tissue engineering applications in any targeted tissue.
Decellularized Neurovascular Bundle for Craniomaxillofacial Reconstruction
Dr. Kaplan is a Reconstructive Plastic Surgeon and Biomedical Engineer with research interests in craniofacial reconstruction using decellularized tissues, and tissue engineering. He is an Associate Research Professor in the NJ Center for Biomaterials at Rutgers University, and an Adjunct Professor in Regulatory Science at the University of Southern California. Dr. Kaplan has held various clinical and research positions across academia and industry, including Senior Medical Director at Allergan (Fortune 500 healthcare) and Vice President of Clinical Sciences at LifeCell (pioneer in decellularizing dermis). He is a founding board member of the non-profits Grossman Burn Foundation, and Look at Us Alliance for Craniofacial Differences.
In traumatic facial injuries, such as large craniomaxillofacial defects and massive burn scarring, quality of life is dependent on restoring form and function. Regeneration within scarred soft tissues and large bony defects are highly dependent on robust vascular supply and sensory-motor reinnervation. Decellularized bone and soft-tissues, such as dermis and nerve grafts, are commercially available for smaller defects, i.e. those that do not require regeneration through a large 3D volume of tissue. For autologous tissues: graft take generally requires proximity of ~5mm to vascular supply, and nerve autografts exceeding 10cm should be vascularized. We therefore hypothesize that decellularized neurovascular bundles (NVBs) can be re- endothelialized and implanted into large areas of relatively avascular, asensate and/or paralyzed scar tissue; and that in so doing, these defects may be successfully reconstructed by techniques that have otherwise thus far remained suitable to smaller defects only. This research aims to make decellularized allogeneic NVBs available so that craniofacial reconstructions may be performed successfully despite the absence of local autograft vessels and nerves. This will be explored in a rodent animal model using perfusion decellularization techniques and whole-organ bioreactors for recellularization.
1. Kaplan HM. Regulating non-viable tissue. Chapter. Section: Regulatory pathways and barriers to implementation of tissue engineering and regenerative medicine. Richmond FJ, Section ed. In: Warburton D, ed. "Encyclopedia of Tissue Engineering and Regenerative Medicine". USA: Elsevier. In Press, Apr 2018.
2. Kaplan HM. Regulating non-viable tissue. Elsevier Reference Module in Biomedical Sciences. USA: Elsevier Online. In Press, Apr 2018.
1. Kaplan HM, Woloszyn DJ, Chueng D, Rupertus N, Njeze OB, Luo J, Gerli M, Lee KB, Ott H, Kohn J. Engineering autologous human neurovascular bundles from decellularized xenogeneic ones for regenerative medicine applications. Proceedings. 10th Symposium on Biologic Scaffolds for Regenerative Medicine. Napa, CA. 3-5 May 2018. (Attached)
2. Kaplan HM, Woloszyn DJ, Chueng D, Rupertus N, Njeze OB, Luo J, Gerli M, Lee KB, Ott H, Kohn J. Engineering autologous human neurovascular bundles from decellularized xenogeneic ones for
reconstructive microsurgery. Proceedings. 2018 Am Soc Reconstr Microsurg (ASRM) Annual Meeting. Phoenix, AZ. 13-16 Jan 2018.
3. Kaplan HM, Woloszyn DJ, Chueng D, Rupertus N, Njeze OB, Luo J, Gerli M, Lee KB, Ott H, Kohn J. Tissue engineering applications for autologous human neurovascular bundles engineered from xenogeneic ones. Proceedings. 2017 NJ Symposium on Biomaterials Science. Iselin, NJ. 23-24 Oct 2017.
4. Rupertus N, Kaplan H, Woloszyn D, Njeze O, Chang W, Kohn J. Designing a perfusion based multiapplication bioreactor system. Poster. 2017 Summer Student Research Symposium. Piscataway, NJ. 11 Aug 2017.
5. Njeze O, Kaplan H, Woloszyn D, Rupertus N, Chang W, Kohn J. Gas exchange within a perfusion bioreactor system. Poster. 2017 Summer Student Research Symposium. Piscataway, NJ. 11 Aug 2017.
6. Kaplan HM. Regenerative Medicine for Reconstructive Surgery Applications. Grand Rounds. Dartmouth-Hitchcock Medical Center. Lebanon, NH. 5 May 2017.
7. Kaplan HM, Chang W, Bhatnagar D, Kohn J. Strategies for bridging large gaps in peripheral nerve regeneration. Proceedings. 2016 NJ Symposium on Biomaterials Science. Iselin, NJ. 24-25 Oct 2016.
8. Kaplan HM, Gerli MFM, Woloszyn DJ, Chueng D, Lee KB, Kohn J, Ott H. Regenerating decellularized Neurovascular Bundles to support engineered and diseased tissues, and hybrid synthetic scaffolds. Proceedings. 9th Symposium on Biologic Scaffolds for Regenerative Medicine. Napa, CA. April 2016.
9. Woloszyn DJ,* Kaplan HM,* Jain I, Nirgudkar N, Richtmyer M, Sotolongo J, Kohn J. Techniques for harvesting and decellularizing Neurovascularized Muscle to replace autologous free flaps: A comparison between immersion and perfusion decellularization. Poster. 9th Symposium on Biologic Scaffolds for Regenerative Medicine. Napa, CA. April 2016. [* Equal Contributions].
10. Kaplan HM. Present and future ECM-derived products in wound care and soft tissue reconstruction. Proceedings. 2015 NJ Symposium on Biomaterials Science. New Brunswick, NJ. 9 Nov 2015.