- [Show abstract] [Hide abstract] ABSTRACT: Engineering the osteochondral junction requires fabrication of a microenvironment that supports both osteogenesis and chondrogenesis. Multiphasic scaffold strategies utilizing a combination of soluble factors and extracellular matrix components are ideally suited for such applications. In this work, the contribution of an osteogenic nanoparticulate mineralized glycosaminoglycan scaffold (MC-GAG) and a dually chondrogenic and osteogenic growth factor, BMP-9, in the differentiation of primary human mesenchymal stem cells (hMSCs) is evaluated. Although 2D cultures demonstrate alkaline phosphatase activity and mineralization of hMSCs induced by BMP-9, MC-GAG scaffolds do not demonstrate significant differences in the collagen I expression, osteopontin expression, or mineralization. Instead, BMP-9 increases expression of collagen II, Sox9, aggrecan (ACAN), and cartilage oligomeric protein. However, the hypertrophic chondrocyte marker, collagen X, is not elevated with BMP-9 treatment. In addition, histologic analyses demonstrate that while BMP-9 does not increase mineralization, BMP-9 treatment results in an increase of sulfated glycosaminoglycans. Thus, the combination of BMP-9 and MC-GAG stimulates chondrocytic and osteogenic differentiation of hMSCs.
- [Show abstract] [Hide abstract] ABSTRACT: Successful bone tissue engineering requires at the minimum sufficient osteoblast progenitors, efficient osteoinductive factors, and biocompatible scaffolding materials. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing osteogenic differentiation of mesenchymal stem cells (MSCs). Here, we investigated the potential use of a biodegradable citrate-based thermosensitive macromolecule, poly(polyethyleneglycol citrate-co-N-isopropylacrylamide) (PPCN) mixed with gelatin (PPCNG) as a scaffold for the delivery of BMP9-stimulated MSCs to promote localized bone formation. The addition of gelatin to PPCN effectively enhanced the cell adhesion and survival properties of MSCs entrapped within the gel in 3D culture. Using the BMP9-transduced MSC line immortalized mouse embryonic fibroblasts (iMEFs), we found that PPCNG facilitated BMP9-induced osteogenic differentiation of iMEFs in vivo and promoted the formation of well-ossified and vascularized trabecular bone-like structures in a mouse model of ectopic bone formation. Histologic evaluation revealed that vascularization of the bony masses retrieved from the iMEFs + PPCNG group was significantly more pronounced than that of the direct cell injection group. Accordingly, vascular endothelial growth factor (VEGF) expression was shown to be significantly higher in the bony masses recovered from the iMEFs + PPCNG group. Taken together, our results suggest that PPCNG may serve as a novel biodegradable and injectable scaffold and carrier for gene and cell-based bone tissue engineering.
- [Show abstract] [Hide abstract] ABSTRACT: Current reconstructive approaches to large craniofacial skeletal defects are often complicated and challenging. Critical-sized defects are unable to heal via natural regenerative processes and require surgical intervention, traditionally involving autologous bone (mainly in the form of nonvascularized grafts) or alloplasts. Autologous bone grafts remain the gold standard of care in spite of the associated risk of donor site morbidity. Tissue engineering approaches represent a promising alternative that would serve to facilitate bone regeneration even in large craniofacial skeletal defects. This strategy has been tested in a myriad of iterations by utilizing a variety of osteoconductive scaffold materials, osteoblastic stem cells, as well as osteoinductive growth factors and small molecules. One of the major challenges facing tissue engineers is creating a scaffold fulfilling the properties necessary for controlled bone regeneration. These properties include osteoconduction, osetoinduction, biocompatibility, biodegradability, vascularization, and progenitor cell retention. This review will provide an overview of how optimization of the aforementioned scaffold parameters facilitates bone regenerative capabilities as well as a discussion of common osteoconductive scaffold materials.
- [Show abstract] [Hide abstract] ABSTRACT: Craniosynostosis, a condition in which the cranial sutures prematurely fuse, can lead to elevated intracranial pressure and craniofacial abnormalities in young children. Currently surgical intervention is the only therapeutic option for patients with this condition. Craniosynostosis has been associated with a variety of different gene mutations and chromosome anomalies. Here we describe three cases of partial deletion of chromosome 19p. Two of the cases present with syndromic craniosynostosis while one has metopic ridging. A review of the genes involved in the rearrangements between the three cases suggests several gene candidates for craniosynostosis. CALR and DAND5, BMP regulators involved in osteoblast differentiation, and MORG1, a mediator of osteoclast dysregulation may play a role in abnormal cranial vault development. Additionally, CACNA1A, a gene that when mutated is associated with epilepsy and CC2D1A, a gene associated with nonsyndromic mental retardation may contribute to additional phenotypic features seen in the patients we describe. In addition, these findings further support the need for genetic testing in cases of syndromic craniosynostosis.
- [Show abstract] [Hide abstract] ABSTRACT: Critical-sized craniofacial defect repair represents a significant challenge to reconstructive surgeons. Many strategies have been employed in an effort to achieve both a functionally and cosmetically acceptable outcome. Bone morphogenetic proteins (BMPs) provide a robust osteoinductive cue to stimulate bony growth and remodeling. Previous studies have suggested that the BMP-9 isoform is particularly effective in promoting osteogenic differentiation of mesenchymal progenitor cells. The aim of this study is to characterize the osteogenic capacity of BMP-9 on calvarial mesenchymal progenitor cell differentiation. Reversibly immortalized murine calvarial progenitor cells (iCALs) were infected with adenoviral vectors encoding BMP-9 or GFP and assessed for early and late stages of osteogenic differentiation in vitro and for osteogenic differentiation via in vivo stem cell implantation studies. Significant elevations in alkaline phosphatase (ALP) activity, osteocalcin (OCN) mRNA transcription, osteopontin (OPN) protein expression, and matrix mineralization were detected in BMP-treated cells compared to control. Specifically, ALP activity was elevated on days 3, 7, 9, 11, and 13 post-infection and OCN mRNA expression was elevated on days 8, 10, and 14 in treated cells. Additionally, treatment groups demonstrated increased OPN protein expression on day 10 and matrix mineralization on day 14 post-infection relative to control groups. BMP-9 also facilitated the formation of new bone in vivo as detailed by gross, microcomputed tomography, and histological analyses. Therefore, we concluded that BMP-9 significantly stimulates osteogenic differentiation in iCALs, and should be considered an effective agent for calvarial tissue regeneration.
- [Show abstract] [Hide abstract] ABSTRACT: Little is known about the role of osteoclasts in cranial suture fusion. Osteoclasts are predominantly regulated by receptor activator of nuclear factor kappa B and receptor activator of nuclear factor kappa B ligand, both of which lead to osteoclast differentiation, activation, and survival; and osteoprotegerin, a soluble inhibitor of receptor activator of nuclear factor kappa B. The authors' work examines the role of osteoprotegerin in this process using knockout technology. Wild-type, osteoprotegerin-heterozygous, and osteoprotegerin-knockout mice were imaged by serial micro-computed tomography at 3, 5, 7, 9, and 16 weeks. Suture density measurements and craniometric analysis were performed at these same time points. Posterofrontal sutures were harvested from mice after the week-16 time point and analyzed by means of histochemistry. Micro-computed tomographic analysis of the posterofrontal suture revealed reduced suture fusion in osteoprotegerin-knockout mice compared with wild-type and heterozygous littermates. Osteoprotegerin deficiency resulted in a statistically significant decrease in suture bone density in knockout mice. There was no reduction in the density of non-suture-containing calvarial bone between wild-type and osteoprotegerin-knockout mice. Histochemistry of suture sections supported these micro-computed tomographic findings. Finally, osteoprotegerin-knockout mice had reduced anteroposterior skull distance at all time points and an increased interorbital distance at the week-16 time point. The authors' data suggest that perturbations in the expression of osteoprotegerin and subsequent changes in osteoclastogenesis lead to alterations in murine cranial and posterofrontal suture morphology.
- [Show abstract] [Hide abstract] ABSTRACT: Modification of the lengthening temporalis myoplasty for reanimation of facial paralysis is presented. A patient experienced traumatic laceration of the left facial nerve resulting in left hemifacial paralysis. Multiple attempts at nerve repair were unsuccessful. For smile restoration, a Labbé procedure was performed. Because of inadequate length, the temporalis tendon could not be directly secured to the modiolus. Therefore, an inferiorly based temporalis fascia flap was recruited from the deep temporal fascia and reflected inferiorly to provide additional length by which the tendon could be secured to the modiolus. This technique provided immediate smile restoration and required no additional donor site.
- [Show abstract] [Hide abstract] ABSTRACT: Bone morphogenetic proteins (BMPs) play a sentinel role in osteoblastic differentiation, and their implementation into clinical practice can revolutionize cranial reconstruction. Preliminary data suggest a therapeutic role of adenoviral gene delivery of BMPs in murine calvarial defect healing. Poor transgene expression inherent in direct adenoviral therapy prompted investigation of cell-based strategies. To isolate and immortalize calvarial cells as a potential progenitor source for osseous tissue engineering. Cells were isolated from murine skulls, cultured, and transduced with a retroviral vector bearing the loxP-flanked SV40 large T antigen. Immortalized calvarial cells (iCALs) were evaluated via light microscopy, immunohistochemistry, and flow cytometry to determine whether the immortalization process altered cell morphology or progenitor cell profile. Immortalized calvarial cells were then infected with adenoviral vectors encoding BMP-2 or GFP and assessed for early and late stages of osteogenic differentiation. Immortalization of calvarial cells did not alter cell morphology as demonstrated by phase contrast microscopy. Mesenchymal progenitor cell markers CD166, CD73, CD44, and CD105 were detected at varying levels in both primary cells and iCALs. Significant elevations in alkaline phosphatase activity, osteocalcin mRNA transcription, and matrix mineralization were detected in BMP-2 treated iCALs compared with GFP-treated cells. Gross and histological analyses revealed ectopic bone production from treated cells compared with controls in an in vivo stem cell implantation assay. We have established an immortalized osteoprogenitor cell line from juvenile calvarial cells that retain a progenitor cell phenotype and can successfully undergo osteogenic differentiation upon BMP-2 stimulation. These cells provide a valuable platform to investigate the molecular mechanisms underlying intramembranous bone formation and to screen for factors/small molecules that can facilitate the healing of osseous defects in the craniofacial skeleton.
- [Show abstract] [Hide abstract] ABSTRACT: Introduction Distraction osteogenesis is a powerful tool in craniomaxillofacial surgery, allowing for large advancements of osteotomized segments in the setting of a restrictive soft tissue envelope. Despite its benefits, distraction can have negative functional consequences. We present a case of a patient with Crouzon syndrome who developed reduced mouth opening capability after a Le Fort III midfacial advancement with rigid external distraction. Technique Radiographic evaluation revealed that the coronoid process was restricting the normal excursion of the mandible by contacting the posterior zygoma. The patient was subsequently treated with a bilateral coronoidectomy via an intraoral approach, which improved his interincisal opening. Maximum interincisal distance was improved from 18 mm to 33 mm following bilateral cornoid resection. Conclusion We report coronoid impingement as a potential complication after Le Fort III distraction. Such a finding suggests the need for a detailed vector analysis in cases undergoing midface advancement with distraction. Post-distraction coronoidectomy is a useful surgical procedure to treat mouth opening limitation due to coronoid impingement against the zygoma after midfacial advancement.
Article: Abstract P69
Article: Abstract P69
- [Show abstract] [Hide abstract] ABSTRACT: Sustained, high level transgene expression in mammalian cells, especially stem cells, may be desired in many cases for studying gene functions. Traditionally, stable transgene expression has been accomplished by using retroviral or lentiviral vectors. However, such viral vector-mediated transgene expression is often at low levels and can be reduced over time due to low copy numbers and/or chromatin remodeling repression. The piggyBac transposon has emerged as a promising non-viral vector system for efficient gene transfer into mammalian cells. Despite its inherent advantages over lentiviral and retroviral systems, piggyBac system has not been widely used, at least in part due to the limited availability of piggyBac vectors with manipulation flexibilities. Here, we seek to optimize piggyBac-mediated transgene expression and generate a more efficient, user-friendly piggyBac system. By engineering a panel of versatile piggyBac vectors and constructing recombinant adenoviruses expressing piggyBac transposase (PBase), we demonstrate that adenovirus-mediated PBase expression significantly enhances the integration efficiency and expression level of transgenes in mesenchymal stem cells and osteosarcoma cells, compared to that obtained from co-transfection of the CMV-PBase plasmid. We further determine the drug selection timeline to achieve optimal stable transgene expression. Moreover, we demonstrate that the transgene copy number of piggyBac-mediated integration is approximately 10 times higher than that mediated by retroviral vectors. Using the engineered tandem expression vector, we show that three transgenes can be simultaneously expressed in a single vector with high efficiency. Thus, these results strongly suggest that the optimized piggyBac system is a valuable tool for making stable cell lines with sustained, high transgene expression.
- [Show abstract] [Hide abstract] ABSTRACT: Fibroblast growth factors (FGF) and their receptors serve many functions in both the developing and adult organism. Humans contain 18 FGF ligands and four FGF receptors (FGFR). FGF ligands are polypeptide growth factors that regulate several developmental processes including cellular proliferation, differentiation, and migration, morphogenesis, and patterning. FGF-FGFR signaling is also critical to the developing axial and craniofacial skeleton. In particular, the signaling cascade has been implicated in intramembranous ossification of cranial bones as well as cranial suture homeostasis. In the adult, FGFs and FGFRs are crucial for tissue repair. FGF signaling generally follows one of three transduction pathways: RAS/MAP kinase, PI3/AKT, or PLCγ. Each pathway likely regulates specific cellular behaviors. Inappropriate expression of FGF and improper activation of FGFRs are associated with various pathologic conditions, unregulated cell growth, and tumorigenesis. Additionally, aberrant signaling has been implicated in many skeletal abnormalities including achondroplasia and craniosynostosis. The biology and mechanisms of the FGF family have been the subject of significant research over the past 30 years. Recently, work has focused on the therapeutic targeting and potential of FGF ligands and their associated receptors. The majority of FGF-related therapy is aimed at age-related disorders. Increased understanding of FGF signaling and biology may reveal additional therapeutic roles, both in utero and postnatally. This review discusses the role of FGF signaling in general physiologic and pathologic embryogenesis and further explores it within the context of skeletal development.
- [Show abstract] [Hide abstract] ABSTRACT: RNA interference (RNAi) denotes sequence-specific mRNA degradation induced by short interfering double-stranded RNA (siRNA) and has become a revolutionary tool for functional annotation of mammalian genes, as well as for development of novel therapeutics. The practical applications of RNAi are usually achieved by expressing short hairpin RNAs (shRNAs) or siRNAs in cells. However, a major technical challenge is to simultaneously express multiple siRNAs to silence one or more genes. We previously developed pSOS system, in which siRNA duplexes are made from oligo templates driven by opposing U6 and H1 promoters. While effective, it is not equipped to express multiple siRNAs in a single vector. Gibson DNA Assembly (GDA) is an in vitro recombination system that has the capacity to assemble multiple overlapping DNA molecules in a single isothermal step. Here, we developed a GDA-based pSOK assembly system for constructing single vectors that express multiple siRNA sites. The assembly fragments were generated by PCR amplifications from the U6-H1 template vector pB2B. GDA assembly specificity was conferred by the overlapping unique siRNA sequences of insert fragments. To prove the technical feasibility, we constructed pSOK vectors that contain four siRNA sites and three siRNA sites targeting human and mouse β-catenin, respectively. The assembly reactions were efficient, and candidate clones were readily identified by PCR screening. Multiple β-catenin siRNAs effectively silenced endogenous β-catenin expression, inhibited Wnt3A-induced β-catenin/Tcf4 reporter activity and expression of Wnt/β-catenin downstream genes. Silencing β-catenin in mesenchymal stem cells inhibited Wnt3A-induced early osteogenic differentiation and significantly diminished synergistic osteogenic activity between BMP9 and Wnt3A in vitro and in vivo. These findings demonstrate that the GDA-based pSOK system has been proven simplistic, effective and versatile for simultaneous expression of multiple siRNAs. Thus, the reported pSOK system should be a valuable tool for gene function studies and development of novel therapeutics.
- [Show abstract] [Hide abstract] ABSTRACT: Hostile sites for cranioplasty occur in patients with a history of radiation, infection, failed cranioplasty, CSF leak or acute infection. We review our series of autologous cranioplasties and present an approach to decision-making for reconstructing these complex defects. Patients with cranioplasty of a hostile cranial site at the University of Chicago between 2003 and 2012 were identified. They were stratified into three groups: chimeric free flap with vascularized bone (the vascular group), non-vascularized bone with local coverage (the non-vascular group) and non-vascularized bone with free flap (the mixed group). The primary outcome measure was a major complication in the year following cranioplasty, identified by flap or bone graft failure. We reviewed 33 cases; 14 "vascular", 13 "non-vascular", and 8 "mixed". There was no difference in flap or bone graft failure rates, which were 7% (1/14) for the vascular group, 8% (1/13) for the non-vascular group, and 0% for the mixed group (p = NS). Overall complication rate was statistically different between the three groups (p = 0.01). The non-vascular group had the lowest complication rate (31%). Based on our data we developed an assessment score (The University of Chicago CRAnial Severity Score of Hostility, CRASSH) for patient and treatment stratification. Vascularized, non-vascularized and mixed reconstructive methods can be used successfully in these challenging situations. We offer the CRASSH to aid in aligning patients with the most appropriate autologous reconstruction method for their hostile cranial sites. Copyright © 2015 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.
- [Show abstract] [Hide abstract] ABSTRACT: The normal growth and development of the skull is a tightly regulated process that occurs along the osteogenic interfaces of the cranial sutures. Here, the borders of the calvarial bones and neighboring tissues above and below, function as a complex. Through coordinated remodeling efforts of bone deposition and resorption, the cranial sutures maintain a state of patency from infancy through early adulthood as the skull continues to grow and accommodate the developing brain’s demands for expansion. However, when this delicate balance is disturbed, a number of pathologic conditions ensue; and if left uncorrected, may result in visual and neurocognitive impairments. A prime example includes craniosynostosis, or premature fusion of one or more cranial and/or facial suture(s). At the present time, the only therapeutic measure for craniosynostosis is surgical correction by cranial vault reconstruction. However, elegant studies performed over the past decade have identified several genes critical for the maintenance of suture patency and induction of suture fusion. Such deeper understandings of the pathogenesis and molecular mechanisms that regulate suture biology may provide necessary insights toward the development of non-surgical therapeutic alternatives for patients with cranial suture defects. In this review, we discuss the intricate cellular and molecular interplay that exists within the suture among its three major components: dura mater, osteoblastic related molecular pathways and osteoclastic related molecular pathways.
- [Show abstract] [Hide abstract] ABSTRACT: The present study is a case report of a 3-year-old girl who was referred to our clinic with the clinical features of cherubism. A locally aggressive tumor was diffusely infiltrating the maxilla and mandible. At 4 years after resection, our patient has not demonstrated any signs of recurrence, which might point to a role for adjunctive chemotherapy, in this case imatinib (Gleevec), for odontogenic myxoma.
- [Show abstract] [Hide abstract] ABSTRACT: Reconstruction of craniofacial defects in children presents several challenges that are not encountered in the adult population. Autologous bone grafts have long been the criterion standard for repairing these defects. Recently, several new materials and techniques have expanded our arsenal of reconstructive options. In this clinical report, we describe the use of both particulate bone grafting and demineralized bone matrix together to repair craniofacial defects encountered in pediatric patients.
- [Show abstract] [Hide abstract] ABSTRACT: The reconstructive goals for myelodysplastic defects are to provide a multilayered, tension-free and well-vascularized closure to prevent cerebrospinal fluid leakage, wound infection or breakdown and to optimize neurologic outcomes. We reviewed our ten-year experience with myelodysplastic defects and our preferred technique for large defects utilizing paraspinous flaps followed by V-Y crescentic rotation advancement flaps. A retrospective chart review was performed on all myelodysplastic defects closed at the University of Chicago Medicine from 2002 to 2012. Twenty-three patients were treated: eight were closed using V-Y crescentic rotation advancement flaps, eight primarily, two with transposition flaps and five with bilateral latissimus dorsi and gluteus maximus myocutaneous flaps. Patient defect characteristics, reconstructive details, follow up time, and wound complications were analyzed. The primary closure group included eight patients. There was one minor complication and two major complications that required debridement and plastic surgery consultation in this group. The transposition group included two patients and had no wound healing issues. The latissimus and gluteus myocutaneous group included five patients and had one minor wound healing issues. The V-Y crescentic group included eight patients. There were four minor wound breakdowns in the lateral donor sites and one major wound complication involving a CSF leak, meningitis and wound breakdown that required debridement. The groups were stratified by size, <5 cm and >5 cm, and further analyzed. Bilateral V-Y crescentic rotation advancement flap is a useful option when confronted with large myelodysplastic defects. It provides a multilayer, tension-free wound closure and spares the gluteus maximus and latissimus dorsi muscle groups.
The Children's Hospital of Philadelphia
Philadelphia, Pennsylvania, United States
- Division of Plastic and Reconstructive Surgery
University of Chicago
Chicago, Illinois, United States
- Department of Surgery