RESUMEN
Recent advances in the understanding of pluripotent stem cell biology and emerging technologies to reprogram somatic cells to a stem cell-like state are helping bring stem cell therapies for a range of human disorders closer to clinical reality. Human pluripotent stem cells (hPSCs) have become a promising resource for regenerative medicine and research into early development because these cells are able to self-renew indefinitely and are capable of differentiation into specialized cell types of all 3 germ layers and trophoectoderm. Human PSCs include embryonic stem cells (hESCs) derived from the inner cell mass of blastocyst-stage embryos and induced pluripotent stem cells (hiPSCs) generated via the reprogramming of somatic cells by the overexpression of key transcription factors. The application of hiPSCs and the finding that somatic cells can be directly reprogrammed into different cell types will likely have a significant impact on regenerative medicine. However, a major limitation for successful therapeutic application of hPSCs and their derivatives is the potential xenogeneic contamination and instability of current culture conditions. This review summarizes recent advances in hPSC culture and methods to induce controlled lineage differentiation through regulation of cell-signaling pathways and manipulation of gene expression as well as new trends in direct reprogramming of somatic cells.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Regeneración Tisular Dirigida/tendencias , Células Madre Pluripotentes/fisiología , Diferenciación Celular/fisiología , Linaje de la Célula , Proliferación Celular , Células Madre Embrionarias/fisiología , Regulación de la Expresión Génica/fisiología , Estratos Germinativos/citología , Humanos , Transducción de Señal/fisiologíaRESUMEN
Current practices to maintain human pluripotent stem cells (hPSCs), which include induced pluripotent stem cells and embryonic stem cells, in an undifferentiated state typically depend on the support of feeder cells such as mouse embryonic fibroblasts (MEFs) or an extracellular matrix such as Matrigel. Culture conditions that depend on these undefined support systems limit our ability to interpret mechanistic studies aimed at resolving how hPSCs interact with their extracellular environment to remain in a unique undifferentiated state and to make fate-changing lineage decisions. Likewise, the xenogeneic components of MEFs and Matrigel ultimately hinder our ability to use pluripotent stem cells to treat debilitating human diseases. Many of these obstacles have been overcome by the development of synthetic coatings and bioreactors that support hPSC expansion and self-renewal within defined culture conditions that are free from xenogeneic contamination. The establishment of defined culture conditions and synthetic matrices will facilitate studies to more precisely probe the molecular basis of pluripotent stem cell self-renewal and differentiation. When combined with three-dimensional cultures in bioreactors, these systems will also enable large-scale expansion for future clinical applications.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Embrionarias/citología , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes/citología , Animales , Reactores Biológicos , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Colágeno , Combinación de Medicamentos , Matriz Extracelular , Células Nutrientes , Humanos , Laminina , Ratones , ProteoglicanosRESUMEN
Human-induced pluripotent stem cells (hiPSCs) may represent an ideal cell source for research and applications in regenerative medicine. However, standard culture conditions that depend on the use of undefined substrates and xenogeneic medium components represent a significant obstacle to clinical translation. Recently, we reported a defined culture system for human embryonic stem cells using a synthetic polymer coating, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH), in conjunction with xenogeneic-free culture medium. Here, we tested the hypothesis that iPSCs could be maintained in an undifferentiated state in this xeno-free culture system and subsequently be differentiated into mesenchymal stem cells (iPS-MSCs). hiPSCs were cultured on PMEDSAH and differentiated into functional MSCs, as confirmed by expression of characteristic MSC markers (CD166+, CD105+, CD90+,CD73+, CD31-, CD34-, and CD45-) and their ability to differentiate in vitro into adipogenic, chondrogenic, and osteoblastic lineages. To demonstrate the potential of iPS-MSCs to regenerate bone in vivo, the newly derived cells were induced to osteoblast differentiation for 4 days and transplanted into calvaria defects in immunocompromised mice for 8 weeks. MicroCT and histologic analyses demonstrated de novo bone formation in the calvaria defects for animals treated with iPS-MSCs but not for the control group. Moreover, positive staining for human nuclear antigen and human mitochondria monoclonal antibodies confirmed the participation of the transplanted hiPS-MSCs in the regenerated bone. These results demonstrate that hiPSCs cultured in a xeno-free system have the capability to differentiate into functional MSCs with the ability to form bone in vivo.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Células Madre Mesenquimatosas/citología , Animales , Procesos de Crecimiento Celular/fisiología , Células Cultivadas , Técnicas Citológicas/métodos , Femenino , Humanos , Huésped Inmunocomprometido , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones , Especificidad por SustratoRESUMEN
Leptin influences bone formation centrally through the hypothalamus and peripherally by acting on osteoblasts or their precursors. However, neither mechanism explains the divergent, gender-specific correlation between leptin and bone mineral density in humans. Although leptin is a potent regulator of pro-inflammatory immune responses, a potential role for leptin as an osteoimmunologic intermediate in bone metabolism has not been tested. Mice with myeloid-specific ablation of the long-form leptin receptor (ObRb) were generated using mice expressing cre-recombinase from the lysoszyme M promoter. At 12 weeks of age, the conditional knockout mice did not display any appreciable phenotype. However, at 52 weeks 2 changes were noted. First, there was a mild increase in liver inflammation. Second, a gender-specific, divergent bone phenotype was observed. Female mice displayed a consistent trend toward decreased trabecular bone parameters including reductions in bone volume fraction, trabecular number, and bone mineral content as well as a significant increase in marrow adipogenesis. Conversely, male mice lacked trabecular changes, but had statistically significant increases in cortical bone volume, thickness, and bone mineral density with equivalent total cortical volume. Since the year 2000, over 25 studies on more than 10,000 patients have sought to determine the correlation between leptin and bone mineral density. The results revealed a gender-specific correlation similar to that observed in our LysM transgenic animals. We hypothesize and show new evidence that regulation of myeloid lineage cells by leptin may facilitate their actions as an osteoimmunologic intermediate and contribute to leptin-regulated bone formation and metabolism in a gender-specific manner.
Asunto(s)
Huesos/metabolismo , Linaje de la Célula , Leptina/metabolismo , Células Mieloides/citología , Células Mieloides/metabolismo , Animales , Huesos/diagnóstico por imagen , Femenino , Fémur/diagnóstico por imagen , Fémur/metabolismo , Integrasas/metabolismo , Masculino , Ratones , Ratones Noqueados , Muramidasa , Receptores de Leptina/deficiencia , Receptores de Leptina/metabolismo , Microtomografía por Rayos XRESUMEN
Osteonecrosis of the jaw (ONJ), a side-effect of bisphosphonate therapy, is characterized by exposed bone that fails to heal within eight weeks. Healing time of oral epithelial wounds is decreased in the presence of amino-bisphosphonates; however, the mechanism remains unknown. We examined human tissue from individuals with ONJ and non-bisphosphonate-treated control individuals to identify changes in oral epithelium and connective tissue. Oral and intravenous bisphosphonate-treated ONJ sites had reduced numbers of basal epithelial progenitor cells, as demonstrated by a 13.8±1.1% and 31.9±5.8% reduction of p63 expression, respectively. No significant differences in proliferation rates, vessel density, or macrophage number were noted. In vitro treatment of clonal and primary oral keratinocytes with zoledronic acid (ZA) inhibited p63, and expression was rescued by the addition of mevalonate pathway intermediates. In addition, both ZA treatment and p63 shRNA knock-down impaired formation of 3D Ex Vivo Produced Oral Mucosa Equivalents (EVPOME) and closure of an in vitro scratch assay. Analysis of our data suggests that bisphosphonate treatment may delay oral epithelial healing by interfering with p63-positive progenitor cells in the basal layer of the oral epithelium in a mevalonate-pathway-dependent manner. This delay in healing may increase the likelihood of osteonecrosis developing in already-compromised bone.
Asunto(s)
Células Madre Adultas/efectos de los fármacos , Conservadores de la Densidad Ósea/efectos adversos , Difosfonatos/efectos adversos , Imidazoles/efectos adversos , Enfermedades Maxilomandibulares/genética , Queratinocitos/efectos de los fármacos , Proteínas de la Membrana/genética , Osteonecrosis/genética , Análisis de Varianza , Estudios de Casos y Controles , Línea Celular Transformada , Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Enfermedades Maxilomandibulares/inducido químicamente , Queratinocitos/metabolismo , Proteínas de la Membrana/biosíntesis , Ácido Mevalónico/metabolismo , Osteonecrosis/inducido químicamente , ARN Interferente Pequeño , Técnicas de Cultivo de Tejidos , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/genética , Ácido ZoledrónicoRESUMEN
Ameloblastin null mice fail to make an enamel layer, but the defects could be due to an absence of functional ameloblastin or to the secretion of a potentially toxic mutant ameloblastin. We hypothesized that the enamel phenotype could be rescued by the transgenic expression of normal ameloblastin in Ambn mutant mice. We established and analyzed 5 transgenic lines that expressed ameloblastin from the amelogenin (AmelX) promoter and identified transgenic lines that express virtually no transgene, slightly less than normal (Tg+), somewhat higher than normal (Tg++), and much higher than normal (Tg+++) levels of ameloblastin. All lines expressing detectable levels of ameloblastin at least partially recovered the enamel phenotype. When ameloblastin expression was only somewhat higher than normal, the enamel covering the molars and incisors was of normal thickness, had clearly defined rod and interrod enamel, and held up well in function. We conclude that ameloblastin is essential for dental enamel formation.
Asunto(s)
Proteínas del Esmalte Dental/genética , Esmalte Dental/patología , Transgenes/genética , Amelogénesis/genética , Amelogenina/análisis , Amelogenina/genética , Animales , Western Blotting , Esmalte Dental/ultraestructura , Proteínas del Esmalte Dental/análisis , Genotipo , Heterocigoto , Incisivo/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Electrónica de Rastreo , Diente Molar/patología , Mutación/genética , Fenotipo , Regiones Promotoras Genéticas/genéticaRESUMEN
Although advances in surgical techniques and bone grafting have significantly improved the functional and cosmetic restoration of craniofacial structures lost because of trauma or disease, there are still significant limitations in our ability to regenerate these tissues. The regeneration of oral and craniofacial tissues presents a formidable challenge that requires synthesis of basic science, clinical science, and engineering technology. Tissue engineering is an interdisciplinary field of study that addresses this challenge by applying the principles of engineering to biology and medicine toward the development of biological substitutes that restore, maintain, and improve normal function. This review will explore the impact of biomaterials design, stem cell biology and gene therapy on craniofacial tissue engineering.
Asunto(s)
Bioingeniería/métodos , Regeneración Ósea/fisiología , Huesos Faciales/fisiología , Cráneo/fisiología , Materiales Biocompatibles/química , Tecnología Biomédica , Terapia Genética/métodos , Humanos , Células Madre/fisiología , Ingeniería de Tejidos/métodosRESUMEN
Experimental data and limited patient experience suggest that rhBMP-2 can be used to regenerate bone in acquired segmental defects of the mandible. Most of these defects are caused by resection of oral squamous cell carcinoma (OSCC) and the biologic effects of rhBMP-2 on these carcinoma cells are unknown. The objective of this study was to determine whether rhBMP-2 produces adverse effects on proliferation and angiogenesis in OSCC, two biologic processes critical to tumor formation. In vitro studies included treating OSCC cells with rhBMP-2 or an adenoviral vector containing the cDNA for BMP-2. In vivo studies involved co-transplantation of OSCC cells with bone marrow stromal cells genetically modified to over express BMP-2, to mimic a clinically relevant scenario for regenerating bone using cell-based therapy in a wound containing microscopic residual disease. Proliferation, as measured by a MTT assay in vitro and tumor growth in vivo was not affected by treatment with BMP-2. Angiogenesis, measured by secretion of the proangiogenic molecules VEGF and IL-8 in vitro and microvessel density in vivo, was not affected. Exposure of OSCC cells to BMP-2 does not stimulate proliferation or angiogenesis. Further studies are needed before using rhBMP-2 for bone tissue engineering in oral cancer-related defects.
Asunto(s)
Proteínas Morfogenéticas Óseas/farmacología , Carcinoma de Células Escamosas/patología , Neoplasias de la Boca/patología , Neovascularización Patológica/patología , Proteínas Recombinantes/farmacología , Factor de Crecimiento Transformador beta/farmacología , Adenoviridae/genética , Animales , Trasplante de Médula Ósea/patología , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/genética , Regeneración Ósea/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN Complementario/genética , Femenino , Vectores Genéticos/genética , Humanos , Interleucina-8/análisis , Ratones , Ratones Mutantes , Ratones Desnudos , Microvasos/efectos de los fármacos , Microvasos/patología , Trasplante de Neoplasias/patología , Proteínas Recombinantes/genética , Células del Estroma/trasplante , Factor de Crecimiento Transformador beta/genética , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular/análisis , Factor de von Willebrand/análisisRESUMEN
Because bone reconstruction in irradiated sites is less than ideal, we applied a regenerative gene therapy method in which a cell-signaling virus was localized to biomaterial scaffolds to regenerate wounds compromised by radiation therapy. Critical-sized defects were created in rat calvariae previously treated with radiation. Gelatin scaffolds containing lyophilized adenovirus encoding BMP-2 (AdBMP-2) or freely suspended AdBMP-2 were transplanted. Lyophilized AdBMP-2 significantly improved bone quality and quantity over free AdBMP-2. Bone mineral density was reduced after radiotherapy. Histological analyses demonstrated that radiation damage led to less bone regeneration. The woven bone and immature marrow formed in the radiated defects indicated that irradiation retarded normal bone development. Finally, we stored the scaffolds with lyophilized AdBMP-2 at -80 degrees C to determine adenovirus stability. Micro-CT quantification demonstrated no significant differences between bone regeneration treated with lyophilized AdBMP-2 before and after storage, suggesting that virus-loaded scaffolds may be convenient for application as pre-made constructs.
Asunto(s)
Proteína Morfogenética Ósea 2/fisiología , Regeneración Ósea/fisiología , Regeneración Tisular Dirigida/métodos , Radioterapia/efectos adversos , Cráneo/efectos de la radiación , Implantes Absorbibles , Adenoviridae/genética , Animales , Densidad Ósea/fisiología , Proteína Morfogenética Ósea 2/administración & dosificación , Proteína Morfogenética Ósea 2/genética , Regeneración Ósea/genética , Portadores de Fármacos , Terapia Genética , Vectores Genéticos/administración & dosificación , Implantes Experimentales , Oseointegración/efectos de los fármacos , Dosis de Radiación , Ratas , Ratas Endogámicas F344 , Procedimientos de Cirugía Plástica/métodos , Cráneo/fisiología , Cráneo/cirugía , Ingeniería de Tejidos/métodos , Andamios del Tejido , Microtomografía por Rayos XRESUMEN
Gene therapy is defined as the treatment of disease by transfer of genetic material into cells. This review will explore methods available for gene transfer as well as current and potential applications for craniofacial regeneration, with emphasis on future development and design. Though non-viral gene delivery methods are limited by low gene transfer efficiency, they benefit from relative safety, low immunogenicity, ease of manufacture, and lack of DNA insert size limitation. In contrast, viral vectors are nature's gene delivery machines that can be optimized to allow for tissue-specific targeting, site-specific chromosomal integration, and efficient long-term infection of dividing and non-dividing cells. In contrast to traditional replacement gene therapy, craniofacial regeneration seeks to use genetic vectors as supplemental building blocks for tissue growth and repair. Synergistic combination of viral gene therapy with craniofacial tissue engineering will significantly enhance our ability to repair and replace tissues in vivo.
Asunto(s)
Regeneración Ósea/genética , Huesos Faciales/fisiología , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos , Animales , Carcinoma de Células Escamosas/terapia , Neoplasias de Cabeza y Cuello/terapia , Humanos , Trasplante de Piel/métodos , Ingeniería de Tejidos , VirusRESUMEN
More than 85% of the global population requires repair or replacement of a craniofacial structure. These defects range from simple tooth decay to radical oncologic craniofacial resection. Regeneration of oral and craniofacial tissues presents a formidable challenge that requires synthesis of basic science, clinical science and engineering technology. Identification of appropriate scaffolds, cell sources and spatial and temporal signals (the tissue engineering triad) is necessary to optimize development of a single tissue, hybrid organ or interface. Furthermore, combining the understanding of the interactions between molecules of the extracellular matrix and attached cells with an understanding of the gene expression needed to induce differentiation and tissue growth will provide the design basis for translating basic science into rationally developed components of this tissue engineering triad. Dental tissue engineers are interested in regeneration of teeth, oral mucosa, salivary glands, bone and periodontium. Many of these oral structures are hybrid tissues. For example, engineering the periodontium requires growth of alveolar bone, cementum and the periodontal ligament. Recapitulation of biological development of hybrid tissues and interfaces presents a challenge that exceeds that of engineering just a single tissue. Advances made in dental interface engineering will allow these tissues to serve as model systems for engineering other tissues or organs of the body. This review will begin by covering basic tissue engineering principles and strategic design of functional biomaterials. We will then explore the impact of biomaterials design on the status of craniofacial tissue engineering and current challenges and opportunities in dental tissue engineering.
Asunto(s)
Boca/cirugía , Ingeniería de Tejidos/métodos , Animales , Materiales Biocompatibles/uso terapéutico , Regeneración Ósea , Procedimientos Quirúrgicos Dermatologicos , Humanos , Mucosa Bucal/cirugía , Articulación Temporomandibular/cirugía , Andamios del Tejido , Diente/cirugíaRESUMEN
BMP2/7 heterodimer expression by adenovirus can stimulate bone formation at subcutaneous sites. In the present study, we evaluate whether this approach will also promote healing of cranial defects. Adenovirus expressing BMP2 or BMP7 (AdBMP2, AdBMP7) was titrated to yield equivalent BMP protein levels after transduction into murine BLK cells. Analysis of conditioned medium showed that BMP2/7 heterodimers have enhanced ability to stimulate alkaline phosphatase and Smad 1,5,8 phosphorylation relative to equivalent amounts of BMP2 or BMP7 homodimers. To measure bone regeneration, we implanted virally transduced BLK cells into critical-sized calvarial defects generated in C57BL6 mice. AdBMP2/7-transduced cells were more effective in healing cranial defects than were cells individually transduced with AdBMP2 or BMP7. Dramatic increases in bone volume fraction, as measured by microCT, as well as fusion of regenerated bone with the defect margins were noted. Thus, the use of gene therapy to express heterodimeric BMPs is a promising potential therapy for healing craniofacial bones.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Regeneración Ósea/fisiología , Terapia Genética/métodos , Regeneración Tisular Dirigida/métodos , Factor de Crecimiento Transformador beta/metabolismo , Cicatrización de Heridas/fisiología , Implantes Absorbibles , Adenoviridae/genética , Animales , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 7 , Proteínas Morfogenéticas Óseas/administración & dosificación , Proteínas Morfogenéticas Óseas/genética , Regeneración Ósea/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Craneotomía , Fibroblastos/citología , Fibroblastos/metabolismo , Esponja de Gelatina Absorbible/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Ratones , Ratones Endogámicos C57BL , Mioblastos/citología , Mioblastos/metabolismo , Osteogénesis/genética , Osteogénesis/fisiología , Andamios del Tejido , Factor de Crecimiento Transformador beta/administración & dosificación , Factor de Crecimiento Transformador beta/genética , Transgenes , Cicatrización de Heridas/genéticaAsunto(s)
Regeneración Ósea/genética , Regeneración Ósea/efectos de la radiación , Anomalías Craneofaciales/terapia , Terapia Genética/métodos , Radioterapia/efectos adversos , Ingeniería de Tejidos/métodos , Animales , Proteína Morfogenética Ósea 7 , Proteínas Morfogenéticas Óseas/genética , Trasplante Óseo/métodos , Células Cultivadas , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/fisiopatología , Vectores Genéticos/uso terapéutico , Hormona Paratiroidea/farmacología , Hormona Paratiroidea/uso terapéutico , Ratas , Procedimientos de Cirugía Plástica/métodos , Factores de Tiempo , Transducción Genética/métodos , Factor de Crecimiento Transformador beta/genética , Resultado del Tratamiento , Cicatrización de Heridas/genética , Cicatrización de Heridas/efectos de la radiaciónRESUMEN
In developing porcine enamel, the space between enamel rods selectively binds lectins and ameloblastin (Ambn) N-terminal antibodies. We tested the hypothesis that ameloblastin N-terminal cleavage products are glycosylated. Assorted Ambn cleavage products showed positive lectin staining by peanut agglutinin (PNA), Maclura pomifera agglutinin (MPA), and Limulus polyphemus agglutinin (LPA), suggesting the presence of an O-linked glycosylation containing galactose (Gal), N-acetylgalactosamine (GalNAc), and sialic acid. Edman sequencing of the lectin-positive bands gave the Ambn N-terminal sequence: VPAFPRQPGTXGVASLXLE. The blank cycles for Pro(11) and Ser(17) confirmed that these residues are hydroxylated and phosphorylated, respectively. The O-glycosylation site was determined by Edman sequencing of pronase-digested Ambn, which gave HPPPLPXQPS, indicating that Ser(86) is the site of the O-linked glycosylation. This modification is within the 15-amino-acid segment (73-YEYSLPVHPPPLPSQ-87) deleted by splicing in the mRNA encoding the 380-amino-acid Ambn isoform. We conclude that only the N-terminal Ambn products derived from the 395-Ambn isoform are glycosylated.
Asunto(s)
Proteínas del Esmalte Dental/genética , Proteínas del Esmalte Dental/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Glicosilación , Lectinas/metabolismo , Fragmentos de Péptidos/metabolismo , Isoformas de Proteínas , Procesamiento Proteico-Postraduccional , Empalme del ARN , PorcinosRESUMEN
A lyophilization method was developed to locally release adenoviral vectors directly from biomaterials for in situ regenerative gene therapy. Adenovirus expressing a beta-galactosidase reporter gene (AdLacZ) was mixed with different excipient formulations and lyophilized on hydroxyapatite (HA) disks followed by fibroblasts culturing and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining, suggesting 1 M sucrose in phosphate-buffered saline had best viability. Adenovirus release studies showed that greater than 30% virus remained on the material surface up to 16 h. Lyophilized adenovirus could be precisely localized in defined patterns and the transduction efficiency was also improved. To determine if the lyophilization formulations could preserve viral bioactivity, the lyophilized AdLacZ was tested after being stored at varying temperatures. Bioactivity of adenovirus lyophilized on HA was maintained for greater than 6 months when stored at -80 degrees C. In vivo studies were performed using an adenovirus encoding BMP-2 (AdBMP-2). AdBMP-2 was lyophilized in gelatin sponges and placed into rat critical-size calvarial defects for 5 weeks. Micro-computed tomography (micro-CT) analysis demonstrated that free-form delivery of AdBMP-2 had only modest effects on bone formation. In contrast, AdBMP-2 lyophilized in gelatin sponges led to more than 80% regeneration of critical-size calvarial defects.
Asunto(s)
Adenoviridae/genética , Proteínas Morfogenéticas Óseas/genética , Regeneración Ósea , Fracturas Óseas/terapia , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Factor de Crecimiento Transformador beta/genética , Adenoviridae/fisiología , Infecciones por Adenoviridae/metabolismo , Animales , Materiales Biocompatibles , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/análisis , Proteínas Morfogenéticas Óseas/metabolismo , Huesos/metabolismo , Huesos/patología , Durapatita , Fracturas Óseas/diagnóstico por imagen , Fracturas Óseas/virología , Liofilización/métodos , Esponja de Gelatina Absorbible , Expresión Génica , Ingeniería Genética , Vectores Genéticos/genética , Implantes Experimentales , Inyecciones , Ratas , Ratas Endogámicas F344 , Tomografía Computarizada por Rayos X , Transducción Genética/métodos , Factor de Crecimiento Transformador beta/análisis , Factor de Crecimiento Transformador beta/metabolismo , beta-Galactosidasa/genéticaRESUMEN
Ameloblastin (AMBN) cleavage products are the most abundant non-amelogenin proteins in the enamel matrix of developing teeth. AMBN N-terminal cleavage products accumulate in the sheath space between enamel rods, while AMBN C-terminal products localize within rods. We tested the hypothesis that MMP-20 is the protease that cleaves AMBN. Glycosylated recombinant porcine AMBN (rpAMBN) was expressed in human kidney 293F cells, and recombinant porcine enamelysin (rpMMP-20) was expressed in bacteria. The purified proteins were incubated together at an enzyme:substrate ratio of 1:100. N-terminal sequencing of AMBN digestion products determined that rpMMP-20 cleaved rpAMBN after Pro(2), Gln(130), Gln(139), Arg(170), and Ala(222). This shows that MMP-20 generates the 23-kDa AMBN starting at Tyr(223), as well as the 17-kDa (Val(1)-Arg(170)) and 15-kDa (Val(1)-Gln(130)) AMBN cleavage products that concentrate in the sheath space during the secretory stage. We conclude that MMP-20 processes ameloblastin in vitro and in vivo.
Asunto(s)
Amelogénesis/fisiología , Proteínas del Esmalte Dental/metabolismo , Metaloproteinasa 20 de la Matriz/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunohistoquímica , Proteínas Recombinantes/metabolismo , Sus scrofaRESUMEN
Craniofacial tissue engineering promises the regeneration or de novo formation of dental, oral, and craniofacial structures lost to congenital anomalies, trauma, and diseases. Virtually all craniofacial structures are derivatives of mesenchymal cells. Mesenchymal stem cells are the offspring of mesenchymal cells following asymmetrical division, and reside in various craniofacial structures in the adult. Cells with characteristics of adult stem cells have been isolated from the dental pulp, the deciduous tooth, and the periodontium. Several craniofacial structures--such as the mandibular condyle, calvarial bone, cranial suture, and subcutaneous adipose tissue--have been engineered from mesenchymal stem cells, growth factor, and/or gene therapy approaches. As a departure from the reliance of current clinical practice on durable materials such as amalgam, composites, and metallic alloys, biological therapies utilize mesenchymal stem cells, delivered or internally recruited, to generate craniofacial structures in temporary scaffolding biomaterials. Craniofacial tissue engineering is likely to be realized in the foreseeable future, and represents an opportunity that dentistry cannot afford to miss.
Asunto(s)
Células Madre Mesenquimatosas , Periodoncio/citología , Regeneración/fisiología , Cráneo/citología , Ingeniería de Tejidos , Implantes Absorbibles , Tejido Adiposo/citología , Adulto , Células Madre Adultas , Animales , Pulpa Dental/citología , Técnicas de Transferencia de Gen , Humanos , Cóndilo Mandibular/citología , Trasplante de Células Madre Mesenquimatosas , Articulación Temporomandibular/citologíaRESUMEN
The aim of this study was to determine if endothelial cells could enhance bone marrow stromal-cell-mediated bone regeneration in an osseous defect. Using poly-lactide-co-glycolide scaffolds as cell carriers, we transplanted bone marrow stromal cells alone or with endothelial cells into 8.5-mm calvarial defects created in nude rats. Histological analyses of blood vessel and bone formation were performed, and microcomputed tomography (muCT) was used to assess mineralized bone matrix. Though the magnitude of the angiogenic response between groups was the same, muCT analysis revealed earlier mineralization of bone in the co-transplantation condition. Ultimately, there was a significant increase (40%) in bone formation in the co-transplantation group (33 +/- 2%), compared with the transplantation of bone marrow stromal cells alone (23 +/- 3%). Analysis of these data demonstrates that, in an orthotopic site, transplanted endothelial cells can influence the bone-regenerative capacity of bone marrow stromal cells.