RESUMO
Huntington's disease (HD) is a rare autosomal dominant neurodegenerative disorder caused by a cytosine-adenine-guanine (CAG) trinucleotide repeat (TNR) expansion within the HTT gene. The mechanisms underlying HD-associated cellular dysfunction in pluripotency and neurodevelopment are poorly understood. We had previously identified downregulation of selected DNA repair genes in HD fibroblasts relative to wild-type fibroblasts, as a result of promoter hypermethylation. Here, we tested the hypothesis that hypomethylation during cellular reprogramming to the induced pluripotent stem cell (iPSC) state leads to upregulation of DNA repair genes and stabilization of TNRs in HD cells. We sought to determine how the HD TNR region is affected by global epigenetic changes through cellular reprogramming and early neurodifferentiation. We find that early stage HD-affected neural stem cells (HD-NSCs) contain increased levels of global 5-hydroxymethylation (5-hmC) and normalized DNA repair gene expression. We confirm TNR stability is induced in iPSCs, and maintained in HD-NSCs. We also identify that upregulation of 5-hmC increases ten-eleven translocation 1 and 2 (TET1/2) protein levels, and show their knockdown leads to a corresponding decrease in the expression of select DNA repair genes. We further confirm decreased expression of TET1/2-regulating miR-29 family members in HD-NSCs. Our findings demonstrate that mechanisms associated with pluripotency induction lead to a recovery in the expression of select DNA repair gene and stabilize pathogenic TNRs in HD.
Assuntos
Reparo do DNA , Epigênese Genética , Doença de Huntington/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Expansão das Repetições de Trinucleotídeos , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Huntington disease (HD) is an autosomal dominantly inherited disease that exhibits genetic anticipation of affected progeny due to expansions of a trinucleotide repeat (TNR) region within the HTT gene. DNA repair machinery is a known effector of TNR instability; however, the specific defects in HD cells that lead to TNR expansion are unknown. We hypothesized that HD cells would be deficient in DNA repair gene expression. To test this hypothesis, we analyzed expression of select DNA repair genes involved in mismatch/loop-out repair (APEX1, BRCA1, RPA1, and RPA3) in patient-derived HD cells and found each was consistently down-regulated relative to wild-type samples taken from unaffected individuals in the same family. Rescue of DNA repair gene expression by 5-azacytidine treatment identified DNA methylation as a mediator of DNA repair gene expression deficiency. Bisulfite sequencing confirmed hypermethylation of the APEX1 promoter region in HD cells relative to control, as well as 5-azacytidine-induced hypomethylation. 5-Azacytidine treatments also resulted in stabilization of TNR expansion within the mutant HTT allele during long-term culture of HD cells. Our findings indicate that DNA methylation leads to DNA repair down-regulation and TNR instability in mitotically active HD cells and offer a proof of principle that epigenetic interventions can curb TNR expansions.
Assuntos
Metilação de DNA/genética , Reparo do DNA/genética , Doença de Huntington/genética , Expansão das Repetições de Trinucleotídeos/genética , Células Cultivadas , Regulação para Baixo , Imunofluorescência , Humanos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Bone grafting procedures are performed to treat wounds incurred during wartime trauma, accidents, and tumor resections. Endogenous mechanisms of repair are often insufficient to ensure integration between host and donor bone and subsequent restoration of function. We investigated the role that bone marrow-derived cells play in bone regeneration and sought to increase their contributions by functionalizing bone allografts with bioactive lipid coatings. Polymer-coated allografts were used to locally deliver the immunomodulatory small molecule FTY720 in tibial defects created in rat bone marrow chimeras containing genetically-labeled bone marrow for monitoring cell origin and fate. Donor bone marrow contributed significantly to both myeloid and osteogenic cells in remodeling tissue surrounding allografts. FTY720 coatings altered the phenotype of immune cells two weeks post-injury, which was associated with increased vascularization and bone formation surrounding allografts. Consequently, degradable polymer coating strategies that deliver small molecule growth factors such as FTY720 represent a novel therapeutic strategy for harnessing endogenous bone marrow-derived progenitors and enhancing healing in load-bearing bone defects.
Assuntos
Transplante Ósseo , Cloridrato de Fingolimode/administração & dosagem , Fatores Imunológicos/administração & dosagem , Aloenxertos , Animais , Células da Medula Óssea/citologia , Osso e Ossos/fisiologia , Linhagem da Célula , Portadores de Fármacos/administração & dosagem , Sistemas de Liberação de Medicamentos , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Ácido Láctico/administração & dosagem , Masculino , Mielopoese/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ácido Poliglicólico/administração & dosagem , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Quimera por Radiação , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Regeneração , Estresse Mecânico , Tíbia/lesões , Tíbia/cirurgia , Microtomografia por Raio-XRESUMO
Mimicking one or more components of the basement membrane (BM) holds great promise for overcoming insufficiencies in tissue engineering therapies. We have electrospun laminin nanofibers (NFs) isolated from the murine Engelbreth-Holm Swarm (EHS) tumor and evaluated them as a scaffold for embryonic stem cell culture. Seeded human embryonic stem cells were found to better maintain their undifferentiated, colony environment when cultured on laminin NFs compared to laminin mats, with 75% remaining undifferentiated on NFs. Mouse embryonic stem cells cultured on 10% laminin-polycaprolactone (PCL) NFs maintained their colony formation for twice as long without passage compared to those on PCL or gelatin substrates. In addition, we have established a protocol for electrospinning reconstituted basement membrane aligned (RBM)-PCL NFs within 10° of angular deviation. Neuron-like PC12 cells show significantly greater attachment (p < 0.001) and percentage of neurite-extending cells in vitro on 10% RBM-PCL NFs when compared to 1% and 0% RBM-PCL NFs (p < 0.015 and p < 0.001, respectively). Together, these results implicate laminin- and RBM-PCL scaffolds as a promising biomimetic substrate for regenerative medicine applications.
RESUMO
Peripheral nerve transection occurs commonly in traumatic injury, causing deficits distal to the injury site. Conduits for repair currently on the market are hollow tubes; however, they often fail due to slow regeneration over long gaps. To facilitate increased regeneration speed and functional recovery, the ideal conduit should provide biochemically relevant signals and physical guidance cues, thus playing an active role in regeneration. To that end, laminin and laminin-polycaprolactone (PCL) blend nanofibers were fabricated to mimic peripheral nerve basement membrane. In vitro assays established 10% (wt) laminin content is sufficient to retain neurite-promoting effects of laminin. In addition, modified collector plate design to introduce an insulating gap enabled the fabrication of aligned nanofibers. The effects of laminin content and fiber orientation were evaluated in rat tibial nerve defect model. The lumens of conduits were filled with nanofiber meshes of varying laminin content and alignment to assess changes in motor and sensory recovery. Retrograde nerve conduction speed at 6 weeks was significantly faster in animals receiving aligned nanofiber conduits than in those receiving random nanofiber conduits. Animals receiving nanofiber-filled conduits showed some conduction in both anterograde and retrograde directions, whereas in animals receiving hollow conduits, no impulse conduction was detected. Aligned PCL nanofibers significantly improved motor function; aligned laminin blend nanofibers yielded the best sensory function recovery. In both cases, nanofiber-filled conduits resulted in better functional recovery than hollow conduits. These studies provide a firm foundation for the use of natural-synthetic blend electrospun nanofibers to enhance existing hollow nerve guidance conduits.
Assuntos
Laminina/farmacologia , Nanofibras/química , Regeneração Nervosa/efeitos dos fármacos , Poliésteres/farmacologia , Nervo Tibial/fisiologia , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Materiais Biocompatíveis/farmacologia , Feminino , Gânglios Espinais/efeitos dos fármacos , Laminina/química , Camundongos , Atividade Motora/efeitos dos fármacos , Nanofibras/ultraestrutura , Condução Nervosa/efeitos dos fármacos , Células PC12 , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Sensação/efeitos dos fármacos , Resistência à Tração/efeitos dos fármacos , Nervo Tibial/efeitos dos fármacos , Alicerces Teciduais/químicaRESUMO
The transforming growth factor-ß (TGF-ß) family of extracellular signaling molecules is heavily involved in developmental events, including patterning, formation, maintenance, and closure of the cranial suture. Several studies have demonstrated that TGF-ßs are temporally and spatially localized to the suture and play a pivotal role in sutural state. These signals are translated into intracellular activity through a family of proteins known as smads. There are 8 known smads, with smads 1, 5, and 8 transducing BMP signals and smads 2 and 3 transducing TGF-ß signals. Dimerization of any of these smads and smad 4 is necessary for phosphorylation and activation. Although many studies have delineated the presence of TGF-ß during suture closure, no studies have determined smad activity. It was hypothesized that smad activity would change during sutural closure. Reverse transcription-polymerase chain reaction was used to determine whether TGF-ß-responsive smads were present in the suture at which point they were immunohistochemically localized. A rat model was used in which the posterior intrafrontal suture fused during neonatal days 16 to 22. Time points before and after this event were analyzed for changes in smad expression and function. It was determined from these experiments that (1) the TGF-ß-responsive smads 2, 3, and 4 are all present in the suture; (2) smads 2 and 4 are distributed in the region of the osteogenic front of the suture; and (3) smad 2/4 activity decreases in areas in which presumptive bone will form. These results add to the knowledge present about sutural development and may provide news targets to which therapeutics can be developed.
Assuntos
Suturas Cranianas/metabolismo , Proteínas Smad Reguladas por Receptor/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Animais Recém-Nascidos , Suturas Cranianas/crescimento & desenvolvimento , Técnicas Imunoenzimáticas , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
OBJECTIVES/HYPOTHESIS: Advances in bone repair have focused on the minimally-invasive delivery of tissue-engineered bone (TEB). A promising injectable biopolymer of chitosan and inorganic phosphates was seeded with mesenchymal stem cells (MSCs) and a bone growth factor (BMP-2), and evaluated in a rat calvarial critical size defect (CSD). Green fluorescent protein (GFP)-labeled MSCs are used to evaluate patterns of cell viability and proliferation. STUDY DESIGN: Prospective, controlled trial in an animal model. METHODS: In 30 male rats, 8-mm calvarial CSDs were created, and divided into five groups of six animals each. In the experimental groups, the defects were injected with either chitosan gel, gel loaded with MSCs (0.3 x 10(6) cells/defect), gel loaded with BMP-2 (2 microg/defect), or gel loaded with both MSC and BMP-2. In the control group, the defect was left untreated. At 4 weeks, in vivo microcomputed tomography (micro-CT) analysis was performed. At 8 weeks, calvarial specimens were examined by micro-CT, histology, and immunohistochemistry. RESULTS: New areas of bone growth were seen in the defects of all treated animals. Micro-CT analysis revealed a significant (P < .001) time-dependent increase in the regeneration of bone volume and bone area in defects treated with gel/MSC/BMP-2 as compared to all other groups. Histological analysis confirmed this difference. GFP-labeled TEB was detected within the areas of new bone, indicating cell viability and contribution to new bone growth by the injected MSC. CONCLUSIONS: This study demonstrates that an injectable form of TEB using a chitosan gel, MSC, and BMP-2 can enhance bone formation in a rat calvarial CSD.
Assuntos
Proteína Morfogenética Óssea 2/administração & dosagem , Transplante de Células-Tronco Mesenquimais/métodos , Procedimentos Cirúrgicos Minimamente Invasivos/métodos , Procedimentos de Cirurgia Plástica/métodos , Crânio/cirurgia , Engenharia Tecidual/métodos , Animais , Regeneração Óssea/fisiologia , Quitosana , Géis , Processamento de Imagem Assistida por Computador , Injeções , Masculino , Fosfatos , Ratos , Ratos Sprague-Dawley , Crânio/patologia , Microtomografia por Raio-XRESUMO
PURPOSE OF REVIEW: This review will cover the basic research performed with adipose stem cells (ASCs) over the past several years as well as pertinent translational research. The properties of ASCs that make them particularly interesting to the transplant surgeon will then be covered. These properties include regeneration of native tissue, support of microvasculature, and immunomodulation. These properties will undoubtedly expand the future utility of these cells. RECENT FINDINGS: Recent literature demonstrates that ASCs are able to differentiate into phenotypes resembling hepatic and pancreatic lineages. In addition, several groups have shown that ASCs possess immunomodulatory properties similar to bone marrow-derived mesenchymal stem cells. Several clinical case reports also suggest that ASCs are an effective treatment option for graft-versus-host disease. SUMMARY: Due to their ability to differentiate into pertinent target lineages, their ability to enhance angiogenesis, and their ability to impact immunologic responses, ASCs may prove clinically useful for the transplant surgeon.
Assuntos
Tecido Adiposo/transplante , Transplante de Células-Tronco Mesenquimais , Transplante de Órgãos , Regeneração , Medicina Regenerativa , Tecido Adiposo/citologia , Tecido Adiposo/imunologia , Animais , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Neovascularização Fisiológica , Transplante de Órgãos/efeitos adversos , Doadores de Tecidos/provisão & distribuiçãoRESUMO
The basement membrane protein, laminin I, has been used broadly as a planar two-dimensional film or in a three-dimensional form as a reconstituted basement membrane gel such as Matrigel to support cellular attachment, growth, and differentiation in vitro. In basement membranes in vivo, laminin exhibits a fibrillar morphology, highlighting the electrospinning process as an ideal method to recreate such fibrous substrates in vitro. Electrospinning was employed to fabricate meshes of murine laminin I nanofibers (LNFs) with fiber size, geometry, and porosity of authentic basement membranes. Purified laminin I was solubilized and electrospun in parametric studies of fiber diameters as a function of polymer solution concentration, collecting distance, and flow rate. Resulting fiber diameters ranged from 90 to 300 nm with mesh morphologies containing beads. Unlike previously described nanofibers (NFs) synthesized from proteins such as collagen, meshes of LNFs retain their structural features when wetted and do not require fixation by chemical crosslinking, which often destroys cell attachment and other biological activity. The LNF meshes maintained their geometry for at least 2 days in culture without chemical crosslinking. PC12 cells extended neurites without nerve growth factor stimulation on LNF substrates. Additionally, LNFs significantly enhance both the rate and quantity of attachment of human adipose stem cells (ASCs) compared to laminin films. ASCs were viable and maintained attachment to LNF meshes in serum-free media for at least 3 days in culture and extended neurite-like processes after 24 h in serum-free media conditions without media additives to induce differentiation. LNF meshes are a novel substrate for cell studies in vitro, whose properties may be an excellent scaffold material for delivering cells in tissue engineering applications in vivo.
Assuntos
Membrana Basal/metabolismo , Laminina/metabolismo , Nanoestruturas/química , Tecido Adiposo/citologia , Animais , Membrana Basal/efeitos dos fármacos , Meios de Cultura , Humanos , Laminina/ultraestrutura , Nanoestruturas/ultraestrutura , Fator de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Células PC12 , Ratos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/ultraestruturaRESUMO
BACKGROUND: Theories regarding the cause of craniosynostosis that are more than 15 years old cite the role that tensional forces play in the normal and abnormal development of the cranial suture. These theories highlight the effect of stress bands originating from the skull base to the vertex, guiding sutural development. METHODS: In this study, the normally fusing posterior intrafrontal suture of the rat was subjected to 3 mN of tensional force for 30 minutes per day. The suture was then assessed for patency, proliferation, apoptosis, and transforming growth factor (TGF)-beta signaling components. RESULTS: Sutures that were subjected to tensional force were histologically patent at the end of 14 days. This was in contrast to sutures that were maintained without force. Proliferative and apoptotic activity was increased also in sutures maintained open artificially. Interestingly, levels of active TGF-beta-signaling components were also increased in force-maintained sutures. CONCLUSIONS: Sutural maintenance by mechanical force is concurrent with modulation of cellular activity and protein expression reminiscent of the open suture. This study demonstrates the dynamic reciprocity existing between biochemical activity and morphologic state. Although it is known that changes in TGF-betas and fibroblast growth factors can cause sutural fusion, this is the first study to demonstrate that abrogation of sutural closure is responsible for growth factor signaling modulation.
Assuntos
Regulação da Expressão Gênica/fisiologia , Crânio/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Fenômenos Biomecânicos , Ratos , Ratos Sprague-Dawley , Crânio/anatomia & histologiaRESUMO
During development and growth of the neurocranium, the dura mater regulates events in the underlying brain and overlying skull by the release of soluble factors and cellular activity. Morphogenesis of the cranial bones and sutures is dependent on tissue interactions with the dura mater, which control the size and shape of bones as well as sutural patency. Development of the brain also involves interactions with dura mater: secretion of stromal derived factor 1 (SDF-1) is a critical event in directing migration of the external granular layer precursors of the cerebellar cortex and the Cajal-Retzius (CR) cells of the cerebral cortex. The dura mater is also required for growth of the hippocampal dentate gyrus. Wnt1Cre/R26R transgenic reporter mice were used to study the origin and fates of the cells of dura mater during head development. The dura mater of mammals is derived entirely from the cranial neural crest. Beginning around neonatal day 10 (N 10), the dura mater is infiltrated by cells derived from paraxial mesoderm, which later come to predominate. Over the course of infancy, the neural crest-derived cells of the dura mater become sequestered in niche-like distribution characteristic of stem cells. Simultaneously, dura mater cells underlying the sagittal suture migrate upward into the mesodermally-derived mesenchyme separating the parietal bones. Although initially the parietal bones are formed entirely from paraxial mesoderm, the cellular composition gradually becomes chimeric and is populated mainly by neural crest-derived cells by N 30. This occurs as a consequence of osteoblastic differentiation at the dura mater interface and intravasation of neural crest-derived osteoclastic and other hematopoietic precursors. The isolated cells of the dura mater are multipotent in vitro, giving rise to osteoblasts, neuronal cells and other derivatives characteristic of cranial neural crest, possibly reflecting the multipotent nature of dura mater cells in vivo.
Assuntos
Dura-Máter/crescimento & desenvolvimento , Animais , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Suturas Cranianas/embriologia , Suturas Cranianas/crescimento & desenvolvimento , Craniossinostoses/embriologia , Craniossinostoses/genética , Dura-Máter/citologia , Dura-Máter/embriologia , Fatores de Crescimento de Fibroblastos/fisiologia , Genes Reporter , Cabeça/embriologia , Cabeça/crescimento & desenvolvimento , Integrases/genética , Camundongos , Camundongos Transgênicos , Células-Tronco Multipotentes/citologia , Mutação , Osso Parietal/embriologia , Osso Parietal/crescimento & desenvolvimento , Transdução de Sinais , Proteína Wnt1/genética , beta-Galactosidase/genéticaRESUMO
This article highlights potential uses for harvested fat and describes the current state of the art regarding adipose stem cells.
Assuntos
Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Células-Tronco/metabolismo , Diferenciação Celular/fisiologia , Matriz Extracelular , HumanosRESUMO
Little is known regarding the biology of fat considering its extensive use clinically in soft tissue implantation. Free-fat transfer is problematic the result of graft site volume loss, appearing histologically as the replacement of mature adipocytes with a fibroblast-like infiltrate. We hypothesize that these histologic changes reflect a dedifferentiation of ischemic mature adipocytes instead of, or in addition to, a more traditional response. To explore this hypothesis, we studied the in vitro morphologic changes of cultured mature human adipocytes isolated from liposuctioned adipose tissue. Most adipocytes over time lost significant amounts of intracellular lipid. Ultimately, these cells lost all lipid, appeared fibroblastic, and proliferated to confluence. Adipogenic induction of such dedifferentiated adipocytes resulted in reaccumulation of intracellular lipid. This study demonstrates that mature adipocytes can be cultured from human liposuctioned fat, they can dedifferentiate into fibroblastic cells, and the fibroblast-like cells can be expanded and turned into lipid-synthesizing adipocytes. Exploration of this cellular plasticity might ultimately yield important insights into free-fat transfer and novel tissue-engineering strategies.
Assuntos
Adipócitos/fisiologia , Adipócitos/transplante , Adulto , Diferenciação Celular , Células Cultivadas , HumanosRESUMO
Adult human subcutaneous adipose tissue contains cells with intriguing multilineage developmental plasticity, much like marrow-derived mesenchymal stem cells. Putative stem or progenitor cells from fat have been given many different names in the literature, reflecting an early and evolving consensus regarding their phenotypic characterization. The study reported here used microarrays to evaluate over 170 genes relating to angiogenesis and extracellular matrix in undifferentiated, early-passage human adipose-derived adherent stromal (hADAS) cells isolated from three separate donors. The hADAS populations unanimously transcribed 66% of the screened genes, and 83% were transcribed by at least two of the three populations. The most highly transcribed genes relate to functional groupings such as cell adhesion, matrix proteins, growth factors and receptors, and proteases. The transcriptome of hADAS cells demonstrated by this work reveals many similarities to published profiles of bone marrow mesenchymal stem cells (MSCs). In addition, flow analysis of over 24 hADAS cell surface proteins (n = 7 donors) both confirms and expands on the existing literature and reveals strong intergroup correlation, despite an inconsistent nomenclature and the lack of standardized protocols for cell isolation and culture. Finally, based on flow analysis and reverse transcription polymerase chain reaction studies, our results suggest that hADAS cells do not express several proteins that are implicated as markers of "stemness" in other stem cell populations, including telomerase, CD133, and the membrane transporter ABCG2.
Assuntos
Tecido Adiposo/citologia , Antígenos de Superfície/análise , Perfilação da Expressão Gênica , Células Estromais/citologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Tecido Adiposo/química , Tecido Adiposo/metabolismo , Adulto , Antígenos de Diferenciação/análise , Diferenciação Celular/fisiologia , Células Cultivadas , Citometria de Fluxo , Humanos , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/química , Células Estromais/metabolismo , Telomerase/genética , Transcrição Gênica/genéticaRESUMO
Autologous bone grafting techniques involve the use of tissues that need to be extracted from healthy sites. This can lead to significant donor site morbidity that causes a one-site defect to become a two-site defect. Bone grafts can be especially difficult to manipulate, because bone is a relatively nonmoldable tissue. Furthermore, the inability of a bone graft to contain a transplantable vascular supply also limits the possible size that such a bone graft can be. Because of these limitations, a graft that was moldable with a vascular supply would possess significant advantages in reconstructive applications. In this research, gene therapy techniques were used to create such a graft. An adenovirus expressing BMP-9 was injected into the latissimus dorsi of a nude animal to cause bony differentiation of that muscle. Differentiation of the muscle to cartilage in bone was measured by reverse transcription polymerase chain reaction and immunohistochemistry to determine the optimal time of flap elevation. After injection of the BMP-9 virus, the animals were biopsied weekly over a 3-week period. Both bone and cartilage markers were discovered in these tissues over the study period. Optimal flap elevation time was established to be 2 weeks after injection of the virus.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Transplante Ósseo/métodos , Técnicas de Transferência de Genes , Músculo Esquelético/citologia , Osteogênese , Animais , Diferenciação Celular , Condrogênese , Fator 2 de Diferenciação de Crescimento , Sialoproteína de Ligação à Integrina , Músculo Esquelético/transplante , Osteopontina , Ratos , Sialoglicoproteínas/biossíntese , Retalhos Cirúrgicos/irrigação sanguínea , Transdução GenéticaRESUMO
There are a variety of recommended methods for harvesting, treating, and utilizing autologous fat grafts. Previous work with the MTT assay illustrated that various preimplantation handling techniques had minimal effect on the viability of fat samples. This assay was used to test the viability of harvested fat samples after being stored for up to 8 days in a variety of conditions. Surprisingly, freezing the fat before assaying also had no measurable detrimental effect, which led us to study this phenomenon in greater detail. The results demonstrated that fat stored at subzero temperatures showed remarkable maintenance of their mitochondrial metabolic activity as compared with fat stored in a 32 degrees C incubator. These data suggest exciting possibilities for storage and banking of human adipose tissue, which would reduce patient cost, discomfort, and time associated with multiple grafting procedures.
Assuntos
Tecido Adiposo , Criopreservação , Sobrevivência de Tecidos , Coleta de Tecidos e Órgãos , Tecido Adiposo/patologia , Tecido Adiposo/transplante , Criopreservação/métodos , Sobrevivência de Enxerto , Humanos , Procedimentos de Cirurgia Plástica , Transplante de Pele , Fatores de Tempo , Coleta de Tecidos e Órgãos/métodosRESUMO
Fibroblast growth factor receptor mutations are associated with and, in fact, cause most syndromes presenting with craniosynostosis. This knowledge has resulted in a shift in the paradigm of suture fusion causation; it was thought previously that abnormal tensional forces arising in the cranial base caused fusion of the vault sutures, but it is now understood that aberrant intercellular signaling in the developing skull leads to abnormal suture morphogenesis. Although the mutations associated with these syndromes are known and the phenotypic consequences are well documented, the pathway from mutation to phenotype has yet to be elucidated. Surgical reconstruction is the primary treatment of craniofacial abnormalities associated with craniosynostotic syndromes such as Crouzon syndrome. In many cases, calvarial vault reshaping is dependent on the quality of the autologous bone available; however, the bone of patients with craniosynostosis syndrome is often more brittle, thinner, and less robust than cranial bone from nonaffected donors. The relation between syndromic craniosynostoses and this bone has not been previously described. In this study, the osteon and blood vessel diameters of calvarial bone from patients with Crouzon syndrome and age- and sex-matched normal calvarial bone are measured. Statistical analysis demonstrates a quantitative and significant difference in the blood vessel diameter but not in the osteon diameter. This finding could be a result of abnormal blood vessel development caused by the fibroblast growth factor receptor mutation occurring before and coincident with bone formation and leading to weakened and fragile bone tissue.
Assuntos
Disostose Craniofacial/genética , Mutação/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Crânio/irrigação sanguínea , Vasos Sanguíneos/patologia , Estudos de Casos e Controles , Suturas Cranianas/anormalidades , Suturas Cranianas/patologia , Disostose Craniofacial/patologia , Craniossinostoses/genética , Craniossinostoses/patologia , Ósteon/irrigação sanguínea , Ósteon/patologia , Humanos , Fenótipo , Transdução de Sinais/genética , Crânio/patologiaRESUMO
BACKGROUND: Surgical experience and anecdotal data on the most effective method of harvesting, preparing, and injecting autologous fat grafts are inconsistent and conflicting. Because the limitation of fat grafting is its resorption, understanding how various handling techniques affect adipocyte survival is crucial to optimizing its long-term survival. OBJECTIVE: We sought to develop a method for assaying fat viability in its clinically used form and then to test several common techniques used in fat grafting for their effects on the viability of the fat. METHODS: We performed the well-established MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] cell survival and proliferation assay on fat, but the colored enzyme-breakdown product could not be released into the supernatant for spectrophotometric analysis. An entirely new protocol was developed that allowed the MTT assay to quantitate the viability of free fat grafts. The assay was able to distinguish between different quantities of live fat and to quantify the decrease in viability when the fat is stored. We subjected the fat to various treatments, including insulin and Triton-X 100 detergent, (Sigma Aldrich, St. Louis, MO) centrifugation, extrusion through different types and sizes of needles, and freezing. RESULTS: With the exception of detergent, which decreased viability, all other treatments had no statistically significant effect on adipocyte survival. Freezing did not result in decreased cell viability. CONCLUSIONS: It is unlikely that variations in the clinical results of free fat grafting are the result of the handling techniques examined in this study.