RESUMO
How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.
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Osso e Ossos/citologia , Células-Tronco Mesenquimais/citologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem/citologia , Linhagem da Célula , Cruzamentos Genéticos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de SinaisRESUMO
Loss of skeletal integrity during ageing and disease is associated with an imbalance in the opposing actions of osteoblasts and osteoclasts1. Here we show that intrinsic ageing of skeletal stem cells (SSCs)2 in mice alters signalling in the bone marrow niche and skews the differentiation of bone and blood lineages, leading to fragile bones that regenerate poorly. Functionally, aged SSCs have a decreased bone- and cartilage-forming potential but produce more stromal lineages that express high levels of pro-inflammatory and pro-resorptive cytokines. Single-cell RNA-sequencing studies link the functional loss to a diminished transcriptomic diversity of SSCs in aged mice, which thereby contributes to the transformation of the bone marrow niche. Exposure to a youthful circulation through heterochronic parabiosis or systemic reconstitution with young haematopoietic stem cells did not reverse the diminished osteochondrogenic activity of aged SSCs, or improve bone mass or skeletal healing parameters in aged mice. Conversely, the aged SSC lineage promoted osteoclastic activity and myeloid skewing by haematopoietic stem and progenitor cells, suggesting that the ageing of SSCs is a driver of haematopoietic ageing. Deficient bone regeneration in aged mice could only be returned to youthful levels by applying a combinatorial treatment of BMP2 and a CSF1 antagonist locally to fractures, which reactivated aged SSCs and simultaneously ablated the inflammatory, pro-osteoclastic milieu. Our findings provide mechanistic insights into the complex, multifactorial mechanisms that underlie skeletal ageing and offer prospects for rejuvenating the aged skeletal system.
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Envelhecimento/patologia , Osso e Ossos/patologia , Senescência Celular , Inflamação/patologia , Nicho de Células-Tronco , Células-Tronco/patologia , Animais , Proteína Morfogenética Óssea 2/metabolismo , Regeneração Óssea , Linhagem da Célula , Feminino , Consolidação da Fratura , Hematopoese , Fator Estimulador de Colônias de Macrófagos/metabolismo , Masculino , Camundongos , Células Mieloides/citologia , Osteoclastos/citologia , RejuvenescimentoRESUMO
OBJECTIVE: The aim of this study was to determine the interaction of full thickness excisional wounds and tumors in vivo. SUMMARY OF BACKGROUND DATA: Tumors have been described as wounds that do not heal due to similarities in stromal composition. On the basis of observations of slowed tumor growth after ulceration, we hypothesized that full thickness excisional wounds would inhibit tumor progression in vivo. METHODS: To determine the interaction of tumors and wounds, we developed a tumor xenograft/allograft (human head and neck squamous cell carcinoma SAS/mouse breast carcinoma 4T1) wound mouse model. We examined tumor growth with varying temporospatial placement of tumors and wounds or ischemic flap. In addition, we developed a tumor/wound parabiosis model to understand the ability of tumors and wounds to recruit circulating progenitor cells. RESULTS: Tumor growth inhibition by full thickness excisional wounds was dose-dependent, maintained by sequential wounding, and relative to distance. This effect was recapitulated by placement of an ischemic flap directly adjacent to a xenograft tumor. Using a parabiosis model, we demonstrated that a healing wound was able to recruit significantly more circulating progenitor cells than a growing tumor. Tumor inhibition by wound was unaffected by presence of an immune response in an immunocompetent model using a mammary carcinoma. Utilizing functional proteomics, we identified 100 proteins differentially expressed in tumors and wounds. CONCLUSION: Full thickness excisional wounds have the ability to inhibit tumor growth in vivo. Further research may provide an exact mechanism for this remarkable finding and new advances in wound healing and tumor biology.
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Neoplasias/patologia , Úlcera/patologia , Ferimentos e Lesões/patologia , Animais , Feminino , Camundongos , Neoplasias/complicações , Úlcera/complicações , Ferimentos e Lesões/complicaçõesRESUMO
The postnatal skeleton undergoes growth, remodeling, and repair. We hypothesized that skeletal progenitor cells active during these disparate phases are genetically and phenotypically distinct. We identified a highly potent regenerative cell type that we term the fracture-induced bone, cartilage, stromal progenitor (f-BCSP) in the fracture callus of adult mice. The f-BCSP possesses significantly enhanced skeletogenic potential compared with BCSPs harvested from uninjured bone. It also recapitulates many gene expression patterns involved in perinatal skeletogenesis. Our results indicate that the skeletal progenitor population is functionally stratified, containing distinct subsets responsible for growth, regeneration, and repair. Furthermore, our findings suggest that injury-induced changes to the skeletal stem and progenitor microenvironments could activate these cells and enhance their regenerative potential.
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Osso e Ossos/patologia , Fraturas Ósseas/patologia , Células-Tronco/citologia , Animais , Animais Recém-Nascidos , Desenvolvimento Ósseo , Calo Ósseo/citologia , Cartilagem/patologia , Proliferação de Células , Separação Celular , Fêmur/patologia , Perfilação da Expressão Gênica , Membro Posterior/efeitos da radiação , Integrina alfa6/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Osteogênese , Fenótipo , Células Estromais/citologiaRESUMO
Nanotechnology represents a major frontier with potential to significantly advance the field of bone tissue engineering. Current limitations in regenerative strategies include impaired cellular proliferation and differentiation, insufficient mechanical strength of scaffolds, and inadequate production of extrinsic factors necessary for efficient osteogenesis. Here we review several major areas of research in nanotechnology with potential implications in bone regeneration: 1) nanoparticle-based methods for delivery of bioactive molecules, growth factors, and genetic material, 2) nanoparticle-mediated cell labeling and targeting, and 3) nano-based scaffold construction and modification to enhance physicochemical interactions, biocompatibility, mechanical stability, and cellular attachment/survival. As these technologies continue to evolve, ultimate translation to the clinical environment may allow for improved therapeutic outcomes in patients with large bone deficits and osteodegenerative diseases. FROM THE CLINICAL EDITOR: Traditionally, the reconstruction of bony defects has relied on the use of bone grafts. With advances in nanotechnology, there has been significant development of synthetic biomaterials. In this article, the authors provided a comprehensive review on current research in nanoparticle-based therapies for bone tissue engineering, which should be useful reading for clinicians as well as researchers in this field.
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Regeneração Óssea , Nanotecnologia/métodos , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Humanos , Nanomedicina/métodos , Nanopartículas/química , Nanopartículas/metabolismo , Medicina Regenerativa/métodos , Coloração e Rotulagem/métodos , Alicerces Teciduais/químicaRESUMO
BACKGROUND: Early fetuses heal wounds without the formation of a scar. Many studies have attempted to explain this remarkable phenomenon. However, the exact mechanism remains unknown. Herein, we examine the predominant cell types of the epidermis and dermis--the keratinocyte and fibroblast--during different stages of fetal development to better understand the changes that lead to scarring wound repair versus regeneration. MATERIALS AND METHODS: Keratinocytes and fibroblasts were harvested and cultured from the dorsal skin of time-dated BALB/c fetuses. Total RNA was isolated and microarray analysis was performed using chips with 42,000 genes. Significance analysis of microarrays was used to select genes with >2-fold expression differences with a false discovery rate<2. Enrichment analysis was performed on significant genes to identify differentially expressed pathways. RESULTS: By comparing the gene expression profile of keratinocytes from E16 versus E18 fetuses, we identified 24 genes that were downregulated at E16. Analysis of E16 and E18 fibroblasts revealed 522 differentially expressed genes. Enrichment analysis showed the top 20 signaling pathways that were downregulated in E16 keratinocytes and upregulated or downregulated in E16 fibroblasts. CONCLUSIONS: Our data reveal 546 differentially expressed genes in keratinocytes and fibroblasts between the scarless and scarring transition. In addition, a total of 60 signaling pathways have been identified to be either upregulated or downregulated in these cell types. The genes and pathways recognized by our study may prove to be essential targets that may discriminate between fetal wound regeneration and adult wound repair.
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Feto/metabolismo , Fibroblastos/metabolismo , Queratinócitos/metabolismo , Transcriptoma , Animais , Células Cultivadas , Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Fator de Crescimento Derivado de Plaquetas/fisiologia , Superóxidos/metabolismo , Via de Sinalização Wnt , beta Catenina/fisiologiaRESUMO
Improvements in medical care, nutrition and social care are resulting in a commendable change in world population demographics with an ever increasing skew towards an aging population. As the proportion of the world's population that is considered elderly increases, so does the incidence of osteodegenerative disease and the resultant burden on healthcare. The increasing demand coupled with the limitations of contemporary approaches, have provided the impetus to develop novel tissue regeneration therapies. The use of stem cells, with their potential for self-renewal and differentiation, is one potential solution. Adipose-derived stem cells (ASCs), which are relatively easy to harvest and readily available have emerged as an ideal candidate. In this review, we explore the potential for ASCs to provide tangible therapies for craniofacial and long bone skeletal defects, outline key signaling pathways that direct these cells and describe how the developmental signaling program may provide clues on how to guide these cells in vivo. This review also provides an overview of the importance of establishing an osteogenic microniche using appropriately customized scaffolds and delineates some of the key challenges that still need to be overcome for adult stem cell skeletal regenerative therapy to become a clinical reality.
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Tecido Adiposo/citologia , Regeneração Óssea , Osteogênese , Transdução de Sinais , Células-Tronco/citologia , Tecido Adiposo/metabolismo , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células-Tronco/metabolismo , Engenharia Tecidual/métodos , Alicerces TeciduaisAssuntos
Carcinoma Basocelular , Pessoas Famosas , Conhecimentos, Atitudes e Prática em Saúde , Aceitação pelo Paciente de Cuidados de Saúde , Neoplasias Cutâneas , Revelação da Verdade , Carcinoma Basocelular/diagnóstico , Carcinoma Basocelular/etiologia , Carcinoma Basocelular/terapia , Humanos , Internet , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/terapiaRESUMO
INTRODUCTION: Vaginectomy is frequently indicated to ensure an adequate resection in rectal cancer. This paper reviews the success, complications, and functional results after various methods of vaginal reconstruction following resection for rectal tumors. METHODS: A systematic review of the literature was performed by using the MEDLINE database, Embase, and the Cochrane library (1990-2010). RESULTS: Eighteen papers were available to review. Fifty percent of all women who received a neovagina following vaginectomy returned to sexual activity postoperatively. The rectus abdominis myocutaneous (RAM) flap remains the most common method of vaginal reconstruction after surgery for colorectal cancer. Advantages include low operative morbidity, a wide arc of rotation, and ease of harvest. Alternatives to flap reconstruction of the vagina have limited indications because of higher morbidity rates. CONCLUSIONS: Preferred techniques for vaginal reconstruction following vaginectomy with colorectal cancer resection include RAM flaps for partial posterior vaginal defects and bilateral Gracilis myocutaneous flaps for complete vaginal excisions.
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Neoplasias Colorretais/cirurgia , Procedimentos de Cirurgia Plástica , Vagina/cirurgia , Feminino , Humanos , Masculino , Prognóstico , Literatura de Revisão como Assunto , Retalhos CirúrgicosRESUMO
BACKGROUND: The debate on how to create ideal facial proportions--once the remit of artists and sculptors--has been reignited during the past 5 decades, with the emergence of aesthetic facial surgery. Classic ideals exist, but few individuals actually satisfy these criteria. Aesthetic ideals are culturally and temporally variable. For centuries, the optimum nasal profile, particularly the supratip break (STB), has long been a matter for debate. Little is published on preferences for the STB. We therefore undertook a population study to determine the preferred angulation of the STB. METHODS: Lateral profile photographs of 10 models were taken under standard conditions in forward gaze. The angulation of the STB was altered in one of the photographs of each model, so that one photograph had a subtle angulation of the STB and the other had a more marked angulation. Participants in the study were shown 2 photographs of each of the 10 models. One hundred male and female participants were recruited to the study and asked to indicate their preference for the nasal tip for each of the models. Statistical analysis was performed using the Wilcoxon signed-rank test to determine significance. RESULTS: Both male and female participants in the study indicated an overall, and statistically significant, preference for lateral profile photographs showing a nasal tip with a very subtle angulation of the STB, compared with a larger angulation of the STB. CONCLUSIONS: This study provides strong evidence that a subtle STB in the white female subject is most favorable.
Assuntos
Estética , Osso Nasal/anatomia & histologia , Nariz/anatomia & histologia , Preferência do Paciente , Rinoplastia/métodos , Adulto , Feminino , Humanos , Irlanda , Masculino , Pessoa de Meia-Idade , Osso Nasal/cirurgia , Nariz/cirurgia , Fotografação , Estudos de Amostragem , Fatores Sexuais , População Branca , Adulto JovemRESUMO
BACKGROUND: The Pfannenstiel incision is used almost invariably in Caesarean section. With Caesarean section rates increasing, the Pfannenstiel is a commonly performed incision. The prevailing recommendation is not to close the peritoneum when closing a Pfannenstiel incision, and peritoneal non-closure does not appear to statistically influence postoperative complication rates. CASE: A 33-year-old woman presented with severe, intermittent lower abdominal pain one year after a Caesarean section. Laparoscopy showed a hernial defect at the Pfannenstiel incision, between the left rectus abdominis muscle and the anterior rectus sheath. Mesh repair was performed with uncomplicated postoperative outcome. CONCLUSION: The current case illustrates that complications specific to non-closure of the peritoneum do arise. We advocate that laparoscopy should be considered for any patient with persistent, severe, or atypical pain following a Pfannenstiel incision.
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Cesárea/efeitos adversos , Hérnia Abdominal/etiologia , Adulto , Cesárea/métodos , Feminino , Hérnia Abdominal/diagnóstico , Hérnia Abdominal/cirurgia , Humanos , Laparoscopia , Complicações Pós-Operatórias/cirurgia , Reto do AbdomeRESUMO
BACKGROUND: Nipple-areolar complex (NAC) reconstruction following curative mastectomy is traditionally performed as a second-stage procedure several months after initial breast reconstruction. The recent literature has documented the increasing popularity of immediate nipple reconstruction carried out simultaneously during autologous reconstruction. The aim of this study was to evaluate the surgical outcomes and patient satisfaction with immediate breast and nipple reconstruction performed in a single stage after skin-sparing mastectomy. METHODS: All patients who underwent a skin-sparing mastectomy with immediate latissimus dorsi flap breast and NAC reconstruction as a single-stage procedure from 2007 to 2015 were included. Patient demographics, oncologic details, and surgical outcomes were recorded. The BREAST-Q questionnaire was administered to patients to assess the impact and effectiveness of this reconstructive strategy. RESULTS: During the study period, 34 breast and NAC reconstructions in 29 patients were performed at Cork University Hospital. The majority of our patient cohort were non-smokers (93.1%) and did not receive adjuvant radiotherapy. Postoperative complications were infrequent, with no cases of partial necrosis or complete loss of the nipple. The response rate to the BREAST-Q was 62% (n=18). Patients reported high levels of satisfaction with the reconstructed breast (62±4), nipple reconstruction (61±4.8), overall outcome (74.3±5), and psychosocial well-being (77.7±3.2). CONCLUSIONS: Skin-sparing mastectomy with immediate nipple reconstruction during autologous latissimus dorsi reconstruction was demonstrated to be a safe and aesthetically reliable procedure in our cohort, yielding high levels of psychological and physical well-being. A single-stage procedure promotes psychosocial well-being involving issues that are intrinsically linked with breast cancer surgery.
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Adipose tissue represents an abundant and easily accessible source of multipotent cells, which may serve as excellent building blocks for tissue engineering. This article presents a newly described protocol for isolating adipose-derived stromal cells (ASCs) from human lipoaspirate, compared to the standard protocol for harvesting ASCs established in 2001. Human ASC isolation is performed using two methods, and resultant cells are compared through cell yield, cell viability, cell proliferation and regenerative potential. The osteogenic and adipogenic potential of ASCs isolated using both protocols are assessed in vitro and gene expression analysis is performed. The focus of this series of protocols is the regenerative potential of both cell populations in vivo. As such, the two in vivo animal models described are fat graft retention (soft tissue reconstruction) and calvarial defect healing (bone regeneration). The techniques described comprise fat grafting with cell assisted lipotransfer, and calvarial defect creation healed with cell-seeded scaffolds. © 2017 by John Wiley & Sons, Inc.
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Adipogenia , Tecido Adiposo , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Osteogênese , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Transplante de Células , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
Diabetes mellitus (DM) is a metabolic disease frequently associated with impaired bone healing. Despite its increasing prevalence worldwide, the molecular etiology of DM-linked skeletal complications remains poorly defined. Using advanced stem cell characterization techniques, we analyzed intrinsic and extrinsic determinants of mouse skeletal stem cell (mSSC) function to identify specific mSSC niche-related abnormalities that could impair skeletal repair in diabetic (Db) mice. We discovered that high serum concentrations of tumor necrosis factor-α directly repressed the expression of Indian hedgehog (Ihh) in mSSCs and in their downstream skeletogenic progenitors in Db mice. When hedgehog signaling was inhibited during fracture repair, injury-induced mSSC expansion was suppressed, resulting in impaired healing. We reversed this deficiency by precise delivery of purified Ihh to the fracture site via a specially formulated, slow-release hydrogel. In the presence of exogenous Ihh, the injury-induced expansion and osteogenic potential of mSSCs were restored, culminating in the rescue of Db bone healing. Our results present a feasible strategy for precise treatment of molecular aberrations in stem and progenitor cell populations to correct skeletal manifestations of systemic disease.
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Fraturas do Fêmur/tratamento farmacológico , Consolidação da Fratura/efeitos dos fármacos , Proteínas Hedgehog/farmacologia , Células-Tronco Mesenquimais/citologia , Nicho de Células-Tronco , Animais , Osso e Ossos/patologia , Proliferação de Células , Separação Celular , Diabetes Mellitus Experimental/patologia , Feminino , Citometria de Fluxo , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Osteogênese , Transdução de SinaisRESUMO
Scaffold-mediated gene delivery holds great promise for tissue regeneration. However, previous attempts to induce bone regeneration using scaffold-mediated non-viral gene delivery rarely resulted in satisfactory healing. We report a novel platform with sustained release of minicircle DNA (MC) from PLGA scaffolds to accelerate bone repair. MC was encapsulated inside PLGA scaffolds using supercritical CO2 , which showed prolonged release of MC. Skull-derived osteoblasts transfected with BMP-2 MC in vitro result in higher osteocalcin gene expression and mineralized bone formation. When implanted in a critical-size mouse calvarial defect, scaffolds containing luciferase MC lead to robust in situ protein production up to at least 60 days. Scaffold-mediated BMP-2 MC delivery leads to substantially accelerated bone repair as early as two weeks, which continues to progress over 12 weeks. This platform represents an efficient, long-term nonviral gene delivery system, and may be applicable for enhancing repair of a broad range of tissues types. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2099-2107, 2016.
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Proteína Morfogenética Óssea 2/administração & dosagem , DNA Circular/metabolismo , Técnicas de Transferência de Genes , Crânio/patologia , Alicerces Teciduais/química , Cicatrização , Animais , Proteína Morfogenética Óssea 2/metabolismo , Luciferases/metabolismo , Camundongos , Camundongos Nus , Osteogênese , Ácido Poliglicólico/química , Regulação para Cima , Microtomografia por Raio-XRESUMO
BACKGROUND: The authors have developed a novel protocol for isolating adipose-derived stem cells from human lipoaspirate. In this study, they compare their new method to a previously published standard protocol. METHODS: Human adipose-derived stem cell isolation was performed using two methods to compare cell yield, cell viability, cell proliferation, and regenerative potential. The new and conventional isolation methods differ in two key areas: the collagenase digestion buffer constituents and the use of an orbital shaker. The osteogenic and adipogenic potential of adipose-derived stem cells isolated using both protocols was assessed in vitro, and gene expression analysis was performed. To assess the ability of the isolated cells to generate bone in vivo, the authors created critical-size calvarial defects in mice, which were treated with adipose-derived stem cells loaded onto hydroxyapatite-coated poly(lactic-co-glycolic acid) scaffolds. To test the ability of the isolated cells to enhance adipogenesis, the cells were added to lipoaspirate and placed beneath the scalp of immunocompromised mice. Fat graft volume retention was subsequently assessed by serial computed tomographic volumetric scanning. RESULTS: The new method resulted in a 10-fold increased yield of adipose-derived stem cells compared with the conventional method. Cells harvested using the new method demonstrated significantly increased cell viability and proliferation in vitro (p < 0.05). New method cells also demonstrated significantly enhanced osteogenic and adipogenic differentiation capacity in vitro (p < 0.05) in comparison with the conventional method cells. Both cell groups demonstrated equivalent osteogenic and adipogenic regenerative potential in mice. CONCLUSIONS: The authors have developed a protocol that maximizes the yield of adipose-derived stem cells derived from lipoaspirate. The new method cells have increased osteogenic and adipogenic potential in vitro and are not inferior to conventional method cells in terms of their ability to generate bone and fat in vivo. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.
Assuntos
Separação Celular/métodos , Células-Tronco Mesenquimais , Gordura Subcutânea/citologia , Adipogenia , Animais , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Feminino , Humanos , Lipectomia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Camundongos , Osteogênese , Engenharia TecidualRESUMO
Bone is a dynamic tissue, with a range of diverse functions, including locomotion, protection of internal organs, and hematopoiesis. Optimum treatment of fractures and/or bone defects requires knowledge of the complex cellular interactions involved with bone healing and remodeling. Emerging data have underscored the importance of osteoclasts in this process, playing a key role both in normal bone turnover and in facilitating bone regeneration. In this review, the authors discuss the basic principles of osteoclast biology, including its cellular origins, its function, and key regulatory mechanisms, in addition to conditions that arise when osteoclast function is altered.
Assuntos
Remodelação Óssea/fisiologia , Reabsorção Óssea/metabolismo , Fraturas Ósseas/metabolismo , Osteoclastos/fisiologia , Animais , Reabsorção Óssea/patologia , Fraturas Ósseas/patologia , Homeostase , HumanosRESUMO
Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.